How can you combine professional-quality research with discovery-based undergraduate education? The UCLA Undergraduate Consortium for Functional Genomics provides the answer

The excitement of scientific research and discovery cannot be fully conveyed by didactic lectures alone. Several recent initiatives and proposals, therefore, have supported a more participatory, discovery-based instruction for undergraduate science education [ 1 , 2 ]. In functional genomics, we have found an ideal platform to simultaneously benefit students and contribute to scientific discovery. The sequencing of eukaryotic genomes has facilitated the identification of complete sets of genes in humans and model genetic organisms. This has allowed many forms of high-throughput analyses of transcriptional profiles, protein interactions, structural motifs, and even genome-wide knock-downs in cell lines or in selected organisms. However, one of the best tools to provide functional information about gene action— obtaining in vivo evidence about the phenotype resulting from heritable loss of function—is difficult and less amenable to high-throughput research. We were able to achieve a large-scale in vivo analysis with a significant number of undergraduate students at UCLA, called the UCLA Undergraduate Consortium for Functional Genomics. This work, a practical manifestation of policy positions proposing discoverybased education, is described in summary form here (and in  Box 1 ) and in detail online at  http://www.bruinfly.ucla.edu . This effort combines professional-quality research with a strategy for research-based undergraduate education.Box 1. Scientific Results The  Drosophila  eye is an intricate neurocrystalline lattice of approximately 800 individual ommatidia arrayed in a very precise order ( Figure 3A ) [ 3 ]. Minor perturbations in ommatidial development can be easily detected, making it a very sensitive system for functional genomic screens [ 3 ]. Our study utilized 1,375 unique recessive lethal transposable element (P-element) insertion stocks from the 2nd and 3rd chromosomes of  Drosophila  to characterize their later role in eye development. To avoid early lethality, the FLP/FRT system was used to generate homozygous mutant tissue specifically in the eye [ 4 ]. Of the mutations analyzed, 501 (36%) displayed a mutant eye phenotype, providing the first genome-wide estimate of the fraction of essential genes that are also involved in eye development. Adult eye phenotypes were classified into three broad classes: rough, cell lethal, and glossy ( Figure 3 ). The genes responsible for these phenotypes were assigned into 19 different functional categories, which are summarized in  Table 1 . Signal transduction components previously established to be important for eye development (e.g,.  EGFR ,  pointed ,  Star ,  tramtrak ,  Delta ) were identified, validating the effectiveness of our screen. In addition, our genomics approach has shown that a number of novel classes of genes are involved in eye development that have not been previously described ( Table 1 ).  Figure 3 Summary of Phenotypes Determined Light (left panels) and scanning electron (right panels) micrographs of mosaic Drosophila eyes. Large homozygous mutant clones are orange (arrowheads); heterozygous tissues are dark red. Examples are shown of lethal mutations that give a (A) wild-type (63.4% of lethal mutations), (B) rough (disordered ommatidia, 18.2%), (C) cell lethal (absence of homozygous mutant tissue, 14.5%), and (D) glossy (loss of lens structure, 3.9%) phenotype. For details on how clones are generated, see  http://www.bruinfly.ucla.edu . Table 1 Summary of Genes Involved in Adult Eye Development Molecular information is available for 315 of the 501 phenotypic mutants. Of these, 291 were identified as unique genes and categorized into 19 classes, based solely on the insertion site data from FlyBase
Box 1. Scientific Results
The  Drosophila  eye is an intricate neurocrystalline lattice of approximately 800 individual ommatidia arrayed in a very precise order ( Figure 3A ) [ 3 ]. Minor perturbations in ommatidial development can be easily detected, making it a very sensitive system for functional genomic screens [ 3 ]. Our study utilized 1,375 unique recessive lethal transposable element (P-element) insertion stocks from the 2nd and 3rd chromosomes of  Drosophila  to characterize their later role in eye development. To avoid early lethality, the FLP/FRT system was used to generate homozygous mutant tissue specifically in the eye [ 4 ]. Of the mutations analyzed, 501 (36%) displayed a mutant eye phenotype, providing the first genome-wide estimate of the fraction of essential genes that are also involved in eye development. Adult eye phenotypes were classified into three broad classes: rough, cell lethal, and glossy ( Figure 3 ). The genes responsible for these phenotypes were assigned into 19 different functional categories, which are summarized in  Table 1 . Signal transduction components previously established to be important for eye development (e.g,.  EGFR ,  pointed ,  Star ,  tramtrak ,  Delta ) were identified, validating the effectiveness of our screen. In addition, our genomics approach has shown that a number of novel classes of genes are involved in eye development that have not been previously described ( Table 1 ).EGFRpointedStartramtrakDeltaSummary of Genes Involved in Adult Eye Development
Molecular information is available for 315 of the 501 phenotypic mutants. Of these, 291 were identified as unique genes and categorized into 19 classes, based solely on the insertion site data from FlyBaseWe have created a novel curriculum with three main components: didactic, computer, and laboratory. The only prerequisite for this course is high school advanced-placement-level biology; all other knowledge necessary for the course is taught within it. Since there are no other prerequisites for the course, a majority of the students enrolled are freshmen and sophomores, enabling us to educate them in this novel way early in their undergraduate career. Approximately 30 students take this course each quarter, and it is offered every quarter through the school year, allowing for a broad impact. In the lecture series, students are exposed to interactive lectures on background material, basic concepts of genetics, research ethics, and career options. For their “midterm,” each student proposes an experiment in a grant proposal formatted according the National Institutes of Health requirements. The “final” is written as a scientific paper summarizing the student's own results. In the computer section, students perform research with a “virtual fly lab” to help them understand more about their crosses in the laboratory section. In addition, they learn about modern genomic resources available on the Internet, and utilize some of the genomics tools available (e.g., BLAST) to help them determine the identity and function of their disrupted genes. The main component of the class, however, is the laboratory portion.In the laboratory, the students perform all the necessary work to manipulate the genotypes of their stocks to determine what effect homozygous mutation of their target genes has in the adult  Drosophila  eye ( Figure 1 ). To accomplish this, the students perform five-generation  Drosophila  crosses that nicely fit into a ten-week quarter. Each student is assigned about ten mutants to work with. During the quarter, students are able to recombine each mutation onto a flippase (FLP) recombination target (FRT) chromosome, generate mutant somatic clones, and record details of the adult eye phenotype with both light and scanning electron microscopic techniques. The students then upload their data into an online database ( http://www.bruinfly.ucla.edu ).Our database contains pictures of the mutant eyes for all of the stocks examined, as well as other information pertinent to that stock, including the gene disrupted, the exact genomic location of the P-element insertion, and whether an excision of the P-element has been performed and its results. A sample Web page of the database is shown in  Figure 2 . Following the introductory course, a small number of students continue to analyze the developmental basis for select mutations in future quarters in more advanced laboratory classes. In these advanced classes, the students perform P-element excision experiments to determine whether the mutant phenotype observed is indeed derived from the P-element. These students have performed 294 excision experiments, the results of which indicate that 72% of the stocks successfully revert to wildtype phenotype when the P-element is removed. Over the last two years, we have educated 138 students in the introductory course. Advanced classes have totaled 96 student-quarters (46 students, each working two or more additional quarters).In summary, discovery-based experiments in functional genomics are well suited for undergraduate education: they actively engage a large number of students in research without compromising their didactic training. The sense of ownership developed from this research amplifies the students' learning experience. For the research community, the online database and the large collection of newly generated FRT-lethal lines represent a valuable resource for future experiments in eye development. Furthermore, the stocks developed can be used to create mutant clones in an investigator's tissue of choice. This novel approach for performing research, for which functional genomics is very amenable, not only encourages many students in new ways of thinking but also generates professional-quality results and resources for the scientific community.Supporting Information Figure S1 A Compilation of the Undergraduates and Some of Their Fly Mutants (1.2 MB JPG). Click here for additional data file.
Supporting Information
A Compilation of the Undergraduates and Some of Their Fly Mutants
(1.2 MB JPG).Click here for additional data file.


Retrovirology has been publishing as an Open Access online journal for approximately six months. In this editorial, I review the reasons for and the advantages of Open Access publishing, update our progress to date, and summarize where we intend to go with this journal.

