Longevity is a multifactorial trait with a genetic contribution, and mitochondrial DNA (mtDNA) polymorphisms were found to be involved in the phenomenon of longevity.To explore the effects of mtDNA haplogroups on the prevalence of extreme longevity (EL), a population based case-control study was conducted in Rugao – a prefecture city in Jiangsu, China. Case subjects include 463 individuals aged ≥95 yr (EL group). Control subjects include 926 individuals aged 60–69 years (elderly group) and 463 individuals aged 40–49 years (middle-aged group) randomly recruited from Rugao. We observed significant reduction of M9 haplogroups in longevity subjects (0.2%) when compared with both elderly subjects (2.2%) and middle-aged subjects (1.7%). Linear-by-linear association test revealed a significant decreasing trend of N9 frequency from middle-aged subjects (8.6%), elderly subjects (7.2%) and longevity subjects (4.8%) (p = 0.018). In subsequent analysis stratified by gender, linear-by-linear association test revealed a significant increasing trend of D4 frequency from middle-aged subjects (15.8%), elderly subjects (16.4%) and longevity subjects (21.7%) in females (p = 0.025). Conversely, a significant decreasing trend of B4a frequency was observed from middle-aged subjects (4.2%), elderly subjects (3.8%) and longevity subjects (1.7%) in females (p = 0.045).Our observations support the association of mitochondrial DNA haplogroups with exceptional longevity in a Chinese population. Individuals vary widely in their health status over their life course and in age at death. Longevity is thought to be a complex a puzzle of interplay of environmental, behavioral, genetic and stochastic factors. There is increasing evidence that exceptional longevity is a familial trait which is at least partially genetic. However, the magnitude of the genetic contribution is still controversial Mitochondrial DNA (mtDNA) is a maternally inherited genome To assess the possible contribution of mtDNA haplogroup-specific mutations to the prevalence of longevity, we therefore performed a population-based case-control study in a Chinese Han population residing in Rugao, Jiangsu Province. It is worth notion that Rugao has long been claimed as a ‘longevity town’ in China since ancient time.According to the investigation of the fifth national census of China conducted in 2001, the population of Rugao was 1,362.5 thousand, and the life expectancy of Rugao is 75.58 years Overall 926 subjects aged 60–69 years (elderly group) were randomly recruited from the resident registry at the local government offices of Rugao. For both genders of the case subjects, two-fold elderly subjects were chosen. To test the consistency of original case-control observations, another cohort of middle-aged control subjects including 463 individuals aged 40–49 years (middle-aged group) was randomly recruited from the resident registry at the local government offices of Rugao, matched for gender with the extreme longevity group.All subjects were contacted at home and examined by physicians previously trained to administer a structured questionnaire. The questionnaire include socioeconomic status, demographic characteristics, personal habits, dietary habits, family disease histories, personal disease histories, surgery histories, menstrual and reproductive histories, hormone use histories (for women only), daily activity histories, and cognitive function appraisals, etc. Physical examinations were accomplished and fasting blood specimens were drawn for laboratory examination . For the purpose of the quality control of the investigation, all the field staff involved in data collection and data handling was trained. All interviews were tape recorded, and 5 percent of the recorded interviews were evaluated for interviewing quality. About 3–5 percent of subjects were re-contacted by phone to evaluate the interviewers' work. Written informed consent was obtained from each participant or a member of his/her immediate family. The Human Ethnics Committee of Fudan University School of Life Sciences approved the research.*, D4, D4a, D4b, D4b2, D4b2b, D5, M12,G*, G2, M7*, M7b, M8*, M8a, C, M9, M10, N9, N9a, A, F*, F1, B*, B5, B5a, B5b, B4a and B4b) found in the Chinese population, as shown in Genomic DNA was extracted by using a method described by Boom et al, with minor modifications 2 , 0.12 µL of FastStart Taq DNA Polymerase and 5.12 µL and 2.6 µL premixed primers for each multiplex amplification with the multiplex PCR cycling conditions: 95°C*4 min - [94°C*30 sec - 60°C (-0.5°C/cycle)*50 sec - 72°C*90 sec]*11cycles –[94°C*30 sec - 55°C*50 sec - 72°C*90 sec]* 24cycles - 72°C*10 min - 4°C*∞, and the final concentrations of each forward and reverse primers for mtDNA SNPs are as follows: for Multiplex 1, 12705 and 12811 are 0.33 µM; 12406 and 12358 are 0.35 µM; 7600 and 10400 are 0.53 µM; 8281 is 0.6 µM; 14569 is 0.67 µM; 9296 is 0.7 µM. For Multiplex 2, 4491, 8020, 8584 and 8684 are 0.2 µM; 5417 and 5465 are 0.25 µM; 1107 is 0.27 µM; 3010, 3970, 4833, 5178, 7196, 9824, 10646, 10873, and 15662 are 0.3 µM; 15235 is 0.37 µM; 3705, 8964, 13263 and 13590 are 0.4 µM; 14979 is 0.47 µM; 1736 is 0.5 µM. After PCR amplification, 0.95 µL of the both two multiplexes amplification products are mixed and incubated with 0.1 µL Exonuclease I  and 2 µL shrimp alkaline phosphatase (SAP)  for 80 min at 37°C followed by 15 min at 75°C for enzyme inactivation. And then, multiplex primer extension reactions were carried out by SNaPshot Multiplex Ready eaction mix , Sequencing Buffer 5 X  and primers extension mix with the multiplex primer extension cycling conditions: 96°C*1 min - [96°C*10 sec - 52°C*5 sec-60°C*30 sec] *28cycles - 4°C*∞ and the final concentrations of each extension primers are also given: 1107, 1736, 3010, 3705, 3970, 4491, 4833, 5178, 7196, 7600, 8020, 8584, 8684, 8964, 9824, 10646, 10873, 12811, 13263, 13590, 15235 and 15662 are 0.16 µM; 5417 is 0.33 µM; 12406 is 0.37 µM; 5465, 12358, 12705 and 14979 are 0.41 µM; 14569 is 0.45 µM; 8281, 9296 and 10400 are 0.49 µM. After the SNaPshot reaction, 6.1 µl of the SNaPshot reaction was treated with 1 U of shrimp alkaline phosphatase  to inactivate the excess ddNTPs. Electrophoresis of final minisequencing products was accomplished on the ABI 3130xl Genetic Analyzer (Applied Biosystems). Genemapper ver.4.0 software was used to analyze the SNaPshot data.We introduced the SNaPshot  minisequencing reaction assay to genotype mitochondrial deoxyribonucleic acid single nucleotide polymorphism (SNPs) Mitochondrial DNA haplogroup frequencies were calculated by counting from the observed genotypes. The Pearson chi-square test and the chi-square test for linear-by-linear association were used to analyze the significances between the prevalence of haplogroups and different age groups. SPSS software (version 10.0) was used.We typed 33 mtSNPs