Qadaffi's lesson Thirty-five years ago, on September 1 st , 1969, I was an eleven year old boy living with my family in Benghazi, Libya. That morning, I awoke to an eerie silence. The normally bustling traffic outside of our apartment in a busy suburb of Benghazi was not to be heard. In fact, there was to be little or no traffic for the next several days. History will recount that day as the first day of Mohmmar Qadaffi's bloodless coup d'etat, seizing power in Libya. What I learned that September week in 1969 was that access to and distribution of information tipped the fate of a state. On that September day, there were in fact two groups of army officers vying to overthrow the then enfeebled Libyan King Idris. Qadaffi, at the time only twenty-seven years old, headed a small group of very junior officers. He, however, had a bold and swift strategy. His first targets were not the dams, the power stations, the police stations...; but the radio stations (in 1969 television had a very minor penetrance in Libya) and newspapers. By dint of controlling the airwaves and printing presses and by freely distributing  his  information during time of chaos, Qadaffi held sway over the minds and the sentiments of the populace. He was thus able to pre-empt the efforts of a larger competing group of more senior army officers. What does Qadaffi's story have to do with Open Access publishing? Imagine how the outcome might have been different in 1969 if broadcast radio was not freely and openly accessible by the masses but was an expensive subscription service limited to a few. In that setting, and perhaps in all settings, information that reaches a little audience at a steep cost is information with little effect.
Qadaffi's lesson
Thirty-five years ago, on September 1 st , 1969, I was an eleven year old boy living with my family in Benghazi, Libya. That morning, I awoke to an eerie silence. The normally bustling traffic outside of our apartment in a busy suburb of Benghazi was not to be heard. In fact, there was to be little or no traffic for the next several days. History will recount that day as the first day of Mohmmar Qadaffi's bloodless coup d'etat, seizing power in Libya.What I learned that September week in 1969 was that access to and distribution of information tipped the fate of a state. On that September day, there were in fact two groups of army officers vying to overthrow the then enfeebled Libyan King Idris. Qadaffi, at the time only twenty-seven years old, headed a small group of very junior officers. He, however, had a bold and swift strategy. His first targets were not the dams, the power stations, the police stations...; but the radio stations (in 1969 television had a very minor penetrance in Libya) and newspapers. By dint of controlling the airwaves and printing presses and by freely distributing  his  information during time of chaos, Qadaffi held sway over the minds and the sentiments of the populace. He was thus able to pre-empt the efforts of a larger competing group of more senior army officers.What does Qadaffi's story have to do with Open Access publishing? Imagine how the outcome might have been different in 1969 if broadcast radio was not freely and openly accessible by the masses but was an expensive subscription service limited to a few. In that setting, and perhaps in all settings, information that reaches a little audience at a steep cost is information with little effect.Retrovirology, freely and openly accessible by all Curiously, the force that drives a coup d'etat is the same that motivates scientists to publish (although I am not suggesting that scientists are like Qadaffi). As scientists, we want everyone who is interested to read what we have done; and ideally we want that information to get to the widest audience in the fastest, most informative, and least expensive manner. Until now, ideal and reality were two entirely different matters. Traditional publishing in print journals reaches a limited audience, is slow, bulky, and expensive. By its very nature (i.e. pages of paper are heavy to mail and expensive to print), there is a real impetus on the part of print publishers to restrict the space available to authors. We are all too familiar with the results: abbreviated presentation of methods and text, the necessity to "inadvertently" not cite many colleagues' publications, and the ubiquitous use of microscopic figure panels that provoke blurred vision and headaches. I daresay that many after having read a short "letter" or a "brief definitive" report have been left scratching heads and wondering how the experiments were done, where did the vectors come from, and why many controls were definitely missing. For the retrovirology community,  Retrovirology  aims to change such "reality".  Retrovirology  is published by BioMed Central, an independent publisher committed to ensuring immediate free access to peer-reviewed biomedical research.  Retrovirology  is Open Access which means that it is universally and freely available online to everyone. You as the author retain copyright, and your paper is guaranteed to be archived in at least one internationally recognised repository [ 1 ]. More importantly, publishing in  Retrovirology  is rapid. For example, a full research article was reviewed and published in less than 20 days [ 2 ]; and a review article was similarly processed in 8 days [ 3 ].  Retrovirology  will also give you all the space and all the colored prints (at no extra charge) to present your findings informatively and attractively. If we don't publish your paper, we will tell you honestly what is scientifically deficient about your work. I promise not to obfuscate myself using annoyingly belittling mantra that my editorial decision was guided by "intense competition for space".
Retrovirology, freely and openly accessible by all
Curiously, the force that drives a coup d'etat is the same that motivates scientists to publish (although I am not suggesting that scientists are like Qadaffi). As scientists, we want everyone who is interested to read what we have done; and ideally we want that information to get to the widest audience in the fastest, most informative, and least expensive manner. Until now, ideal and reality were two entirely different matters. Traditional publishing in print journals reaches a limited audience, is slow, bulky, and expensive. By its very nature (i.e. pages of paper are heavy to mail and expensive to print), there is a real impetus on the part of print publishers to restrict the space available to authors. We are all too familiar with the results: abbreviated presentation of methods and text, the necessity to "inadvertently" not cite many colleagues' publications, and the ubiquitous use of microscopic figure panels that provoke blurred vision and headaches. I daresay that many after having read a short "letter" or a "brief definitive" report have been left scratching heads and wondering how the experiments were done, where did the vectors come from, and why many controls were definitely missing.For the retrovirology community,  Retrovirology  aims to change such "reality".  Retrovirology  is published by BioMed Central, an independent publisher committed to ensuring immediate free access to peer-reviewed biomedical research.  Retrovirology  is Open Access which means that it is universally and freely available online to everyone. You as the author retain copyright, and your paper is guaranteed to be archived in at least one internationally recognised repository [ 1 ]. More importantly, publishing in  Retrovirology  is rapid. For example, a full research article was reviewed and published in less than 20 days [ 2 ]; and a review article was similarly processed in 8 days [ 3 ].  Retrovirology  will also give you all the space and all the colored prints (at no extra charge) to present your findings informatively and attractively. If we don't publish your paper, we will tell you honestly what is scientifically deficient about your work. I promise not to obfuscate myself using annoyingly belittling mantra that my editorial decision was guided by "intense competition for space".RetrovirologyRetrovirologyRetrovirologyRetrovirologyRetrovirologyJudge a book by its cover? This is my second editorial for  Retrovirology  readers [ 4 ].  Retrovirology  has been publishing on-line now for 6 months. While a first editorial can speak of dreams and aspirations, the burden of a second editorial is to demonstrate results and progress. Aided by an international cohort of extremely capable editors (Monsef Benkirane, Ben Berkhout, Masahiro Fujii, Michael Lairmore, Andrew Lever, and Mark Wainberg) and sixty editorial board members [ 5 ], I am indeed encouraged and gratified by  Retrovirology's  initial achievements. As of this writing, we have published 22 papers. These are good papers; and importantly, these papers are being widely-read. I can say with some pride that half (11 out of 22; see Table  1 ) of our published papers have been read more than 1,000 times each. The remaining papers are not far behind; our most recently published paper, barely 1 week on-line, has already had more than 200 readers. Given that retrovirology is a relatively small community of researchers, my personal experience of twenty years in this field tells me that these are very respectable numbers. Table 1 Access statistics for the top 11  Retrovirology  articles. Article Access Count 1 Early steps of retrovirus replicative cycle  Sébastien Nisole, Ali Saïb  Retrovirology  2004,  1 : 9 1890 2 Establishment of a novel CCR5 and CXCR4 expressing CD4+ cell line which is highly sensitive to HIV and suitable for high-throughput evaluation of CCR5 and CXCR4 antagonists  Katrien Princen, Sigrid Hatse, Kurt Vermeire, Erik De Clercq, Dominique Schols  Retrovirology  2004,  1 : 2 1602 3 Evolution of the HIV-1 envelope glycoproteins with a disulfide bond between gp120 and gp41  Rogier W Sanders, Martijn M Dankers, Els Busser, Michael Caffrey, John P Moore, Ben Berkhout  Retrovirology  2004,  1 : 3 1332 4 Use of a multi-virus array for the study of human viral and retroviral pathogens: gene expression studies and ChIP-chip analysis  Elodie Ghedin, Anne Pumfery, Cynthia de la Fuente, Karen Yao, Naomi Miller, Vincent Lacoste, John Quackenbush, Steven Jacobson, Fatah Kashanchi  Retrovirology  2004,  1 : 10 1313 5 Increased mortality associated with HTLV-II infection in blood donors: a prospective cohort study  Jennie R Orland, Baoguang Wang, David J Wright, Catharie C Nass, George Garratty, James W Smith, Bruce Newman, Donna M Smith, Edward L Murphy,  Retrovirology  2004,  1 : 4 1209 6 Retrovirology  and young Turks...   Kuan-Teh Jeang  Retrovirology  2004,  1 : 1 1140 7 HIV-1 gene expression: lessons from provirus and non-integrated DNA  Yuntao Wu  Retrovirology  2004,  1 : 13 1103 8 Multi-faceted, multi-versatile microarray: simultaneous detection of many viruses and their expression profiles  Biehuoy Shieh, Ching Li  Retrovirology  2004,  1 : 11 1097 9 Two discrete events, human T-cell leukemia virus type I Tax oncoprotein expression and a separate stress stimulus, are required for induction of apoptosis in T-cells  Takefumi Kasai, Kuan-Teh Jeang  Retrovirology  2004,  1 : 7 1088 10 Apoptosis of uninfected cells induced by HIV envelope glycoproteins  Barbara Ahr, Véronique Robert-Hebmann, Christian Devaux, Martine Biard-Piechaczyk  Retrovirology  2004,  1 : 12 1076 11 HIV CTL escape: at what cost?  Stephen M Smith  Retrovirology  2004,  1 : 8 1022 Despite that many of you would argue to me that you would rather publish in  Cell ,  Science , and  Nature  rather than  Retrovirology . I wouldn't dispute you on that point. On the other hand, I would ask you to pause and think about a refrain that I am sure all of you have taught your children, "Don't judge a book by its cover!" Please, don't sell your own work short; and please don't belittle the objectivity of your colleagues. If you have a wonderful piece of work, do you truly need the cover of  Cell  to establish that point? On the other hand, if you have a horrible study, do you think that hiding it behind the cover of  Nature  would fool many of your colleagues for long? There was a time in the print age when display space on reading room shelves was limited and perhaps justifiably dominated by "the big three". Today,  Retrovirology  will get your paper listed in PubMed within 48 hours of acceptance for publication. Anyone in this electronic era whose reading habits are not guided by PubMed in one way or another is probably someone who you may not care to read your study. In short,  Retrovirology  will promise your immediacy of publication and visibility for your work. Your papers will be well-read by your colleagues. Beyond that, I believe, and I think you do too, the impact of your work should rest on its merits and not on our or any other journal's cover.
Judge a book by its cover?
This is my second editorial for  Retrovirology  readers [ 4 ].  Retrovirology  has been publishing on-line now for 6 months. While a first editorial can speak of dreams and aspirations, the burden of a second editorial is to demonstrate results and progress. Aided by an international cohort of extremely capable editors (Monsef Benkirane, Ben Berkhout, Masahiro Fujii, Michael Lairmore, Andrew Lever, and Mark Wainberg) and sixty editorial board members [ 5 ], I am indeed encouraged and gratified by  Retrovirology's  initial achievements. As of this writing, we have published 22 papers. These are good papers; and importantly, these papers are being widely-read. I can say with some pride that half (11 out of 22; see Table  1 ) of our published papers have been read more than 1,000 times each. The remaining papers are not far behind; our most recently published paper, barely 1 week on-line, has already had more than 200 readers. Given that retrovirology is a relatively small community of researchers, my personal experience of twenty years in this field tells me that these are very respectable numbers.RetrovirologyRetrovirologyRetrovirology'sAccess statistics for the top 11  Retrovirology  articles.RetrovirologyArticleAccess CountEarly steps of retrovirus replicative cycleRetrovirology1Establishment of a novel CCR5 and CXCR4 expressing CD4+ cell line which is highly sensitive to HIV and suitable for high-throughput evaluation of CCR5 and CXCR4 antagonistsRetrovirology1Evolution of the HIV-1 envelope glycoproteins with a disulfide bond between gp120 and gp41Retrovirology1Use of a multi-virus array for the study of human viral and retroviral pathogens: gene expression studies and ChIP-chip analysisRetrovirology1Increased mortality associated with HTLV-II infection in blood donors: a prospective cohort studyRetrovirology1Retrovirology  and young Turks...RetrovirologyRetrovirology1HIV-1 gene expression: lessons from provirus and non-integrated DNARetrovirology1Multi-faceted, multi-versatile microarray: simultaneous detection of many viruses and their expression profilesRetrovirology1Two discrete events, human T-cell leukemia virus type I Tax oncoprotein expression and a separate stress stimulus, are required for induction of apoptosis in T-cellsRetrovirology1Apoptosis of uninfected cells induced by HIV envelope glycoproteinsRetrovirology1HIV CTL escape: at what cost?Retrovirology1Despite that many of you would argue to me that you would rather publish in  Cell ,  Science , and  Nature  rather than  Retrovirology . I wouldn't dispute you on that point. On the other hand, I would ask you to pause and think about a refrain that I am sure all of you have taught your children, "Don't judge a book by its cover!" Please, don't sell your own work short; and please don't belittle the objectivity of your colleagues. If you have a wonderful piece of work, do you truly need the cover of  Cell  to establish that point? On the other hand, if you have a horrible study, do you think that hiding it behind the cover of  Nature  would fool many of your colleagues for long? There was a time in the print age when display space on reading room shelves was limited and perhaps justifiably dominated by "the big three". Today,  Retrovirology  will get your paper listed in PubMed within 48 hours of acceptance for publication. Anyone in this electronic era whose reading habits are not guided by PubMed in one way or another is probably someone who you may not care to read your study. In short,  Retrovirology  will promise your immediacy of publication and visibility for your work. Your papers will be well-read by your colleagues. Beyond that, I believe, and I think you do too, the impact of your work should rest on its merits and not on our or any other journal's cover.CellScienceNatureRetrovirologyCellNatureRetrovirologyRetrovirologyOpen Access is not free lunch Besides teaching about book covers, I think the other concept that we teach our children is that "There is no free lunch!" At the end of the day, anything worth doing has a price tag. I, my colleague editors, and all the editorial board members at  Retrovirology  work for free for the journal. We can do this because we collect a salary elsewhere doing research. On the other hand, we do have a dedicated staff based at the publisher in London. No one would question that the salaries for these professionals as well as other publication costs must be borne. To fund these costs, from October 1, 2004, authors of articles accepted for publication will be asked to pay a single all-inclusive article-processing charge (APC) of US$600; there are no additional charges beyond this sum.  Retrovirology  will not levy additional page or colour charges on top of this fee. You are free to publish any number of colour figures and photographs, at no extra cost. I support the need for APC, because without this  Retrovirology  cannot be published. I think most of you will agree based on your experience with other publishers that US$600 is more than modest and reasonable. As a point of comparison, the Public Library of Science has set up two new Open Access journals, and has set their APCs at US$1500, fully two and one-half times our charge, for each accepted article [ 6 ]. Nonetheless, I realize that any APC fee can present an unacceptable financial hardship for some authors. These authors should feel free to contact me in confidence, and I will consider fee-waiver requests on a case-by-case basis. In closing, let me thank those of you who have read, submitted and reviewed papers for  Retrovirology . More than anything else,  Retrovirology  is not my journal as much as it is your journal. I welcome your advice, and I look forward to hearing from you.
Open Access is not free lunch
Besides teaching about book covers, I think the other concept that we teach our children is that "There is no free lunch!" At the end of the day, anything worth doing has a price tag. I, my colleague editors, and all the editorial board members at  Retrovirology  work for free for the journal. We can do this because we collect a salary elsewhere doing research. On the other hand, we do have a dedicated staff based at the publisher in London. No one would question that the salaries for these professionals as well as other publication costs must be borne. To fund these costs, from October 1, 2004, authors of articles accepted for publication will be asked to pay a single all-inclusive article-processing charge (APC) of US$600; there are no additional charges beyond this sum.  Retrovirology  will not levy additional page or colour charges on top of this fee. You are free to publish any number of colour figures and photographs, at no extra cost.RetrovirologyRetrovirologyI support the need for APC, because without this  Retrovirology  cannot be published. I think most of you will agree based on your experience with other publishers that US$600 is more than modest and reasonable. As a point of comparison, the Public Library of Science has set up two new Open Access journals, and has set their APCs at US$1500, fully two and one-half times our charge, for each accepted article [ 6 ]. Nonetheless, I realize that any APC fee can present an unacceptable financial hardship for some authors. These authors should feel free to contact me in confidence, and I will consider fee-waiver requests on a case-by-case basis.RetrovirologyIn closing, let me thank those of you who have read, submitted and reviewed papers for  Retrovirology . More than anything else,  Retrovirology  is not my journal as much as it is your journal. I welcome your advice, and I look forward to hearing from you.RetrovirologyRetrovirologyAbbreviations APC = article-processing charge.
Abbreviations
APC = article-processing charge.


In 1994, the landmark "Cairo Conference" on population and development promised reproductive health for all. Ten years later, what has been achieved?

About a decade ago, I went wandering around Cairo's City of the Dead. This might sound like a grim bit of tourism, but my connection to that vast necropolis runs deep—quite literally, as my family is buried there. After visiting their grave, I rambled through the city's dusty alleyways, past hundreds of years of history. Yet what I remember most about that day was not one of the many magnificent tombs, but a simple brick building with a sign, of all things, for a family planning clinic.I was certainly surprised by my discovery; in retrospect, I should not have been. That part of Cairo is home to hundreds of thousands of people for whom looking after the dead is a way of life. Their fertility invigorates the funereal air: the caretaker of my family's tomb, for example, had a blooming family of his own living near the grave. Where better to offer family planning than in a place so poor that reproduction seemed more a matter of fate than choice?The Cairo Conference That visit is a fitting metaphor for the field of reproductive health as a whole. Ten years ago, officials, experts, and activists from 179 countries also came to Cairo for the International Conference on Population and Development (ICPD). The conference produced a 20-year plan of action that focused on universal access to reproductive health services, including family planning and sexual health; reducing infant, child, and maternal mortality; better education, especially for girls; equality between men and women; and sustainable development. The ICPD's key achievement was to reorient thinking on reproduction away from narrowly defined, government-dictated population control to a broader appreciation of reproductive and sexual well-being within health care systems, a view driven by individual choice and rights, not official priorities. “The Cairo Conference was a peak moment,” says Sally Ethelston, vice president for communications at Population Action International, one member of a consortium of non-governmental organisations launching a report card to mark the anniversary of the Cairo Conference in early September. “There were times when people were excited that they had accomplished something, and you could see it on their faces.” Today, however, the mood is very different. While progress has been made on some of the plan's targets, effort has faltered on others. And the conference “camaraderie” that Ethelston describes has given way to conflict between faith and science, over abortion and condoms. Like signs of life in the City of the Dead, the Cairo Conference gave birth to great expectations, some of which have already expired.
The Cairo Conference
That visit is a fitting metaphor for the field of reproductive health as a whole. Ten years ago, officials, experts, and activists from 179 countries also came to Cairo for the International Conference on Population and Development (ICPD). The conference produced a 20-year plan of action that focused on universal access to reproductive health services, including family planning and sexual health; reducing infant, child, and maternal mortality; better education, especially for girls; equality between men and women; and sustainable development.The ICPD's key achievement was to reorient thinking on reproduction away from narrowly defined, government-dictated population control to a broader appreciation of reproductive and sexual well-being within health care systems, a view driven by individual choice and rights, not official priorities. “The Cairo Conference was a peak moment,” says Sally Ethelston, vice president for communications at Population Action International, one member of a consortium of non-governmental organisations launching a report card to mark the anniversary of the Cairo Conference in early September. “There were times when people were excited that they had accomplished something, and you could see it on their faces.”Today, however, the mood is very different. While progress has been made on some of the plan's targets, effort has faltered on others. And the conference “camaraderie” that Ethelston describes has given way to conflict between faith and science, over abortion and condoms. Like signs of life in the City of the Dead, the Cairo Conference gave birth to great expectations, some of which have already expired.Baby Steps Towards Cairo's Goals So, how far has the developing world come towards meeting the ICPD goals? There has certainly been progress on institutional reform in some countries, according to a recent survey of national policies by the United Nations Population Fund (UNFPA) [ 1 ]. For example, more than a third of the 151 countries questioned have introduced legislation on reproductive rights, and almost half have expanded their primary health care services to include family planning.
 The birth of a baby on August 15, 2000, brought India's population to one billion (Photo: Raghu Rai, on behalf of the David and Lucile Packard Foundation) But translating policy into action has been difficult. Overall, the picture is one of patchy success, according to Susheela Singh, director of research at The Alan Guttmacher Institute, a nongovernmental research organisation. Official statistics, as limited as they are for many aspects of reproductive and sexual health, show mixed results. On a positive note, global population growth has slowed to roughly 77 million people a year [ 2 ]. But while fertility rates have fallen in some developing countries, such as Mexico, they remain stubbornly high in others, such as Ethiopia [ 3 ]. Over the past decade, contraceptive use has grown, but so has demand, and there are now an estimated 201 million women in developing countries whose need for modern birth control goes unmet, resulting in 60 million unintended pregnancies a year [ 4 ]. Progress on legalising abortion has been slow, and an estimated 19 million abortions a year still occur under unsafe conditions [ 5 ]. Despite growing awareness of sexually transmitted disease, the annual number of sexually transmitted infections remains worryingly high at 340 million worldwide [ 6 ]. While infant mortality rates have improved somewhat, maternal mortality figures have barely budged. An estimated 529,000 women still die every year from complications of pregnancy and childbirth. The highest rates are in sub-Saharan Africa, where, on average, 920 women die for every 100,000 live births, compared with 24 deaths per 100,000 live births in Europe [ 7 ]. This is all the more distressing, says Vivien Tsu, senior programme officer at the Program for Appropriate Technology in Health, because these women's lives could be saved through straightforward measures and basic technologies, such as access to skilled midwives, simple drugs like magnesium sulphate for eclampsia and oxytocin for post-partum bleeding, cellular phones to call for help, and transportation to emergency obstetric centres.
Baby Steps Towards Cairo's Goals
So, how far has the developing world come towards meeting the ICPD goals? There has certainly been progress on institutional reform in some countries, according to a recent survey of national policies by the United Nations Population Fund (UNFPA) [ 1 ]. For example, more than a third of the 151 countries questioned have introduced legislation on reproductive rights, and almost half have expanded their primary health care services to include family planning.But translating policy into action has been difficult. Overall, the picture is one of patchy success, according to Susheela Singh, director of research at The Alan Guttmacher Institute, a nongovernmental research organisation. Official statistics, as limited as they are for many aspects of reproductive and sexual health, show mixed results. On a positive note, global population growth has slowed to roughly 77 million people a year [ 2 ]. But while fertility rates have fallen in some developing countries, such as Mexico, they remain stubbornly high in others, such as Ethiopia [ 3 ]. Over the past decade, contraceptive use has grown, but so has demand, and there are now an estimated 201 million women in developing countries whose need for modern birth control goes unmet, resulting in 60 million unintended pregnancies a year [ 4 ]. Progress on legalising abortion has been slow, and an estimated 19 million abortions a year still occur under unsafe conditions [ 5 ]. Despite growing awareness of sexually transmitted disease, the annual number of sexually transmitted infections remains worryingly high at 340 million worldwide [ 6 ].While infant mortality rates have improved somewhat, maternal mortality figures have barely budged. An estimated 529,000 women still die every year from complications of pregnancy and childbirth. The highest rates are in sub-Saharan Africa, where, on average, 920 women die for every 100,000 live births, compared with 24 deaths per 100,000 live births in Europe [ 7 ]. This is all the more distressing, says Vivien Tsu, senior programme officer at the Program for Appropriate Technology in Health, because these women's lives could be saved through straightforward measures and basic technologies, such as access to skilled midwives, simple drugs like magnesium sulphate for eclampsia and oxytocin for post-partum bleeding, cellular phones to call for help, and transportation to emergency obstetric centres.Obstacles to Reproductive Health So why hasn't more been achieved? One problem is certainly money. The 1994 Cairo Conference estimated the cost of implementing programmes for family planning, maternal health, and prevention of sexually transmitted diseases, as well as data collection and analysis in developing countries, at $18.5 billion by 2005—or $24.3 billion in today's dollars. The goal was to mobilise one-third of the money from donor nations, and the rest from developing countries themselves [ 8 ]. Last year, global spending on reproductive health and services reached $14.7 billion, according to estimates from UNFPA, the Joint United Nations Programme on HIV/AIDS, and the Netherlands Interdisciplinary Demographic Institute [ 8 ]. Encouragingly, investment has increased since 2001, when the momentum of ICPD seemed to falter and international spending fell to $9 billion. But this is still wide of the mark. While developing countries have failed to meet their conference commitments, it is donor countries that are most remiss: rich country contributions reached an estimated $2.3 billion in 2003 [ 8 ], a far cry from the conference target of $6.1 billion (or $8.1 billion in today's dollars) by 2005. Reproductive health is not alone in waiting for donors to give generously. For all the rhetoric at international summits, few rich countries have lived up to their lofty pledges of debt relief and of dedicating 0.7% of their gross domestic product to overseas development assistance. But as Steve Sinding, head of the International Planned Parenthood Federation (IPPF), points out, there are other reasons too for the shortfall. In the past donor interest was largely stimulated by fears of a population crisis. When the Cairo Conference reframed issues in terms of women's health and reproductive rights, rather than an impending population explosion, Sinding argues, the “demographic rationale” was lost, taking funding with it. Moreover, there are other issues competing for international funding, most notably AIDS. At the time of the Cairo Conference, 20 million people were infected with HIV; today the number has grown to an estimated 38 million [ 9 ]. AIDS threatens to derail the Cairo Conference plan of action. Through maternal-to-child transmission, and wide-scale orphaning, HIV threatens to reverse small successes at reducing infant and child mortality. By killing off teachers and sapping household incomes, AIDS is sabotaging education. By killing off scarce medical workers and overwhelming fragile health care systems, the disease is compromising reproductive health services. Gender equity is undermined, as women and girls bear the brunt of the epidemic, as caregivers, breadwinners, or patients themselves. Roughly half of the money spent on reproductive health last year went towards HIV/AIDS. And billions more is on the way, from the likes of the Global Fund to Fight AIDS, Tuberculosis, and Malaria and the United States President's Emergency Plan for AIDS Relief, which promises $15 billion over five years to HIV/AIDS programmes [ 10 ]. But much of this money is going into AIDS-specific programmes that do not address reproductive health more broadly. Even as the world is gearing up to scale up AIDS prevention and treatment to millions worldwide, few of the agencies involved come from the world of reproductive and sexual health. This is a pity because it means that HIV/AIDS programmes are not making use of valuable infrastructure and expertise already on the ground in places where AIDS hits hardest. Given that 57% of HIV infections in sub-Saharan Africa are among women [ 9 ], and that, for many of them, family planning clinics are their sole contact with the formal health care system, it seems odd not to integrate such services into the wider battle against HIV. Such centres can offer not only HIV testing and counselling, as well as condoms (against the double whammy of unwanted pregnancy and HIV infection), but also a broad-based message of good sexual health that can help protect against HIV and other sexually transmitted diseases. Moreover, pre- and ante-natal care provide an opportunity to stop mother-to-child transmission of HIV in its tracks.