which defined 28 haplogroups in the exceptional longevity group, elderly group, and middle-aged group. The genotyping success rates ranged from 99.5% to 100%. The prevalence of the aforementioned 28 haplogroups ranged from 1.9% to 23% in the elderly control subjects .Significant reduction of M9 haplogroups was observed in EL subjects (0.2%) when compared with both elderly subjects (2.2%) and middle-aged subjects (1.7%) . Linear-by-linear association test revealed a significant decreasing trend of N9 frequency from middle-aged subjects (8.6%), elderly subjects (7.2%) and EL subjects (4.8%) (p = 0.018) In subsequent analysis stratified by gender, linear-by-linear association test revealed a significant increasing trend of D4 frequency from middle-aged subjects (15.8%), elderly subjects (16.4%) and EL subjects (21.7%) in females (p = 0.025) . ConversIn the present study, we conducted a population-based case-control study to explore the effects of mtDNA haplogroups on the phenomenon of extremely long life in a Chinese population. Crucial to our approach is the strong survival advantage of the extremely longevity people at Rugao . The enrichment in the frequency of certain genetic variants at this extremely old people probably reflects a selection effect that may enhance the likelihood of survival. We did observe the enrichment of certain haplogroups in Rugao population, such as D4 haplogroup. Interestingly, there is a reduction of M9, N9, and B4a in these long lived subjects.Haplogroups D4 is one of the most characteristic mtDNA lineages found in Northeast Asian populations Haplogroups M9 and N9, two major subclusters directly sprout from the basal M and N trunks, respectively, have originated from Southeast Asia conceivably and are prevailing over central or northern China In genetic association studies on longevity, the gender difference is common, where different polymorphisms of mtDNA were associated with longevity only in males Recently, haplogroup association studies have been used to define the role of mtDNA mutations in complex diseases and longevity. In this approach, case and control mtDNA sequence sets are assigned to haplogroups, and the distribution of haplogroups is compared. A significant difference is interpreted to indicate that the mtDNA ‘background’ has an effect on expression or penetrance of the phenotypes. Given the inconsistency results among the different studies, as observed in Rugao population and Japanese In summary, we observed the involvement of mtDNA haplogroups in the prevalence of longevity by using a carefully matched population based case-control design in Rugao, China. The results should lead to increasing understanding of the role of genome maintenance in longevity. Although we selected two control groups to verify our discovery, our finding also needs to be replicated by other independent studies.


Escherichia coli using a heterologous mevalonate pathway derived from yeast (Saccharomyces cerevisiae), though the reconstructed mevalonate pathway was limited at a particular enzymatic step.Artemisinin derivatives are the key active ingredients in Artemisinin combination therapies (ACTs), the most effective therapies available for treatment of malaria. Because the raw material is extracted from plants with long growing seasons, artemisinin is often in short supply, and fermentation would be an attractive alternative production method to supplement the plant source. Previous work showed that high levels of amorpha-4,11-diene, an artemisinin precursor, can be made in Staphylococcus aureus, more than doubling production. Amorpha-4,11-diene titers were further increased by optimizing nitrogen delivery in the fermentation process. Successful cultivation of the improved strain under carbon and nitrogen restriction consistently yielded 90 g/L dry cell weight and an average titer of 27.4 g/L amorpha-4,11-diene.By combining improvements in the heterologous mevalonate pathway with a superior fermentation process, commercially relevant titers were achieved in fed-batch fermentations. Yeast genes for HMG-CoA synthase and HMG-CoA reductase (the second and third enzymes in the pathway) were replaced with equivalent genes from Production of >25 g/L amorpha-4,11-diene by fermentation followed by chemical conversion to artemisinin may allow for development of a process to provide an alternative source of artemisinin to be incorporated into ACTs. Plasmodium falciparum and P. vivax. The most virulent form of malaria is caused by P. falciparum which has become resistant to almost all currently used drug therapies Artemisia annua and combined with one or more other antimalarial drugs to produce ACTs. In 2005 ACTs were recommended for the first-line treatment of uncomplicated falciparum malaria by the World Health Organization (WHO) A. annua are such that a substantial increase in the amount of plant cultivated would be needed to satisfy the forecasted global demand for artemisinin A. annua.Malaria causes more than one million deaths annually, with the highest mortality in children younger than five years of age Escherichia coli. Our work focuses on improving heterologous production of the artemisinin precursor amorpha-4,11-diene, which can be converted to artemisinin via chemical transformation.Historically, heterologous production of small molecules has been hampered by the challenges in expressing complex functional pathways for the production of the non-native molecules. New tools in rapid gene synthesis and metabolite analysis have promoted the field of synthetic biology, which promises advances in pathway re-construction, and strives toward pathway and genome optimization. Recent success in reconstituting heterologous pathways in microorganisms for high-level production of small molecules has demonstrated the feasibility of achieving titers in the g/L range. Examples include production of polyketides such as 6-deoxyerythronolide B S. cerevisiae was engineered in E. coli for the production of amorpha-4,11-diene  and the catalytic domain of HMG-CoA reductase ) were derived from yeast Staphylococcus aureus, doubling amorpha-4,11-diene production. Amorpha-4,11-diene producing E. coli strains were grown in a defined, glucose restricted fed-batch process which achieved cell densities of 90 g/L dry cell weight. Titers were further improved in this process by simultaneously restricting ammonia and carbon. Nitrogen consumption rates depended on the components of the pathway that were expressed, thus the process was modified to accommodate the high production strain containing the modified mevalonate pathway. An amorpha-4,11-diene titer of greater than 25 g/L was achieved in a robust and reproducible fermentation process.Recently, the expression of a synthetic amorpha-4,11-diene synthase gene along with the mevalonate isoprenoid pathway native to 11-diene  [9]. Proet al.E. coli