 Condom distribution in Soweto, South Africa (Photo: Arjen van de Merwe, Population Concern) Where once family planning was the darling of international donors, HIV is now the cause célèbre. “There's a lot of resentment about the spotlight moving on,” says Kevin O'Reilly, a former reproductive health specialist now at the department of HIV/AIDS at the World Health Organization. However, there are now attempts to bring the two together. Meetings earlier this year in Switzerland, New York, and Bangkok have led to calls to action to strengthen links between programmes addressing HIV/AIDS and sexual and reproductive health. While this should help in the battle against AIDS, the money which flows to AIDS should also benefit reproductive health.
Obstacles to Reproductive Health
So why hasn't more been achieved? One problem is certainly money. The 1994 Cairo Conference estimated the cost of implementing programmes for family planning, maternal health, and prevention of sexually transmitted diseases, as well as data collection and analysis in developing countries, at $18.5 billion by 2005—or $24.3 billion in today's dollars. The goal was to mobilise one-third of the money from donor nations, and the rest from developing countries themselves [ 8 ].Last year, global spending on reproductive health and services reached $14.7 billion, according to estimates from UNFPA, the Joint United Nations Programme on HIV/AIDS, and the Netherlands Interdisciplinary Demographic Institute [ 8 ]. Encouragingly, investment has increased since 2001, when the momentum of ICPD seemed to falter and international spending fell to $9 billion. But this is still wide of the mark. While developing countries have failed to meet their conference commitments, it is donor countries that are most remiss: rich country contributions reached an estimated $2.3 billion in 2003 [ 8 ], a far cry from the conference target of $6.1 billion (or $8.1 billion in today's dollars) by 2005.Reproductive health is not alone in waiting for donors to give generously. For all the rhetoric at international summits, few rich countries have lived up to their lofty pledges of debt relief and of dedicating 0.7% of their gross domestic product to overseas development assistance. But as Steve Sinding, head of the International Planned Parenthood Federation (IPPF), points out, there are other reasons too for the shortfall. In the past donor interest was largely stimulated by fears of a population crisis. When the Cairo Conference reframed issues in terms of women's health and reproductive rights, rather than an impending population explosion, Sinding argues, the “demographic rationale” was lost, taking funding with it.Moreover, there are other issues competing for international funding, most notably AIDS. At the time of the Cairo Conference, 20 million people were infected with HIV; today the number has grown to an estimated 38 million [ 9 ]. AIDS threatens to derail the Cairo Conference plan of action. Through maternal-to-child transmission, and wide-scale orphaning, HIV threatens to reverse small successes at reducing infant and child mortality. By killing off teachers and sapping household incomes, AIDS is sabotaging education. By killing off scarce medical workers and overwhelming fragile health care systems, the disease is compromising reproductive health services. Gender equity is undermined, as women and girls bear the brunt of the epidemic, as caregivers, breadwinners, or patients themselves.Roughly half of the money spent on reproductive health last year went towards HIV/AIDS. And billions more is on the way, from the likes of the Global Fund to Fight AIDS, Tuberculosis, and Malaria and the United States President's Emergency Plan for AIDS Relief, which promises $15 billion over five years to HIV/AIDS programmes [ 10 ]. But much of this money is going into AIDS-specific programmes that do not address reproductive health more broadly. Even as the world is gearing up to scale up AIDS prevention and treatment to millions worldwide, few of the agencies involved come from the world of reproductive and sexual health.This is a pity because it means that HIV/AIDS programmes are not making use of valuable infrastructure and expertise already on the ground in places where AIDS hits hardest. Given that 57% of HIV infections in sub-Saharan Africa are among women [ 9 ], and that, for many of them, family planning clinics are their sole contact with the formal health care system, it seems odd not to integrate such services into the wider battle against HIV. Such centres can offer not only HIV testing and counselling, as well as condoms (against the double whammy of unwanted pregnancy and HIV infection), but also a broad-based message of good sexual health that can help protect against HIV and other sexually transmitted diseases. Moreover, pre- and ante-natal care provide an opportunity to stop mother-to-child transmission of HIV in its tracks.Where once family planning was the darling of international donors, HIV is now the cause célèbre. “There's a lot of resentment about the spotlight moving on,” says Kevin O'Reilly, a former reproductive health specialist now at the department of HIV/AIDS at the World Health Organization. However, there are now attempts to bring the two together. Meetings earlier this year in Switzerland, New York, and Bangkok have led to calls to action to strengthen links between programmes addressing HIV/AIDS and sexual and reproductive health. While this should help in the battle against AIDS, the money which flows to AIDS should also benefit reproductive health.Ideological Battles Arguably the most formidable obstacle to that union, and indeed further progress in improving reproductive health, is ideology. Since the Cairo Conference, a fierce battle has emerged between religious conservatives who eschew abortion and condoms in favour of abstinence and fidelity, and more liberal voices who argue for a full armamentarium to tackle these problems. The clash is loudest in the field of HIV/AIDS, where the President's Emergency Plan for AIDS Relief allocates a third of its funding for disease prevention to programmes focusing on abstinence and fidelity; public health experts argue that such an approach is ineffective at best, and dangerous at worst, without an equal emphasis on the availability of condoms for all. But the clash resounds in the wider arena of reproductive health as well. Four years ago, the ICPD's central target—access to reproductive services for all by 2015—failed to make it into the Millennium Development Goals, largely because of political nervousness. But as Kofi Annan, United Nations secretary-general, has pointed out, progress on the other key targets, such as eradication of poverty and hunger, will not be achieved without a focus on women's rights, education, reproductive health, and family planning. The fight between conservatives and liberals is clearest in the case of the US, which is the world's leading bilateral donor on reproductive health, spending $429 million this year [ 11 ]. However, this money comes with strings attached, says Françoise Girard, a reproductive rights lawyer in New York. Some of these are subtle. For example, Girard points to American pressure on several Asian and Latin American governments—during recent regional meetings to mark the anniversary of the Cairo Conference—not to re-affirm their commitment to the ICPD plan of action, with its emphasis on a full suite of reproductive rights and services. Other strings are more obvious. In 2001, George W. Bush reinstated the Mexico City Policy, otherwise known as the “Global Gag Rule”, which denies US family planning assistance—including money and contraceptive supplies—to any non-American group unless it certifies that it neither performs nor endorses abortion. IPPF, Marie Stopes International, and their local affiliates have been hard hit by the Rule, scaling back services in Kenya, Ghana, and elsewhere that offered essential health care to thousands of women and children. Then there is the Kemp-Kasten Amendment, a piece of US legislation which prohibits US assistance to any organisation as deemed by the President that “supports or participates in the management of a program of coercive abortion or involuntary sterilization.” At the behest of conservative supporters, President Bush has used the amendment to withhold $34 million in annual congressional appropriations to the UNFPA for the past three years. The UNFPA says that the $34 million could have been used to prevent 2 million unintended pregnancies, 800,000 induced abortions, 4,700 maternal deaths, and 77,000 infant and child deaths. The White House accuses UNFPA of abetting coercive reproductive practices in China—a claim which the UNFPA strenuously denies. Moreover, a number of international delegations, including one from the US State Department in 2002, have investigated the UNFPA's activities in China and failed to find evidence to support such allegations. Fortunately, other donors are stepping in to fill the breach: earlier this year, for example, the United Kingdom announced it would raise its contribution to the UNFPA to £80 million over the next four years, as well as increase its support to IPPF by a third. But even if the shortfall is made up, the ill will such clashes have engendered cannot be so easily salved.
Ideological Battles
Arguably the most formidable obstacle to that union, and indeed further progress in improving reproductive health, is ideology. Since the Cairo Conference, a fierce battle has emerged between religious conservatives who eschew abortion and condoms in favour of abstinence and fidelity, and more liberal voices who argue for a full armamentarium to tackle these problems. The clash is loudest in the field of HIV/AIDS, where the President's Emergency Plan for AIDS Relief allocates a third of its funding for disease prevention to programmes focusing on abstinence and fidelity; public health experts argue that such an approach is ineffective at best, and dangerous at worst, without an equal emphasis on the availability of condoms for all.But the clash resounds in the wider arena of reproductive health as well. Four years ago, the ICPD's central target—access to reproductive services for all by 2015—failed to make it into the Millennium Development Goals, largely because of political nervousness. But as Kofi Annan, United Nations secretary-general, has pointed out, progress on the other key targets, such as eradication of poverty and hunger, will not be achieved without a focus on women's rights, education, reproductive health, and family planning.The fight between conservatives and liberals is clearest in the case of the US, which is the world's leading bilateral donor on reproductive health, spending $429 million this year [ 11 ]. However, this money comes with strings attached, says Françoise Girard, a reproductive rights lawyer in New York. Some of these are subtle. For example, Girard points to American pressure on several Asian and Latin American governments—during recent regional meetings to mark the anniversary of the Cairo Conference—not to re-affirm their commitment to the ICPD plan of action, with its emphasis on a full suite of reproductive rights and services.Other strings are more obvious. In 2001, George W. Bush reinstated the Mexico City Policy, otherwise known as the “Global Gag Rule”, which denies US family planning assistance—including money and contraceptive supplies—to any non-American group unless it certifies that it neither performs nor endorses abortion. IPPF, Marie Stopes International, and their local affiliates have been hard hit by the Rule, scaling back services in Kenya, Ghana, and elsewhere that offered essential health care to thousands of women and children.Then there is the Kemp-Kasten Amendment, a piece of US legislation which prohibits US assistance to any organisation as deemed by the President that “supports or participates in the management of a program of coercive abortion or involuntary sterilization.” At the behest of conservative supporters, President Bush has used the amendment to withhold $34 million in annual congressional appropriations to the UNFPA for the past three years. The UNFPA says that the $34 million could have been used to prevent 2 million unintended pregnancies, 800,000 induced abortions, 4,700 maternal deaths, and 77,000 infant and child deaths.The White House accuses UNFPA of abetting coercive reproductive practices in China—a claim which the UNFPA strenuously denies. Moreover, a number of international delegations, including one from the US State Department in 2002, have investigated the UNFPA's activities in China and failed to find evidence to support such allegations.Fortunately, other donors are stepping in to fill the breach: earlier this year, for example, the United Kingdom announced it would raise its contribution to the UNFPA to £80 million over the next four years, as well as increase its support to IPPF by a third. But even if the shortfall is made up, the ill will such clashes have engendered cannot be so easily salved.A Call for Strong Leadership Getting it right on reproductive health cannot wait another decade. The largest generation of young people in history—a whopping 1.2 billion aged 10–19 years—is entering adulthood [ 1 ]. They are making their sexual debut at ever earlier ages, against a backdrop of rising sexually transmitted diseases and growing social conservatism, which makes clear information, frank discussion, and free choice on abortion, contraception, and sexual health extremely difficult. More than ever, reproductive health needs strong leaders in rich and poor countries alike to mobilise both money and political commitment. Reproduction is a sexy subject; it is time the world again paid it the attention it deserves. Useful Links
 The Cairo Conference:
 http://www.iisd.ca/cairo.html

 Population Action International:
 www.popact.org

 UNFPA:
 www.unfpa.org

 Program for Appropriate Technology in Health:
 www.path.org

 The Alan Guttmacher Institute:
 www.guttmacher.org

 The Joint United Nations Programme on AIDS:
 www.unaids.org

 Netherlands Interdisciplinary Demographic Institute:
 www.nidi.nl

 IPPF:
 www.ippf.org

 Global Fund to Fight AIDS, Tuberculosis, and Malaria:
 www.theglobalfund.org

 The World Health Organization HIV/AIDS Programme:
 www.who.int/hiv/en
A Call for Strong Leadership
Getting it right on reproductive health cannot wait another decade. The largest generation of young people in history—a whopping 1.2 billion aged 10–19 years—is entering adulthood [ 1 ]. They are making their sexual debut at ever earlier ages, against a backdrop of rising sexually transmitted diseases and growing social conservatism, which makes clear information, frank discussion, and free choice on abortion, contraception, and sexual health extremely difficult. More than ever, reproductive health needs strong leaders in rich and poor countries alike to mobilise both money and political commitment. Reproduction is a sexy subject; it is time the world again paid it the attention it deserves.Useful Links
The Cairo Conference:
 http://www.iisd.ca/cairo.htmlThe Cairo Conference:Population Action International:
 www.popact.orgPopulation Action International:UNFPA:
 www.unfpa.orgUNFPA:Program for Appropriate Technology in Health:
 www.path.orgProgram for Appropriate Technology in Health:The Alan Guttmacher Institute:
 www.guttmacher.orgThe Alan Guttmacher Institute:The Joint United Nations Programme on AIDS:
 www.unaids.orgThe Joint United Nations Programme on AIDS:Netherlands Interdisciplinary Demographic Institute:
 www.nidi.nlNetherlands Interdisciplinary Demographic Institute:IPPF:
 www.ippf.orgIPPF:Global Fund to Fight AIDS, Tuberculosis, and Malaria:
 www.theglobalfund.orgGlobal Fund to Fight AIDS, Tuberculosis, and Malaria:The World Health Organization HIV/AIDS Programme:
 www.who.int/hiv/enThe World Health Organization HIV/AIDS Programme:


Background It is well known that cytotoxic factors, such as lipopolysaccharides, derange nitrogen metabolism in hepatocytes and nitric oxide (NO) is involved among the other factors regulating this metabolic pathway. Hepatocytes have been shown to express large levels of NO following exposure to endotoxins, such as bacterial lipopolysaccharide and/or cytokines, such as tumour necrosis factor-α (TNFα), interleukin-1. The control role of arginine in both urea and NO biosynthesis is well known, when NO is synthesized from arginine, by the NOS reaction, citrulline is produced. Thus, the urea cycle is bypassed by the NOS reaction. Many authors demonstrated in other cellular types, like cardiomyocytes, that bradykinin caused the increase in reactive oxygen species (ROS) generation. The simultaneous increase of NO and ROS levels could cause peroxynitrite synthesis, inducing damage and reducing cell viability. The aim of this research is to study the effect of bradykinin, a proinflammatory mediator, on cell viability and on urea production in cultures of rat hepatocytes.
Background
It is well known that cytotoxic factors, such as lipopolysaccharides, derange nitrogen metabolism in hepatocytes and nitric oxide (NO) is involved among the other factors regulating this metabolic pathway. Hepatocytes have been shown to express large levels of NO following exposure to endotoxins, such as bacterial lipopolysaccharide and/or cytokines, such as tumour necrosis factor-α (TNFα), interleukin-1. The control role of arginine in both urea and NO biosynthesis is well known, when NO is synthesized from arginine, by the NOS reaction, citrulline is produced. Thus, the urea cycle is bypassed by the NOS reaction. Many authors demonstrated in other cellular types, like cardiomyocytes, that bradykinin caused the increase in reactive oxygen species (ROS) generation. The simultaneous increase of NO and ROS levels could cause peroxynitrite synthesis, inducing damage and reducing cell viability. The aim of this research is to study the effect of bradykinin, a proinflammatory mediator, on cell viability and on urea production in cultures of rat hepatocytes.Results Hepatocytes were treated with bradykinin, that stimulates nitric oxide synthase (NOS). NO release was determined using 4,5 diaminofluorescein diacetate (DAF-2DA), as fluorescent indicator of NO. Addition of the NOS inhibitor, N g -nitro-L-arginine methyl ester (L-NAME), to the culture medium inhibited the increase of NO production. Exposure of hepatocytes to bradykinin 0,1 mM for 2 hours resulted in a significant decrease of urea synthesis. Cell viability, instead, showed a significant decrease 24 hours after the end of bradykinin treatment as determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium (MTT) assay. L-NAME addition recovered urea production and cell viability at control values.
Results
Hepatocytes were treated with bradykinin, that stimulates nitric oxide synthase (NOS). NO release was determined using 4,5 diaminofluorescein diacetate (DAF-2DA), as fluorescent indicator of NO. Addition of the NOS inhibitor, N g -nitro-L-arginine methyl ester (L-NAME), to the culture medium inhibited the increase of NO production. Exposure of hepatocytes to bradykinin 0,1 mM for 2 hours resulted in a significant decrease of urea synthesis. Cell viability, instead, showed a significant decrease 24 hours after the end of bradykinin treatment as determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium (MTT) assay. L-NAME addition recovered urea production and cell viability at control values.Conclusion The findings suggest that the cell toxicity, after bradykinin treatment, effectively depends upon exposure to increased NO levels and the effects are prevented by L-NAME. The results show also that the increased NO synthesis induces a reduced urea production, that is another index of cell damage.
Conclusion
The findings suggest that the cell toxicity, after bradykinin treatment, effectively depends upon exposure to increased NO levels and the effects are prevented by L-NAME. The results show also that the increased NO synthesis induces a reduced urea production, that is another index of cell damage.