strain W3110 transformed with plasmids pMevT, pMBIS and pADS E. coli DH1 E. coli DH1. To achieve greater volumetric productivity than Newman et al.600 of ∼30 (20 hours after inoculation). The exponential glucose feed continued until a maximum rate of 31 g/h was achieved, at which point the feed was reduced to 11.7 g/h (3/8 of the maximum) and held constant. Maximum cell density was attained 100 hours after inoculation at an OD600 of 260 and the corresponding titer was measured at 6.5 g/L amorpha-4,11-diene . The ammonia in the medium was allowed to decrease to zero though nitrogen flow into the bioreactor was still positive due to base addition. The carbon consumption in process B was similar to that of process A (data not shown). Once the initial glucose was depleted, the glucose concentration was maintained at zero, thus preventing any accumulation of acetate in the culture . Ammoniu600 . Similar600 . This coet al.E. coli. The wild-type lac promoter was also replaced with the two-fold stronger lacUV5 promoter S. aureus encoded by mvaAEnterococcus faecalis encoded by mvaEmvaE, the entire codon-optimized MevT operon was replaced with E. faecalis genes mvaS , and B66 ,  was selected for further study. In view of the superiority of the S. aureus HMGR for the production of amorpha-4,11-diene in E. coli we wished to test the effect of substituting the yeast HMGS with its S. aureus equivalent encoded by mvaS. This was accomplished by site-directed mutagenesis of pAM41 to substitute the yeast HMGS with S. aureus mvaS, generating pAM52 , , pMBIS as mvaA), . The higng pAM52 . Amorphang pAM52  was highProduction of amorpha-4,11-diene was compared between strains B32 and B86 in bioreactors using process A. While growth of the two strains was comparable, strain B86 produced 2.5-times more amorpha-4,11-diene than B32, reaching 16.5 g/L at 150 hours . The ammE. coli, strain B86 was tested in process B. With no ammonium sulfate in the feed, the ammonia concentration was undetectable at 42 hours, but only remained restricted for 24 hours . An average titer of 27.4±2.1 g/L amorpha-4,11-diene was attained from these runs . It may be that the acetoacetyl-CoA thiolase activity of mvaE or the HMGS encoded by E. faecalis mvaS limits flux through the mevalonate pathway, though the production of 47 g/L mevalonate by a strain expressing mvaE and mvaSStrain improvement was an integral component of enhancing the production of amorpha-4,11-diene. Pitera MGR>mvaE . This reS. aureus mvaA) we replaced the yeast HMGS with S. aureus mvaSS. aureus resulted in a significant increase in amorpha-4,11-diene production from the heterologous mevalonate pathway.In an effort to further improve production of amorpha-4,11-diene from strain B66 . An alternative method of microbial production of amorpha-4,11-diene or artemisinic acid is the use of engineered yeast (Saccharomyces cerevisiae). Ro et al.CYP71AV1 in this yeast strain resulted in production of 115 mg/L artemisinic acid. The strain described by Ro et al.E. coli combined with chemical oxidation and transformation to artemisinin is one option for the semi-synthetic production of artemisinin, which will need to be compared for feasibility of commercial production with a yeast process capable of producing lower concentrations (2.5 g/L) of artemisinic acid. A second source of artemisinin to stabilize the price and increase the supply of ACTs for the treatment of malaria in the developing world is highly desirable, and this work describes progress towards an option for the provision of such an alternative supply.The development of a strain of E. coli DH1 was used as the isoprenoid expression strain. The B32 strain contained plasmids encoding the heterologous mevalonate pathway previously described by Martin et al.E. coli codon-optimized MevT operon is described by Anthony et al.et al.EcoRI+HindIII digested pAM29 Enterococcus faecalis mvaS was amplified from genomic DNA of E. faecalis ATCC 700802 using primers TATAGAATCTTAAGGAGGATATTTAGATGACAATTGGGATTGATAAAATTAG and TTTGGATCCTTAGTTTCGATAAGAGCGAACGG with Phusion™ DNA polymerase  and the following conditions: 1 cycle 98°C, 30 s; 30 cycles 98°C, 30 s/ 55°C, 20 s/ 72°C, 90 s; 1 cycle 72°C, 10 m. E. faecalis mvaE was similarly amplified with primers TATGGATCCTAAGGAGGATATTTAGATGAAAACAGTAGTTATTATTGATGC and AGCTAAGCTTTTATTGTTTTCTTAAATCATTTAAAATAGC. Amplicons were ligated into SmaI digested pBluescript II KS+ (Stratagene) and the DNA sequence verified. E. faecalis mvaS and mvaE were excised from pBluescript II KS+ with EcoRI+BamHI and BamHI+HindIII respectively and the excised amplicons were ligated into EcoRI+HindIII digested pAM29 Staphylococcus aureus mvaA (HMGR) to generate pAM41. S. aureus mvaA was amplified from genomic DNA of Staphylococcus aureus subsp. aureus ATCC 700699D with the primers GCTACTAGTAGGAGGAAAACATCATGCAAAGTTTAGATAAGAATTTCCG and GCTTCTAGACTATTGTTGTCTAATTTCTTGTAAAATGCG using the same reaction conditions used to amplify E. faecalis mvaE and mvaS. The amplified mvaA fragment was digested with SpeI and ligated into the SpeI+HincII digested pAM25 so as to create pAM41 with the operon atoB-ERG13-mvaA. mvaS was similarly amplified with the primers GAACTGAAGATCTAGGAGGAAAGCAAAATGACAATAGGTATCGACAAAATAAACT and TTGCATGATGTTTTCCTCCTACTAGTTACTCTGGTCTGTGATATTCGCGAAC. ERG13 in the atoB-ERG13-mvaA operon of pAM41 was replaced with mvaS by the method of Geiser et al.atoB-mvaS-mvaA operon in pAM52.E. coli strain B32 or B86 into a flask containing 50 mL of M9 medium. M9 medium contained (per L) 8 g glucose, 12.8 g Na2HPO4·7H2O, 3 g KH2PO4, 0.5 g NaCl, 1 g NH4Cl, 2 mmol MgSO4, 0.1 mmol CaCl2, 0.1 µg thiamine, 100 mmol MOPS buffer pH 7.4, 3.7 µg (NH3)6Mo7O24·4 H2O, 25 µg H3BO4, 7.1 µg CoCl2, 2.4 µg CuSO4, 16 µg MnCl2, 2.8 µg ZnSO4, and 0.28 mg FeSO4. B32 cultures also contained 5 µg/mL tetracycline, 100 µg/mL carbenicillin and 34 µg/mL chloramphenicol and B86 cultures also contained 5 µg/mL tetracycline, 100 µg/mL carbenicillin, and 50 µg/mL kanamycin. Cultures were incubated overnight at 37°C and 250 rpm overnight and subcultured the following morning into the same medium containing the same antibiotics to an OD600 of ∼1.0 and allowed to grow to an OD600 of 3–5 at 37°C and 250 rpm. The cells were used to inoculate a 2 L bioreactor at 5% (v/v).Seed cultures for fed-batch cultivation were prepared by inoculating 1 mL of frozen cells (20% (v/v) glycerol) of 600 while dodecane samples were removed at intervals for measurement of amorph-4,11-diene as described below.For shake flask production experiments 20 ml of M9 medium in 125 ml shake flasks was inoculated with frozen cells as described above. Following overnight growth at 30°C the culture was used to inoculate 140 ml of M9 medium containing 1 mM IPTG and 20 ml dodecane in a 500 ml flask at a 1∶100 dilution. Cell growth was monitored by OD2PO4, 15.7 g K2HPO4·3H2O, 2 g (NH4)2SO4, 1.7 g citric acid, and 8.4 mg EDTA was prepared and sterilized in a 2 L Applikon Bioconsole ADI 1025 vessel at 121°C for 30 minutes. Post sterile additions for a batch medium were prepared as concentrated stocks, filter sterilized, and injected into the bioreactor through a septum on the head plate. They consisted of 15 g glucose, 1.2 g MgSO4·7H2O, 4.5 mg thiamine HCl, and 10 mL of batch trace metal solution containing 0.25 g/L CoCl2·6H2O, 1.5 g/L MnCl2·4H2O, 0.15 g/L CuCl2·2H2O, 0.3 g/L H3BO4, 0.25 g/L Na2MoO4·2H2O, 1.3 g/L Zn(CH3COO)2·2H2O, and 10 g/L Fe(III)citrate hydrate. Antibiotics  were added to B86 processes for plasmid retention. For strain B32 plasmid stability was >95% without antibiotics in the production medium (data not shown). Cultivation was carried out at 30°C and the pH was adjusted at 7.0 using ammonium hydroxide. The airflow and the initial agitation rate were set at 1 v/v/m and 700 rpm, respectively. The dissolved oxygen tension was controlled at 40% of saturation using an agitation cascade and oxygen enrichment. Biospumex antifoam 200 K was used to control foam. Once the initial glucose (15 g/L) was exhausted and the dissolved oxygen spiked, an exponential feed with a 6 hour doubling time was initiated. The feed solution consisted of 650 g/L glucose, 12 g/L MgSO4·7H2O, 10.7 g/L (NH4)2SO4, 13 mg/L EDTA, and 10 mL/L of feed trace metal solution that was composed of 0.4 g/L CoCl2·6H2O, 2.35 g/L MnCl2·4H2O, 0.25 g/L CuCl2·2H2O, 0.5 g/L H3BO4, 0.4 g/L Na2MoO4·2H2O, 1.6 g/L Zn(CH3COO)2·2H2O, and 4 g/L Fe(III)citrate hydrate. (NH4)2SO4 was taken out from the feed for nitrogen restricted runs. The fermenter software (BioXpert) was programmed to calculate feed rates according to the following equation:0 is initial glucose concentration, μ is specific growth rate, and t0 is time of glucose depletion from the batch medium. After reaching a maximum feed rate of 31 g glucose/h, the feed rate was reduced to 11.7 g glucose/h and held constant for the remainder of the run. The culture was induced with 1 mM IPTG at an OD600 of 30 and 10% (v/v) dodecane was added to the culture immediately after induction to capture amorpha-4,11-diene in the organic phase. The pH was maintained at 7 with the automatic addition of ammonium hydroxide and/or sodium hydroxide as described in the results. The fed-batch fermentation process was cultivated for 6–7 days.The process used for production of amorpha-4,11-diene was based on the glucose process described by Korz et. al. The ammonia, glucose, and acetate concentrations were measured off-line using Bioprofile 300 analyzer  according to the manufacturer's instructions. Where the concentrations of the analytes were outside the range of the instrument, samples were diluted into Nova buffer which contained per liter 6 g HEPES acid, 0.64 g LiCl, and 0.46 g LiOH.600 was measured in a Genesys 10vis spectrophotometer at 600 nm. The relationship between dry cell weight and OD600 was measured in the following manner. Samples were taken throughout high density E. coli processes and OD600 was measured for each sample (the OD600 measurements ranged between 0.5 and 200). To measure dry cell weight, 1.5 mL of well mixed broth was centrifuged in weighed eppendorf tubes. The supernatants were removed from the samples and discarded. The cell pellets were washed once with water. The samples were centrifuged again and the water was removed. The washed cell pellets were dried in an oven at 80 C for at least three days. The samples were weighed, and the tube weight subtracted. The dry cell weight was calculated by dividing the sample weight (g) by the sample volume (0.0015 L), and graphing the relationship. OD600 of 1 was found to be equivalent to 0.4 g/L dry cell weight.ODm/z 189, and the molecular ion, m/z 204, were used to monitor AD and TC. To expedite run times, the temperature program and column matrix were modified to achieve optimal resolution and the shortest overall runtime (5.43 min). A 1 µL sample was split 1∶20 and was separated using a HP-5MS column  with helium as the carrier gas. The temperature program for the analysis was follows: the column was initially held at 100°C for 0.75 min, followed by a temperature gradient of 65°C/min to a temperature of 170°C, where the gradient was decreased to 50°C/min until the temperature reached 300°C. The column was held at 300°C for 1 min to elute all remaining components. Under these conditions, TC and AD elute at 3.37 and 3.49 min respectively.The production of amorpha-4,11-diene (AD) was monitored by gas chromatography/mass spectrometry (GC/MS), using trans-caryophyllene  as an internal standard. AD was extracted from the cell pellet by diluting 25 µL of well mixed whole cell broth with 975 µL of methanol, followed by mixing, and dilution of 10 µL of the methanol extract into 990 µL of ethyl acetate containing TC (10 ppm v/v). AD standards were prepared by diluting purified AD into the same ethyl acetate containing TC to concentrations between 0.63–10 mg/L. The standards and ethyl acetate-extracted samples were analyzed on an Agilent 6890N gas chromatograph equipped with an Agilent 5975 mass spectrometer (GC/MS) in single-ion monitoring (SIM) mode. The fragment ion,


The results indicated epimuqubilin A (1) possessed potent NO inhibitory activity against lipopolysaccharide (LPS)-induced nitric oxide release with an IC50 value of 7.4 μM, a level three times greater than the positive control, L-NG-monomethyl arginine citrate, followed by 6 , whereas other compounds exhibited only modest activity  a monocyclic carbon skeleton framework was essential for activity, (2) free acids gave higher activity, (3) the orientation of H3-22 with an equatorial position increased activity, and (4) a bicyclic structure reduced activity. This is the first report of a norsesterterpene peroxide with NO–inhibitory activity. In addition, compounds 1–7 were also evaluated for their inhibitory activities in the yeast glycogen synthase kinase-3β assay. In summary, several norsesterterpene peroxides showed novel biological activities of inhibition in NO production, suggesting that these might provide leads for anti-inflammatory or cancer chemopreventive agents.Seven norsesterterpene peroxides: epimuqubilin A (activity . These c Sesterterpenes isolated from marine organisms are often modified by the loss or addition of one or more carbon units. Norsesterterpene peroxides are characterized with a 2-substituted propionic acid or methyl propionate group attached to a 1,2-dioxane ring at the C-3 position.Sigmosceptrella D21+55.9 ; IR (film) νmax 3450, 2925, 1710, 1578, 1320 cm−1; ESIMS m/z 393.30 [M + H]+.Muqubilone B (2): Colorless viscous oil; [α]D21+52.5 ; IR (film) νmax 3422, 2953, 1700, 1498, 1321 cm−1; ESIMS m/z 425.30 [M + H]+.Unnamed cyclic peroxide ester (3): Colorless viscous oil; [α]D21−57.8 ; IR (film) νmax 2967, 1670, 1530, 1134 cm−1; ESIMS