Background It is well known that cytotoxic factors, such as lipopolysaccharides, derange nitrogen metabolism in hepatocytes and nitric oxide (NO) is involved among the other factors regulating this metabolic pathway [ 1 ]. NO is a free radical that is involved in many cellular events. In the biological systems NO has an halflife long lasting few seconds. It is an oxidation intermediate, therefore is both an oxidant and a reducing agent of metabolic products. Its biosynthesis is mainly performed by converting L-arginine to L-citrulline. L-arginine analogues, such as N g -nitro-L-arginine methyl ester (L-NAME), act as false substrates and are selective inhibitors of NO synthesis. NO synthase (NOS) is either a constitutive or inducible enzyme. The endothelial isoform (e-NOS) and the neuronal isoform (n-NOS) are constitutive. The inducible form of the enzyme (i-NOS), has the main property to be not regulated by intracellular calcium concentration and Ca 2+ -calmodulin complex, unlike the constitutive form [ 2 ]. It is known that iNOS is expressed by many cell types including macrophages, smooth muscle cells and hepatocytes [ 3 ]. Hepatocytes have been shown to express large levels of NO following exposure to endotoxins, such as bacterial lipopolysaccharide and/or cytokines, such as tumour necrosis factor-α (TNFα), interleukin-1 [ 4 , 5 ]. NO may posses both cytoprotective and cytotoxic properties, depending on the amount and the isoform of NOS by which it is produced [ 6 ]. NO generally mediates beneficial responses, but becomes deleterious when coexistence with enhanced superoxide formation leads to the synthesis of peroxynitrite, a potent oxidant and nitrating agent [ 7 ]. According to this hypothesis, authors studied the effect of bradykinin, a proinflammatory mediator kinin, on cell viability and on urea production in cultures of rat hepatocytes. Kinins exert numerous physiological and pathological actions; they partecipate in vascular and cellular events that accompany the inflammatory processes. In pathological states, kinins are thought to be implicated in inflammatory diseases and in haemorrhagic and endotoxic shock [ 8 ]. To demonstrate the decrease of cell viability and urea production by bradykinin, the authors studied its effects on NO production. The measurements of NO release from hepatocytes were investigated by using a NO-specific fluorescence indicator, 4,5 diaminofluorescein diacetate (DAF-2DA) [ 9 ].
Background
It is well known that cytotoxic factors, such as lipopolysaccharides, derange nitrogen metabolism in hepatocytes and nitric oxide (NO) is involved among the other factors regulating this metabolic pathway [ 1 ]. NO is a free radical that is involved in many cellular events. In the biological systems NO has an halflife long lasting few seconds. It is an oxidation intermediate, therefore is both an oxidant and a reducing agent of metabolic products. Its biosynthesis is mainly performed by converting L-arginine to L-citrulline. L-arginine analogues, such as N g -nitro-L-arginine methyl ester (L-NAME), act as false substrates and are selective inhibitors of NO synthesis. NO synthase (NOS) is either a constitutive or inducible enzyme. The endothelial isoform (e-NOS) and the neuronal isoform (n-NOS) are constitutive. The inducible form of the enzyme (i-NOS), has the main property to be not regulated by intracellular calcium concentration and Ca 2+ -calmodulin complex, unlike the constitutive form [ 2 ]. It is known that iNOS is expressed by many cell types including macrophages, smooth muscle cells and hepatocytes [ 3 ]. Hepatocytes have been shown to express large levels of NO following exposure to endotoxins, such as bacterial lipopolysaccharide and/or cytokines, such as tumour necrosis factor-α (TNFα), interleukin-1 [ 4 , 5 ]. NO may posses both cytoprotective and cytotoxic properties, depending on the amount and the isoform of NOS by which it is produced [ 6 ]. NO generally mediates beneficial responses, but becomes deleterious when coexistence with enhanced superoxide formation leads to the synthesis of peroxynitrite, a potent oxidant and nitrating agent [ 7 ]. According to this hypothesis, authors studied the effect of bradykinin, a proinflammatory mediator kinin, on cell viability and on urea production in cultures of rat hepatocytes. Kinins exert numerous physiological and pathological actions; they partecipate in vascular and cellular events that accompany the inflammatory processes. In pathological states, kinins are thought to be implicated in inflammatory diseases and in haemorrhagic and endotoxic shock [ 8 ]. To demonstrate the decrease of cell viability and urea production by bradykinin, the authors studied its effects on NO production. The measurements of NO release from hepatocytes were investigated by using a NO-specific fluorescence indicator, 4,5 diaminofluorescein diacetate (DAF-2DA) [ 9 ].Results Effect of bradykinin treatment on NO production The amounts of released NO were measured using DAF-2DA, that specifically reacts with the oxidized form of NO, producing the fluorescent triazolofluorescein [ 9 ]. NO determination was performed after 2 hours of incubation in the presence of bradykinin (0.01 mM and 0.1 mM). As shown in figure  1  the treatment with 0.01 mM bradykinin did not produce NO increase compared to control, but 0.1 mM bradykinin increased significantly the NO release. In contrast no appreciable NO release was observed during the same period in hepatocytes cultured with 0.1 mM bradykinin and 1.68 mM L-NAME. Effect of bradykinin treatment on urea production To evaluate urea synthesis after bradykinin treatment, the hepatocytes were treated with 1 mM NH 4 Cl for 2 h. Figure  2  shows that only the treatment with 0.1 mM bradykinin significantly decreased urea production and that the treatment with 0.1 mM bradykinin and 1.68 mM L-NAME did not produce a significant urea level decrease in comparison to control. Effect of bradykinin treatment on cell viability To determine the effects of bradykinin on cell viability, the hepatocytes were exposed to bradykinin (either 0.01 mM or 0.1 mM) for an incubation time of 2 hours. In one experimental series, the cell viability was determined by MTT test after 2 hours of incubation. In a second one, culture medium containing bradykinin was removed and replaced with the same fresh medium at 2 hours after the addition of bradykinin, and then cell viability was measured 24 hours after the end of bradykinin treatment. The MTT test after 2 hours of incubation does not indicate any significant viability difference in treated hepatocyte cultures in comparison to control (figure  3A ). By MTT test after 24 h (figure  3B ), a significant lowering of viability is observed in bradykinin 0.1 mM treated hepatocytes in comparison to control. The decrease was significantly reduced by the simultaneous treatment with L-NAME 1.68 mM even if always significantly lower than in control. Cell viability was validated by Trypan blue exclusion test (Table  1 ).
Results
Effect of bradykinin treatment on NO production The amounts of released NO were measured using DAF-2DA, that specifically reacts with the oxidized form of NO, producing the fluorescent triazolofluorescein [ 9 ]. NO determination was performed after 2 hours of incubation in the presence of bradykinin (0.01 mM and 0.1 mM). As shown in figure  1  the treatment with 0.01 mM bradykinin did not produce NO increase compared to control, but 0.1 mM bradykinin increased significantly the NO release. In contrast no appreciable NO release was observed during the same period in hepatocytes cultured with 0.1 mM bradykinin and 1.68 mM L-NAME.
Effect of bradykinin treatment on NO production
The amounts of released NO were measured using DAF-2DA, that specifically reacts with the oxidized form of NO, producing the fluorescent triazolofluorescein [ 9 ]. NO determination was performed after 2 hours of incubation in the presence of bradykinin (0.01 mM and 0.1 mM). As shown in figure  1  the treatment with 0.01 mM bradykinin did not produce NO increase compared to control, but 0.1 mM bradykinin increased significantly the NO release. In contrast no appreciable NO release was observed during the same period in hepatocytes cultured with 0.1 mM bradykinin and 1.68 mM L-NAME.Effect of bradykinin treatment on urea production To evaluate urea synthesis after bradykinin treatment, the hepatocytes were treated with 1 mM NH 4 Cl for 2 h. Figure  2  shows that only the treatment with 0.1 mM bradykinin significantly decreased urea production and that the treatment with 0.1 mM bradykinin and 1.68 mM L-NAME did not produce a significant urea level decrease in comparison to control.
Effect of bradykinin treatment on urea production
To evaluate urea synthesis after bradykinin treatment, the hepatocytes were treated with 1 mM NH 4 Cl for 2 h. Figure  2  shows that only the treatment with 0.1 mM bradykinin significantly decreased urea production and that the treatment with 0.1 mM bradykinin and 1.68 mM L-NAME did not produce a significant urea level decrease in comparison to control.Effect of bradykinin treatment on cell viability To determine the effects of bradykinin on cell viability, the hepatocytes were exposed to bradykinin (either 0.01 mM or 0.1 mM) for an incubation time of 2 hours. In one experimental series, the cell viability was determined by MTT test after 2 hours of incubation. In a second one, culture medium containing bradykinin was removed and replaced with the same fresh medium at 2 hours after the addition of bradykinin, and then cell viability was measured 24 hours after the end of bradykinin treatment. The MTT test after 2 hours of incubation does not indicate any significant viability difference in treated hepatocyte cultures in comparison to control (figure  3A ). By MTT test after 24 h (figure  3B ), a significant lowering of viability is observed in bradykinin 0.1 mM treated hepatocytes in comparison to control. The decrease was significantly reduced by the simultaneous treatment with L-NAME 1.68 mM even if always significantly lower than in control. Cell viability was validated by Trypan blue exclusion test (Table  1 ).
Effect of bradykinin treatment on cell viability