m/z 407.32 [M + H]+.Epimuqubilin B (4): Colorless viscous oil; [α]D21+ 15.8 ; IR (film) νmax 3445, 2929, 1685, 1540, 1130 cm−1; ESIMS m/z 423.31 [M + H]+.Sigmosceptrellin A methyl ester (5): Colorless viscous oil; [α]D21+57.9 ; IR (film) νmax 2935, 1665, 1434, 1150 cm−1; ESIMS m/z 407.32 [M + H]+.Sigmosceptrellin A (6): Colorless viscous oil; [α]D21+51.3 ; IR (film) νmax 3380, 2960, 1723, 1560, 1140 cm−1; ESIMS m/z 393.30 [M + H]+.Sigmosceptrellin B methyl ester (7): Colorless viscous oil; [α]D21−55.4 ; IR (film) νmax 2960, 1660, 1560, 1150 cm−1; ESIMS m/z 407.32 [M + H]+.5 cells/well and incubated for 24 h at 37°C in a humidified atmosphere containing 5% CO2. The cells were treated with samples dissolved in phenol red-free DMEM for 30 min followed by 1 μg/mL of LPS treatment for 20 h. The amount of NO in the cultured medium was measured by the Griess reagent. The standard curve was created by using known concentrations of sodium nitrite, and the absorbance was measured at 540 nm. To evaluate the cytotoxic effect of samples in RAW 264.7 cells in the assay condition, SRB assay was performed. Briefly, after the fixation with 10% trichloroacetic acid (TCA), cells were stained with 0.4% SRB solution in 1% acetic acid followed by dissolving bound SRB in 10 mM Tris-buffer. The optical density was determined at 515 nm.The inhibitory effects of samples on NO production were evaluated in LPS-activated murine macrophage RAW 264.7 cells, using a method modified from that previously reported . Brieflygsk-3 null mutant confers a temperature-sensitive phenotype (growth defect at 37ºC), that can be suppressed by the expression of cloned mammalian GSK-3β , was grown in 5 mL of liquid media of SC minus uracil, shaking, at 37ºC for 2 days. Four hundred-μL of the yeast culture was added to 100 mL of SC minus uracil agar . To perform the assay, compounds and extracts at a concentration of 80 μg per disk were dispensed on filter disks, and placed on plates containing yeast seeded into SC minus uracil media. The Petri dishes were incubated at both 25ºC and 37ºC for 72 hours. Three phenotypes are observed on yeast plates. A clear zone of inhibition (ZOI) at 37ºC and no ZOI at 25ºC indicate that the agent tested inhibits the GSK-3β pathway. The clear zones of inhibition at both 37 and 25ºC of equal width indicate that the agents are cytotoxic. Plates that have a negative phenotype are plates that have no observable ZOI. Active compounds were then tested at lower concentrations . The assays were performed in duplicate. GSK-3β inhibitor , and staurosporine were used as positive controls.Disruption of four yeast GSK-3 homologs  in n GSK-3β . Inhibit


The high fermentation cost of l-lactic acid is another limitation for PLA polymers to compete with conventional plastics.The demand for lactic acid has been increasing considerably because of its use as a monomer for the synthesis of polylactic acid (PLA), which is a promising and environment-friendly alternative to plastics derived from petrochemicals. Optically pure Bacillus sp. strain 2–6 for production of l-lactic acid was isolated at 55°C from soil samples. Its thermophilic characteristic made it a good lactic acid producer because optically pure l-lactic acid could be produced by this strain under open condition without sterilization. In 5-liter batch fermentation of Bacillus sp. 2–6, 118.0 g/liter of l-lactic acid with an optical purity of 99.4% was obtained from 121.3 g/liter of glucose. The yield was 97.3% and the average productivity was 4.37 g/liter/h. The maximum l-lactic acid concentration of 182.0 g/liter was obtained from 30-liter fed-batch fermentation with an average productivity of 3.03 g/liter/h and product optical purity of 99.4%.A Bacillus sp. strain 2–6, high concentration of optically pure l-lactic acid could be produced efficiently in open fermentation without sterilization, which would lead to a new cost-effective method for polymer-grade l-lactic acid production from renewable resources.With the newly isolated  Because the physical properties and stability of PLA depend on the isomeric composition of lactic acid, the optically pure lactic acid is essential for polymerization Lactic acid, also named 2-hydroxypropanoic acid, is a versatile chemical widely used in food, cosmetic, pharmaceutical, textile and chemical industries. Recently, its application has been extended to the plastics industry, where polylactic acid (PLA) is utilized as a biodegradable and biocompatible plastic material l(+) or d(−) only) or a racemic mixture (dl) of lactic acid using different organisms Lactic acid can be produced by either chemical synthesis or fermentation. Chemical synthesis provides only the racemic lactic acid, whereas, fermentation technology can produce single desired stereoisomer . The enantiomeric excess (ee) value of l-lactic acid produced by this strain was compared with those by some Lactobacillus species under same conditions , while activities of l-lactate dehydrogenase were detected in all the three strains . Effects of several inorganic nitrogen sources and trace elements on l-lactic acid production were investigated and the results showed that NaNO3, NH4Cl and Mg2+ had positive effect on l-lactic acid production when YE (5 g/liter) was used as the sole nitrogen source (BacillusX1) and YE (X2) had significant positive influence on l-lactic acid production l-lactic acid reached its maximum of 115.0 g/liter at X1 = 7 g/liter, X2 = 12 g/liter. Central composite design was carried out around this point and the maximum value of l-lactic acid (116.7 g/liter) was attained at soy peptide and YE concentrations of 5.1 g/liter (x1 = −0.4680) and 14.3 g/liter (x2 = −0.3768), respectively  and YE (X2) concentrations at 1.2 g/liter and 12.6 g/liter resulted in 110.9 g/liter of l-lactic acid production, amounting to 95.0% of the theoretical maximum yield (The dosage of soy peptide and YE were further optimized by testing the cost-effectiveness of the medium. Soy peptide (um yield . This mel-lactic acid production by Bacillus sp. 2–6 was obtained (g/liter): glucose 97–133, YE 12.6, soy peptide 1.2, cottonseed protein 3, NaNO3 1, NH4Cl 1.According to the results of the statistically designed experiments and the investigation on initial glucose concentration, a cost-effective medium for Bacillus sp. 2–6. Fermentation profiles suggested two distinct phases due to decoupling of growth from lactic acid production around 15 h at which the dry cell weight reached the maximum of 13.2 g/liter  were 5.41 g/liter/h and 3.07 g/liter/h, respectively. The formation of l-lactic acid finished at 27 h when the residual glucose was completely consumed, and the l-lactic acid titer climbed to its maximum at 118.0 g/liter. The yield was 97.3% of the theoretical value and no other organic acids could be detected. The average productivity from 0 to 27 h was 4.37 g/liter/h. At 0 h, the ee value of l-lactic acid was 62.1%, indicating that there was 0.35 g/liter d-lactic acid mainly from the medium. The ee value dramatically increased to 88.9% after 3 h and then reached 99.4% at 18 h : glucose 150, YE 20, CaCO3 75. After 48 h of incubation at 55°C without agitation, the strain that produced the most l-lactic acid, designated as 2–6, was selected for further analysis.Soil samples were collected from various areas, including farmland, gardens and lands near milk factories. Approximately 2 g of each was enriched in 50 ml of nutrient liquid medium and incubated at 55°C without agitation for 6 h. An aliquot of the broth was plated on nutrient agar medium containing (g/liter): glucose 50, YE 10, CaCOL. casei DSM 20011, L. plantarum DSM 20205, L. pentosus DSM 20314 and Lactobacillus sp. DSM 20605 were studied in this work. These strains were maintained on MRS agar slants containing (g/liter): peptone 10, ‘Lab-Lemco’ powder 8, YE 4, glucose 20, triammonium citrate 2, sodium acetate 5, K2HPO4 2, MgSO4·7H2O 0.2, MnSO4·H2O 0.05, CaCO3 10. The pH was adjusted to 6.5. The slants were incubated at 50°C or 37°C and the fully grown slants were stored at 4°C.Strain 2–6, 3 20 (GSY medium). The seed culture was prepared as follows: a loop of cells from the fully-grown slant was inoculated into 50 ml of the above sterile medium in 300-ml Erlenmeyer flasks and incubated for 12 h at 50°C without agitation. Then the seed culture was inoculated into Erlenmeyer flasks or bioreactors (inoculum volume at 10%) for lactic acid production.The medium for inoculation contained (g/liter): glucose 70, soybean peptone 5, YE 10, CaCOBacillus sp. 2–6 was tested using the medium contained (g/liter): glucose 100, YE 20, CaCO3 60. Fermentations were conducted at 45°C, 50°C, 55°C and 60°C, respectively, and samples were taken after 48 h of fermentation.The optimal fermentation temperature of 3 70. The initial glucose concentrations studied were at 97 g/liter, 133 g/liter, 186 g/liter, 220 g/liter and 254 g/liter. Samples were taken every 12 h and the concentrations of residual glucose and l-lactic acid were determined. All the fermentations mentioned above were conducted at 50°C in 300-ml Erlenmeyer flasks containing 100 ml media without agitation.For studying initial glucose concentration, the following medium was used (g/liter): YE 20, CaCOBacillus sp. 2–6, L. pentosus DSM 20314 and L. plantarum DSM 20205, cells grown in GSY medium were harvested during the exponential phase. Then they were washed and resuspended in 0.1 M potassium phosphate buffer (pH 7.4) and disrupted by sonication in an ice bath. The disrupted cells were centrifuged for 15 min at 10,000×g, and the supernatant was used as the crude cell extract. d-Lactate dehydrogenase (EC 1.1.1.28) was assayed photometrically (340 nm) by reactions coupled to NAD reduction as described Bacillus sp. 2–6 and 37°C for Lactobacillus strains. One unit (1 U) was defined as the amount of enzyme accumulating 1 µmol of NADH per minute. Protein concentrations were determined as described previously To assay the enzymatic activities in d-lactate or dl-lactate) separately with gentle shaking for 60 min. Cell extracts of Bacillus sp. 2–6, L. pentosus DSM 20314 and L. plantarum DSM 20205 were concentrated and used for the native PAGE.Active staining of NAD-dependent lactate dehydrogenases after native PAGE was performed according to the previous report 3. All of the experiments in this part were conducted in 300-ml Erlenmeyer flasks containing 100 ml of each medium with shaking at 100 rpm on a rotary shaker at 50°C. Samples were taken after 24 h and the concentrations of l-lactic acid were determined.Statistical approaches including Plackett-Burman design, steepest ascent design and central composite design were used in this study F was calculated based on the following values: μ = 0.51/h or 0.15/h, V0 = 3 liters, Yx/s = 0.1, Si = 800 g/liter. In all fed-batch fermentations, temperature was controlled at 50°C during the first 36 h and then shifted to 55°C gradually (0.2°C/h) to increase the solubility of calcium lactate and maintain the liquid state of the broth. Agitation was maintained at 200 rpm for batch and fed-batch fermentations. The pH was controlled at 5.6 by automatically adding CaCO3 slurry and no aeration was used in all batches. The preparation of pre-culture was the same as that in flask experiments and the inoculum volume was 10% (v/v). Fermentations were conducted open and without sterilization.Batch fermentations of strain 2–6 were carried out in a 5-liter bioreactor  containing 3 liters of the optimal medium and the temperature was maintained at 50°C. Fed-batch fermentations were performed in the 5-liter bioreactor containing 3 liters of the medium and a 30-liter bioreactor  containing 20 liters of the medium. In the pulse feeding strategy, when the residual glucose concentration was below 20 g/liter, glucose powder (200 g) was added to the 5-liter bioreactor. Constant glucose-concentration feeding strategy was performed by pumping glucose solution (800 g/liter) into the bioreactor to maintain the residual glucose concentration at about 20 g/liter. Exponential feeding strategy was performed according that reported previously l-lactic acid were measured using an SBA-40C biosensor analyzer . Dry cell weight was calculated from the optical density (OD620 nm) with a linear correlation factor (1 OD620 nm = 0.565 g dry cell weight/liter). CaCO3 particles were eliminated by adding hydrochloric acid before measuring OD620 nm. High performance liquid chromatography (HPLC) analysis of l/d-lactic acid was performed on an Agilent 1100 series  equipped with a chiral column  and a tunable UV detector at 254 nm. The mobile phase was 2 mM CuSO4 at a flow rate of 0.5 ml/min (25°C). The optical purity of l-lactic acid was described as ee value which was defined as 2SO4. The flow rate was 0.5 ml/min, and chromatographic peaks were detected in line at 205 nm. The column temperature was maintained at 55°C.The concentrations of residual glucose and Table S1Comparison of L-lactic acid optical purity produced by different bacteria(0.03 MB DOC)Click here for additional data file.Supplementary Information S1Statistical optimization of fermentation medium.(0.14 MB DOC)Click here for additional data file.Figure S1Y) as a function of soy peptide (x1) and YE (x2). ▴ represents the location where the maximized L-lactic acid concentration occurred; • represents raw data point.The response surface plot (A) and the corresponding contour plot (B) of L-lactic acid concentration ((1.17 MB TIF)Click here for additional data file.Figure S2Z as a function of soy peptide (X1) and YE (X2) concentrations. ▴ represents the location where the maximized Z occurred.The response surface plot (A) and the corresponding contour plot (B) of (1.21 MB TIF)Click here for additional data file.