To determine the effects of bradykinin on cell viability, the hepatocytes were exposed to bradykinin (either 0.01 mM or 0.1 mM) for an incubation time of 2 hours. In one experimental series, the cell viability was determined by MTT test after 2 hours of incubation. In a second one, culture medium containing bradykinin was removed and replaced with the same fresh medium at 2 hours after the addition of bradykinin, and then cell viability was measured 24 hours after the end of bradykinin treatment. The MTT test after 2 hours of incubation does not indicate any significant viability difference in treated hepatocyte cultures in comparison to control (figure  3A ). By MTT test after 24 h (figure  3B ), a significant lowering of viability is observed in bradykinin 0.1 mM treated hepatocytes in comparison to control. The decrease was significantly reduced by the simultaneous treatment with L-NAME 1.68 mM even if always significantly lower than in control. Cell viability was validated by Trypan blue exclusion test (Table  1 ).Discussion The role of NO as mediator of hepatic injury after endotoxic shock remains controversial [ 16 ]. Increased NO production in response to cytokines has been demonstrated in cultured hepatocytes [ 17 ]. Laskin et al. [ 18 ] demonstrated that the induction of acute endotoxemia, caused an increase in NO production in the liver. This was associated with expression of inducible nitric oxide synthase (iNOS) messenger m-RNA in hepatocytes. Also our data showed an increase of NO production after 2 hours treatment of culture with 0.1 mM bradykinin in an arginine supplemented medium, as substrate for the synthesis of NO. The simultaneous treatment with L-NAME, a known inhibitor of NOS, blocked the increase of NO production. In this work we analyzed the urea synthesis after bradykinin treatment. Urea synthesis was decreased after 2 hours treatment with bradykinin 0.1 mM and the simultaneous treatment with L-NAME leaves urea biosynthesis unaltered. These data can be attributed to the control role of arginine in both urea and NO biosynthesis. When NO is synthesized from arginine, by the NOS reaction, cytrulline, an intermediate of urea cycle, is produced. Thus, the urea cycle is bypassed by the NOS reaction [ 1 ]. Whether NO exerts cytotoxic or cytoprotective action remains unclear [ 6 ]. We also found a significant decrease of viability, at long term, in hepatocytes subjected to bradykinin treatment. The simultaneous treatment of hepatocytes with L-NAME improves cell viability even if control levels are not restored. The data show that the increased NO production plays a role in liver damage induction, that follows the proinflammatory mediator treatment. The hepatocellular injury attributed to NO may be due either to its direct cytotoxicity or its reaction with superoxide to produce the toxic nitrogen metabolite peroxynitrite [ 19 ]. Oldenburg et al. [ 20 ], demonstrated in other cell types, like cardiomyocytes, that bradykinin caused the increase in reactive oxygen species (ROS) generation. At last, our results show that the increased NO synthesis induces a reduced urea production, that is an index of cell damage. The simultaneous treatment of liver cell cultures with L-NAME decreases NO levels and sustains overall biosynthesis activities and cell viability.
Discussion
The role of NO as mediator of hepatic injury after endotoxic shock remains controversial [ 16 ]. Increased NO production in response to cytokines has been demonstrated in cultured hepatocytes [ 17 ]. Laskin et al. [ 18 ] demonstrated that the induction of acute endotoxemia, caused an increase in NO production in the liver. This was associated with expression of inducible nitric oxide synthase (iNOS) messenger m-RNA in hepatocytes. Also our data showed an increase of NO production after 2 hours treatment of culture with 0.1 mM bradykinin in an arginine supplemented medium, as substrate for the synthesis of NO. The simultaneous treatment with L-NAME, a known inhibitor of NOS, blocked the increase of NO production. In this work we analyzed the urea synthesis after bradykinin treatment. Urea synthesis was decreased after 2 hours treatment with bradykinin 0.1 mM and the simultaneous treatment with L-NAME leaves urea biosynthesis unaltered. These data can be attributed to the control role of arginine in both urea and NO biosynthesis. When NO is synthesized from arginine, by the NOS reaction, cytrulline, an intermediate of urea cycle, is produced. Thus, the urea cycle is bypassed by the NOS reaction [ 1 ]. Whether NO exerts cytotoxic or cytoprotective action remains unclear [ 6 ]. We also found a significant decrease of viability, at long term, in hepatocytes subjected to bradykinin treatment. The simultaneous treatment of hepatocytes with L-NAME improves cell viability even if control levels are not restored. The data show that the increased NO production plays a role in liver damage induction, that follows the proinflammatory mediator treatment. The hepatocellular injury attributed to NO may be due either to its direct cytotoxicity or its reaction with superoxide to produce the toxic nitrogen metabolite peroxynitrite [ 19 ]. Oldenburg et al. [ 20 ], demonstrated in other cell types, like cardiomyocytes, that bradykinin caused the increase in reactive oxygen species (ROS) generation. At last, our results show that the increased NO synthesis induces a reduced urea production, that is an index of cell damage. The simultaneous treatment of liver cell cultures with L-NAME decreases NO levels and sustains overall biosynthesis activities and cell viability.Conclusions In summary, we conclude that 0.1 mM bradykinin treatment induces an increase of NO levels and reduction of urea synthesis in the hepatocytes. This increased NO production mediates, after 24 hours, cell toxicity as shown by MTT test. In contrast, the administration of the NOS inhibitor L-NAME protects against cell damage and increases urea levels, suggesting that NO plays a key role in the bradykinin-induced liver damage.
Conclusions
In summary, we conclude that 0.1 mM bradykinin treatment induces an increase of NO levels and reduction of urea synthesis in the hepatocytes. This increased NO production mediates, after 24 hours, cell toxicity as shown by MTT test. In contrast, the administration of the NOS inhibitor L-NAME protects against cell damage and increases urea levels, suggesting that NO plays a key role in the bradykinin-induced liver damage.Methods Materials Unless otherwise specified, all chemicals were obtained from Sigma (St. Louis, MO, USA). Isolation and culture of rat hepatocytes Hepatocytes were isolated from male rats, Wistar strain, (180 to 200 gbw), by a modification of the method of Seglen [ 10 ]. All procedures on the animals were performed according to the CEE directive n. 86/609 on animal experimentation. Rats were anesthetized with diethylether, the pre-perfusion of the liver in situ was performed at a rate of 20–30 ml/min with Ca 2+ -free Hanks balanced salt solution. The liver was then excised and the digestion was carried out by adding 0.05% (w/v) collagenase (type IV) in Hanks balanced salt solution supplemented with CaCl 2 ·H 2 O (0.0186 g/L) at a flux rate of 40 ml/min. At this point liver was transferred to a square plate containing 100 ml of RPMI 1640 medium supplemented with 200 mM L-glutamine, 20 ml/L essential amino acid solution and 10 ml/L non-essential amino acid solution, 1% antibiotic antimycotic stabilized solution and 100 μM L-arginine (incomplete medium). The cells were dispersed by gentle distruption with a stainless steel comb. After filtration through 200 μm Nytal mesh, parenchymal cells (hepatocytes) were separated from nonparenchymal cells (endothelial cells, Kupffer cells and stellate cells) by centrifugation at 50 g in Eppendorf Centrifuge 5810R at 4°C for 2 minutes and then washed twice in washing buffer [ 11 ]. Then the cells were resuspended in the same medium and filtered through 63 μm Nytal mesh. The viability of the cells was more than 80%, as estimated by trypan blue dye exclusion test [ 12 ]. After cell counting the cells were diluited at a concentration of 5 × 10 5  cells/ml with incomplete medium supplemented with 2% fetal calf serum, 0.1 U/ml insulin and 10 -6  M dexamethasone (complete medium). The hepatocytes were then plated in 24 well-plates coated with rat tail collagen at the final cell density of 2.5 × 10 5  cells per well and incubated at 37°C in an humidified atmosphere of 5% CO 2  and 95% air. After 6 hours incubation, the medium was changed and replaced with incomplete medium to remove dead cells. To verify the isolation method efficiency, the acid fosfatase activity per mg of proteins was evaluated. According to literature data, the specific activity of acid fosfatase in nonparenchimal cells is 1,7 folds the same activity in parenchimal cells [ 13 ]. Treatment After 24 hours of culture the hepatocytes were exposed either to bradykinin (0.01 mM and 0.1 mM) or bradykinin 0.1 mM supplemented with L-NAME 1.68 mM [ 14 ]. Determination of NO from hepatocytes DAF-2DA (Alexis Biochemicals, Lausen, Switzerland) was dissolved in DMSO (1 mg/0.45 ml) and diluted to 10 μM in phosphate buffer (0.1 M, pH 7.4). Then the cells were either incubated in phosphate buffer containing 10 μM DAF-2DA, bradykinin (0.01 mM and 0.1 mM) and bradykinin 0.1 mM supplemented with L-NAME 1.68 mM. After 2 hours of incubation in this reaction mixture, the fluorescence from the reaction of DAF-2DA with NO released from hepatocytes was measured with Perkin-Elmer MPF-44B Spectrofluorimeter calibrated for excitation at 495 nm and emission at 515 nm. Results were expressed as a percentage of the fluorescence of the samples in comparison to control. Determination of urea synthesis To determine the effects of bradykinin on urea production, cells were treated either with bradykinin (0.01 mM and 0.1 mM) and cotreated with bradykinin 0.1 mM and L-NAME 1.68 mM. At the same media 1 mM NH 4 Cl was added. After 2 hours urea levels in the media were measured by spectrophotometric method using Urea Color 2 Kit (Sclavo Diagnostics, Siena, Italia) measuring absorbance at 600 nm and blank sample with the same NH 4 Cl final concentration was used. Urea synthesis was calculated as ng urea per cell per hour. Determination of cell viability Cell viability was determined by MTT test method [ 15 ] and confirmed by Trypan blue exclusion test [ 12 ]. MTT (5 mg/ml) was dissolved in RPMI-1640 without phenol red. The solution is filtered through a 0.2 μm filter and stored at 2–8°C for frequent use. To determine the effects of bradykinin on cell viability, cells were either treated with bradykinin (0.01 mM and 0.1 mM) and cotreated with bradykinin 0.1 mM and L-NAME 1.68 mM for a 2 h period. After that cells were used either immediately or after an additional 24 h incubation period in incomplete medium. For the determination of cell viability, the medium has been discarded and MTT solution was added and incubated for 3 hours. At the end of the incubation period the MTT solution was removed and the cells and dye cristals were dissolved by adding dimethylsulfoxide (DMSO). Absorbance was measured at 570 nm in a Shimadzu UV-2100 Spectrophotometer and the results were expressed as a percentage of the absorbance of the samples in comparison to control. Statistical analysis At least four independent determinations of each parameter were compared to control using Student's T-test. Differences were considered significant when p < 0.05 was obtained.