Nitrosylcobalamin (NO-Cbl) is a chemotherapeutic pro-drug derived from vitamin B12 that preferentially delivers nitric oxide (NO) to tumor cells, based upon increased receptor expression. NO-Cbl induces Apo2L/TRAIL-mediated apoptosis and inhibits survival signaling in a variety of malignant cell lines. Chemotherapeutic agents often simultaneously induce an apoptotic signal and activation of NF-κB, which has the undesired effect of promoting cell survival. The specific aims of this study were to 1) measure the anti-tumor effects of NO-Cbl alone and in combination with conventional chemotherapeutic agents, and to 2) examine the mechanism of action of NO-Cbl as a single agent and in combination therapy.Using anti-proliferative assays, electrophoretic mobility shift assay (EMSA), immunoblot analysis and kinase assays, we demonstrate an increase in the effectiveness of chemotherapeutic agents in combination with NO-Cbl as a result of suppressed NF-κB activation.Eighteen chemotherapeutic agents were tested in combination with NO-Cbl, in thirteen malignant cell lines, resulting in a synergistic anti-proliferative effect in 78% of the combinations tested. NO-Cbl pre-treatment resulted in decreased NF-κB DNA binding activity, inhibition of IκB kinase (IKK) enzymatic activity, decreased AKT activation, increased caspase-8 and PARP cleavage, and decreased cellular XIAP protein levels.The use of NO-Cbl to inhibit survival signaling may enhance drug efficacy by preventing concomitant activation of NF-κB or AKT. A major obstacle to conventional chemotherapy is the unwanted activation of survival signaling which leads to acquired resistance and decreased therapeutic efficacy. Nuclear factor kappa-B (NF-κB)NF-κB is a family of heterodimers: NF-κB1 (p50/p105), NF-κB2 (p52/p100), REL, RELA (p65/NF-κB3) in mammals, Dosal, Dif, and Relish in Drosophilia Constitutive activation of NF-κB has been implicated in the development of chemo-resistance in several human carcinoma cell linesAkt, a serine/threonine kinase that mediates survival signaling, functions as an oncogene and is implicated in resistance to chemotherapyWe have previously demonstrated the anti-tumor activity of nitrosylcobalamin (NO-Cbl) as a single agent and in combination with biological therapies such as IFN-βWe have previously shown that NO-Cbl suppressed Apo2L/TRAIL- and TNF-α- mediated IKK activation with subsequent decreased phosphorylation of IκBα and inhibition of NF-κB DNA binding activityTo test the hypothesis that NO-Cbl might potentiate the anti-neoplastic effects of various chemotherapeutic agents, we measured antiproliferative effects in thirteen cell lines using 18 chemotherapeutic agents . We usedEMSA was utilized to examine the effects of NO-Cbl on NF-κB activation by chemotherapeutic agents . We examDoxorubicin treatment resulted in NF-κB activation in all four cell lines. NO-Cbl inhibited NF-κB DNA binding activity in A375 cells by 40.71% (95% CI = 40.52% to 40.87%), MCF-7 cells by 46.99% (95% CI = 46.41% to 47.64%), SW480 cells by 7.52% (95% CI = 5.71% to 9.14%), and OVCAR-3 cells by 40.13% (95% CI = 39.30% to 41.08%).Cisplatin treatment resulted in low-level NF-κB activation in A375 cells. NO-Cbl inhibited cisplatin-induced activation by 41.54% (95% CI = 40.77% to 42.41%) in A375 cells. Cisplatin did not activate NF-κB in OVCAR-3 cells.5-FU activated NF-κB in each cell line treated. NO-Cbl inhibited NF-κB activation in A375 cells by 6.89% (95% CI = 6.43% to 7.28%), SW480 cells by 38.14% (95% CI = 34.91% to 40.69%), and OVCAR-3 cells by 18.34% (95% CI = 17.90% to 18.72%).Etoposide activated NF-κB in A375 and OVCAR-3 cells. Pre-treatment with NO-Cbl inhibited NF-κB DNA binding activity by 29.70% (95% CI = 28.79% to 30.69%) in A375 cells, and 30.37% (95% CI = 30.00% to 30.67%) in OVCAR-3 cells.Paclitaxel treatment of MCF-7 and OVCAR-3 cells induced NF-κB activation in both cell lines. NO-Cbl inhibited paclitaxel-induced activation by 47.28% (95% CI = 46.23% to 48.43%) and 43.40% (95% CI = 42.48% to 44.47%) respectively, in MCF-7 and OVCAR-3 cells.IκB kinase (IKK) mediates phosphorylation of IκBα, marking it for eventual polyubiquitination and proteolysis thereby resulting in NF-κB activationDoxorubicin activated IKK in MCF-7, SW480 and OVCAR-3 cells. NO-Cbl decreased IKK activity in MCF-7, SW480 and OVCAR-3 cells by 41.70% (95% CI = 39.65% to 44.47%), 45.22% (95% CI = 41.43% to 50.07%) and 50.69% (95% CI = 48.39% to 53.57%), respectively.Cisplatin treatment resulted in minor activation of IKK only in OVCAR-3 cells. NO-Cbl was effective at inhibiting this activation by 17.06% (95% CI = 15.48% to 19.06%). 5-FU treatment resulted in IKK activation in SW480 and OVCAR-3 cells. NO-Cbl inhibited 5-FU induced IKK activation in SW480 cells by 18.85% (95% CI = 17.20% to 21.05%), and OVCAR-3 cells by 34.12% (95% CI = 31.21% to 37.74%). Paclitaxel did not activate IKK in MCF-7 or OVCAR-3 cells. Treatment of OVCAR-3 cells with etoposide resulted in IKK activation which was inhibited by NO-Cbl by 71% (95% CI = 70.22% to 71.96%).Next we examined the role of the pro-survival protein AKT and the effects of NO-Cbl on its activation , using tDoxorubicin increased the phosphorylation of AKT in MCF-7 and SW480 cells which was inhibited by NO-Cbl by 83.77% (95% CI = 81.43% to 86.21%) and 52.60% (95% CI = 49.62% to 55.04%), respectively. Treatment of MCF-7 cells with paclitaxel resulted in increased phosphorylation of AKT which was inhibited by 43.74% (95% CI = 42.01% to 45.00%) following NO-Cbl. Treatment of SW480 cells with 5-FU increased AKT activation which was inhibited by NO-Cbl by 32.50% (95% CI = 31.28% to 33.61%). Although increased phosphorylation of AKT did not occur with any of the chemotherapeutic agents in OVCAR-3 cells, which exhibited a high basal level of phosphorylated AKT, NO-Cbl reduced the high basal AKT phosphorylation levels.X-linked mammalian inhibitor of apoptosis (XIAP), one of the most potent inhibitors of apoptosis, is a NF-κB-induced geneConsistent with AKT phosphorylation in A375 cells, XIAP protein expression was enhanced following doxorubicin, cisplatin, 5-FU, and etoposide treatment. NO-Cbl blunted XIAP expression by 20.12% (95% CI = 14.64% to 23.79%) with doxorubicin, 19.40% (95% CI = 17.06% to 22.00%) with cisplatin, 21.12% (95% CI = 20.89% to 21.31%) with 5-FU, and 18.62% (95% CI = 17.57% to 20.09%) with etoposide.In addition, doxorubicin increased XIAP expression in MCF-7, SW480, and OVCAR-3 cells; expression was inhibited with NO-Cbl treatment by 44.96% (95% CI = 39.66% to 47.85%), 12.16% (95% CI = 10.57% to 13.79%), and 64.90% (95% CI = 60.46% to 66.85%), respectively. 