Methods
Materials Unless otherwise specified, all chemicals were obtained from Sigma (St. Louis, MO, USA).
Materials
Unless otherwise specified, all chemicals were obtained from Sigma (St. Louis, MO, USA).Isolation and culture of rat hepatocytes Hepatocytes were isolated from male rats, Wistar strain, (180 to 200 gbw), by a modification of the method of Seglen [ 10 ]. All procedures on the animals were performed according to the CEE directive n. 86/609 on animal experimentation. Rats were anesthetized with diethylether, the pre-perfusion of the liver in situ was performed at a rate of 20–30 ml/min with Ca 2+ -free Hanks balanced salt solution. The liver was then excised and the digestion was carried out by adding 0.05% (w/v) collagenase (type IV) in Hanks balanced salt solution supplemented with CaCl 2 ·H 2 O (0.0186 g/L) at a flux rate of 40 ml/min. At this point liver was transferred to a square plate containing 100 ml of RPMI 1640 medium supplemented with 200 mM L-glutamine, 20 ml/L essential amino acid solution and 10 ml/L non-essential amino acid solution, 1% antibiotic antimycotic stabilized solution and 100 μM L-arginine (incomplete medium). The cells were dispersed by gentle distruption with a stainless steel comb. After filtration through 200 μm Nytal mesh, parenchymal cells (hepatocytes) were separated from nonparenchymal cells (endothelial cells, Kupffer cells and stellate cells) by centrifugation at 50 g in Eppendorf Centrifuge 5810R at 4°C for 2 minutes and then washed twice in washing buffer [ 11 ]. Then the cells were resuspended in the same medium and filtered through 63 μm Nytal mesh. The viability of the cells was more than 80%, as estimated by trypan blue dye exclusion test [ 12 ]. After cell counting the cells were diluited at a concentration of 5 × 10 5  cells/ml with incomplete medium supplemented with 2% fetal calf serum, 0.1 U/ml insulin and 10 -6  M dexamethasone (complete medium). The hepatocytes were then plated in 24 well-plates coated with rat tail collagen at the final cell density of 2.5 × 10 5  cells per well and incubated at 37°C in an humidified atmosphere of 5% CO 2  and 95% air. After 6 hours incubation, the medium was changed and replaced with incomplete medium to remove dead cells. To verify the isolation method efficiency, the acid fosfatase activity per mg of proteins was evaluated. According to literature data, the specific activity of acid fosfatase in nonparenchimal cells is 1,7 folds the same activity in parenchimal cells [ 13 ].
Isolation and culture of rat hepatocytes
Hepatocytes were isolated from male rats, Wistar strain, (180 to 200 gbw), by a modification of the method of Seglen [ 10 ]. All procedures on the animals were performed according to the CEE directive n. 86/609 on animal experimentation. Rats were anesthetized with diethylether, the pre-perfusion of the liver in situ was performed at a rate of 20–30 ml/min with Ca 2+ -free Hanks balanced salt solution. The liver was then excised and the digestion was carried out by adding 0.05% (w/v) collagenase (type IV) in Hanks balanced salt solution supplemented with CaCl 2 ·H 2 O (0.0186 g/L) at a flux rate of 40 ml/min. At this point liver was transferred to a square plate containing 100 ml of RPMI 1640 medium supplemented with 200 mM L-glutamine, 20 ml/L essential amino acid solution and 10 ml/L non-essential amino acid solution, 1% antibiotic antimycotic stabilized solution and 100 μM L-arginine (incomplete medium). The cells were dispersed by gentle distruption with a stainless steel comb. After filtration through 200 μm Nytal mesh, parenchymal cells (hepatocytes) were separated from nonparenchymal cells (endothelial cells, Kupffer cells and stellate cells) by centrifugation at 50 g in Eppendorf Centrifuge 5810R at 4°C for 2 minutes and then washed twice in washing buffer [ 11 ]. Then the cells were resuspended in the same medium and filtered through 63 μm Nytal mesh. The viability of the cells was more than 80%, as estimated by trypan blue dye exclusion test [ 12 ]. After cell counting the cells were diluited at a concentration of 5 × 10 5  cells/ml with incomplete medium supplemented with 2% fetal calf serum, 0.1 U/ml insulin and 10 -6  M dexamethasone (complete medium). The hepatocytes were then plated in 24 well-plates coated with rat tail collagen at the final cell density of 2.5 × 10 5  cells per well and incubated at 37°C in an humidified atmosphere of 5% CO 2  and 95% air. After 6 hours incubation, the medium was changed and replaced with incomplete medium to remove dead cells.To verify the isolation method efficiency, the acid fosfatase activity per mg of proteins was evaluated. According to literature data, the specific activity of acid fosfatase in nonparenchimal cells is 1,7 folds the same activity in parenchimal cells [ 13 ].Treatment After 24 hours of culture the hepatocytes were exposed either to bradykinin (0.01 mM and 0.1 mM) or bradykinin 0.1 mM supplemented with L-NAME 1.68 mM [ 14 ].
Treatment
After 24 hours of culture the hepatocytes were exposed either to bradykinin (0.01 mM and 0.1 mM) or bradykinin 0.1 mM supplemented with L-NAME 1.68 mM [ 14 ].Determination of NO from hepatocytes DAF-2DA (Alexis Biochemicals, Lausen, Switzerland) was dissolved in DMSO (1 mg/0.45 ml) and diluted to 10 μM in phosphate buffer (0.1 M, pH 7.4). Then the cells were either incubated in phosphate buffer containing 10 μM DAF-2DA, bradykinin (0.01 mM and 0.1 mM) and bradykinin 0.1 mM supplemented with L-NAME 1.68 mM. After 2 hours of incubation in this reaction mixture, the fluorescence from the reaction of DAF-2DA with NO released from hepatocytes was measured with Perkin-Elmer MPF-44B Spectrofluorimeter calibrated for excitation at 495 nm and emission at 515 nm. Results were expressed as a percentage of the fluorescence of the samples in comparison to control.
Determination of NO from hepatocytes
DAF-2DA (Alexis Biochemicals, Lausen, Switzerland) was dissolved in DMSO (1 mg/0.45 ml) and diluted to 10 μM in phosphate buffer (0.1 M, pH 7.4). Then the cells were either incubated in phosphate buffer containing 10 μM DAF-2DA, bradykinin (0.01 mM and 0.1 mM) and bradykinin 0.1 mM supplemented with L-NAME 1.68 mM. After 2 hours of incubation in this reaction mixture, the fluorescence from the reaction of DAF-2DA with NO released from hepatocytes was measured with Perkin-Elmer MPF-44B Spectrofluorimeter calibrated for excitation at 495 nm and emission at 515 nm. Results were expressed as a percentage of the fluorescence of the samples in comparison to control.Determination of urea synthesis To determine the effects of bradykinin on urea production, cells were treated either with bradykinin (0.01 mM and 0.1 mM) and cotreated with bradykinin 0.1 mM and L-NAME 1.68 mM. At the same media 1 mM NH 4 Cl was added. After 2 hours urea levels in the media were measured by spectrophotometric method using Urea Color 2 Kit (Sclavo Diagnostics, Siena, Italia) measuring absorbance at 600 nm and blank sample with the same NH 4 Cl final concentration was used. Urea synthesis was calculated as ng urea per cell per hour.
Determination of urea synthesis
To determine the effects of bradykinin on urea production, cells were treated either with bradykinin (0.01 mM and 0.1 mM) and cotreated with bradykinin 0.1 mM and L-NAME 1.68 mM. At the same media 1 mM NH 4 Cl was added. After 2 hours urea levels in the media were measured by spectrophotometric method using Urea Color 2 Kit (Sclavo Diagnostics, Siena, Italia) measuring absorbance at 600 nm and blank sample with the same NH 4 Cl final concentration was used. Urea synthesis was calculated as ng urea per cell per hour.Determination of cell viability Cell viability was determined by MTT test method [ 15 ] and confirmed by Trypan blue exclusion test [ 12 ]. MTT (5 mg/ml) was dissolved in RPMI-1640 without phenol red. The solution is filtered through a 0.2 μm filter and stored at 2–8°C for frequent use. To determine the effects of bradykinin on cell viability, cells were either treated with bradykinin (0.01 mM and 0.1 mM) and cotreated with bradykinin 0.1 mM and L-NAME 1.68 mM for a 2 h period. After that cells were used either immediately or after an additional 24 h incubation period in incomplete medium. For the determination of cell viability, the medium has been discarded and MTT solution was added and incubated for 3 hours. At the end of the incubation period the MTT solution was removed and the cells and dye cristals were dissolved by adding dimethylsulfoxide (DMSO). Absorbance was measured at 570 nm in a Shimadzu UV-2100 Spectrophotometer and the results were expressed as a percentage of the absorbance of the samples in comparison to control.
Determination of cell viability
Cell viability was determined by MTT test method [ 15 ] and confirmed by Trypan blue exclusion test [ 12 ]. MTT (5 mg/ml) was dissolved in RPMI-1640 without phenol red. The solution is filtered through a 0.2 μm filter and stored at 2–8°C for frequent use. To determine the effects of bradykinin on cell viability, cells were either treated with bradykinin (0.01 mM and 0.1 mM) and cotreated with bradykinin 0.1 mM and L-NAME 1.68 mM for a 2 h period. After that cells were used either immediately or after an additional 24 h incubation period in incomplete medium. For the determination of cell viability, the medium has been discarded and MTT solution was added and incubated for 3 hours. At the end of the incubation period the MTT solution was removed and the cells and dye cristals were dissolved by adding dimethylsulfoxide (DMSO). Absorbance was measured at 570 nm in a Shimadzu UV-2100 Spectrophotometer and the results were expressed as a percentage of the absorbance of the samples in comparison to control.Statistical analysis At least four independent determinations of each parameter were compared to control using Student's T-test. Differences were considered significant when p < 0.05 was obtained.
Statistical analysis
At least four independent determinations of each parameter were compared to control using Student's T-test. Differences were considered significant when p < 0.05 was obtained.Authors' contributions SS: Fluorimetric analysis and overall statistical analysis of data MG: Director of research MS: Spectrophotometric analysis CR: Primary hepatocyte cultures and characterization All authors read and approved the final manuscript
Authors' contributions
SS: Fluorimetric analysis and overall statistical analysis of dataMG: Director of researchMS: Spectrophotometric analysisCR: Primary hepatocyte cultures and characterizationAll authors read and approved the final manuscript