5-FU treatment increased XIAP expression in SW480 cells which was inhibited with NO-Cbl by 13.66% (95% CI = 11.87% to 15.75%). NO-Cbl treatment inhibited basal XIAP expression when combined with every chemotherapeutic agent in all cell lines examined.A375 cells were stimulated with doxorubicin, followed by assessment of AKT phosphorylation, XIAP expression, and caspase-8 and PARP cleavage. . NO-Cbl Concordantly, caspase-8 activity was enhanced when NO-Cbl was combined with doxorubicin at 8, 12, 16 and 24 h by 41.39% (95% CI = 27.98% to 47.95%), 36.82% (95% CI = 32.79% to 39.87%), 17.54% (95% CI = 12.22% to 23.98%), and 25.28% (95% CI = 7.88% to 34.92%), respectively. PARP cleavage was increased at all time points following treatment with NO-Cbl and doxorubicin. NO-Cbl enhanced the cleavage of PARP by 82.94% (95% CI = 81.27% to 84.14%) at 8 h, 49.36% (95% CI = 41.51% to 58.74%) at 12 h, 64.67% (95% CI = 60.68% to 67.69%) at 16 h, and by 35.81% (95% CI = 33.93% to 36.54%) at 24 h.Next, we examined the expression of XIAP in OVCAR-3 cells treated with etoposide and NO-Cbl . ExpressIn vitro combination therapy with NO-Cbl on tumor cells in culture exhibited positive effects in inhibiting cell proliferation and decreasing cell survival signaling  compared to controls. Tumors from mice treated with etoposide were smaller by 27.30%  compared to control tumors. Mice receiving the combination of NO-Cbl and etoposide displayed tumors that were inhibited by 99.01%  compared to controls. In two mice, tumors disappeared completely and did not recur 60 days after treatment cessation.ignaling –6. Tpalpable . After 2In a syngeneic P388 murine leukemia model, cells were inoculated into the peritoneal cavities of DBA/2 mice. Daily drug treatments began on day 2 following inoculation . UntreatNitric oxide (NO) is a ubiquitous multi-faceted signaling molecule critical to many physiological and pathological processesA major advantage of the pro-drug NO-Cbl is its tumor-specific accumulation due to higher transcobalamin receptor (TCII-R) expression in tumor cells compared to normal tissuesNO inhibits survival signaling via inhibition of NF-κB The activation of NF-κB is a highly complex process. There are cell type-specific variations in NF-κB activation that differ between cell lines as well as activation differences among known NF-κB-activators. Taxol treatment of pancreatic cancer cell lines induces formation of several different NF-κB/IκB complexes, suggesting the presence of multiple up-stream activators of NF-κBOur data demonstrates consistent inhibition of NF-κB by NO-Cbl, irrespective of the survival pathways induced by a variety of chemotherapeutic agents. We demonstrate increased NF-κB DNA binding induced by almost every chemotherapeutic agent across all cell lines. Doxorubicin, etoposide, and paclitaxel induced NF-κB activation in all cell lines examined . InteresConversely, AKT was activated by cisplatin, 5-FU, and etoposide in A375 cells. NO-Cbl markedly reduced AKT phosphorylation following all treatments . Thus, Ain vivo  , consistin vivo  is consi1 and 15dPGJ2) inhibit IKK by covalently modifying a critical cysteine residue (C179) within the activation loop We are continuing to study the mechanism by which NO-Cbl inhibits NF-κB activation. This likely involves S-nitrosylation Therefore, NO-Cbl likely nitrosylates redox-sensitive residues in its target proteins, causing inhibition of NF-κB-mediated survival signaling and this appears to also be the case with AKT-mediated survival signaling. We have shown that NO-Cbl maximizes the anti-tumor effectiveness of primary chemotherapeutic agents. The use of NO-Cbl to inhibit survival signaling may prevent drug resistance, a common occurrence in multiple cycle chemotherapy, and may improve response rates and enhance drug efficacy.Nitrosylcobalamin was synthesized as previously described2 at 37°C in a humidified incubator. Cells were confirmed as mycoplasma free using a commercially available kit .Human tumor cell lines  were grown in RPMI 1640 and Dulbecco's modified Eagle medium as appropriate  supplemented with 10% fetal bovine serum  and 1% Antibiotic-Antimycotic  according to ATCC recommendations. Cells were maintained in 5% COexp) were compared with initial absorbance readings representing the starting cell numbers (Aini). To determine the starting cell number, an additional 96-well plate was seeded with cells and fixed at the beginning of the experiment. The absorbances derived from the initial plate and from the untreated cells at the end of the growth period (Afin) were defined as 0% and 100% growth, respectively. The percentage control growth (100%×[Aexp–Aini]/[Afin–Aini]) was expressed as a percentage of untreated controls.Cells were harvested with 0.5% trypsin/0.53 mM EDTA, washed with PBS and resuspended in media containing 10% FBS. Cells were plated in 96-well plates in 0.2 mL aliquots containing 2,000 cells. Cells were allowed to adhere to the plate for 4 h and then NO-Cbl was added in different dilutions to the assay plate. Replicates of eight were performed for each treatment. After 16 h, chemotherapeutic agents were added at different concentrations. Growth was monitored by the sulforhodamine B  colorimetric assay(nu/nu) mice  were inoculated with NIH-OVCAR-3 tumors. There were four experimental groups , n = 10. Tumor cells (2×106) were inoculated into flanks in the mid-axillary line. NO-Cbl was given once daily  and etoposide  was administered once weekly, starting on day 2. Tumor volume was measured three times a week using the formula for a prolate spheroid: (4/3) πab2 where 2a = major axis, 2b = minor axis. Formalin-fixed sections were processed by the Cleveland Clinic Histology Core. Sections were stained with hematoxylin and eosin and evaluated for pathologic changes in a blinded fashion. For syngeneic studies, five week-old DBA/2 male mice (Taconic) were inoculated with P388 murine leukemia cells. There were four experimental groups , n = 10. Tumor cells (105) were inoculated i.p.. NO-Cbl was given twice daily  and doxorubicin  was administered once weekly, starting on day 2. Animals were monitored for ninety days. The Institutional Animal Care and Use Committee at the Cleveland Clinic Foundation approved all procedures for animal experimentation.Five week-old NCR male athymic nude homozygous Cells were pre-treated with NO-Cbl (300 µM) for 16 h followed by doxorubicin , cisplatin , 5 flurouracil , etoposide , or paclitaxel . As a positive control for NF-κB induction, cells were treated with TNF-α . Whole cell lysates were prepared in 1× high salt lysis buffer , 10% glycerol, 0.5 mM NaOrthovanadate, 1mM PMSF, 0.5% NP40 and 1× protease inhibitor cocktail set I  and 1× phosphatase inhibitor cocktail set II  for subsequent immunoblotting studies. Total cell protein (80 µg per sample) was resolved by electrophoresis on a 10% Bis-TRIS gel  resolved at 160 V for 1 hour and transferred to a PVDF membrane  with the use of a wet transfer apparatus (Bio-Rad) for 30 min at 100 V . The membranes were incubated in blocking buffer  for 1 h at room temperature and then incubated overnight with primary antibody  diluted in Starting Block. After washing in 1× TBST  membranes were incubated with horseradish peroxidase-conjugated goat secondary antibodies (Biorad) diluted in Starting Block for 1 h at room temperature. The protein bands were visualized by enhanced chemiluminescence . Antibodies against glyceraldehyde-3-phosphate dehydrogenase  were used to ensure equal loading.GGCGACTTTCCCAGGC 3′)  was end-labeled with γ32P-dATP , using T4 polynucleotide kinase . DNA binding reactions were performed in 28 µL reaction volume for 30 min on ice containing 20 µg protein, 100 mM HEPES, 3.0 mM EDTA, 50% glycerol, 3 mM DTT, 25 mM MgCl2, 20 mM TRIS, pH 7.90, 5 ug Poly [d(I-C)] and labeled probe. Complexes were separated from the free probe on 6% non-denaturing polyacrylamide gels in 0.5× TBE buffer at 195 V for 2 h. Gels were dried and exposed to film. To verify that the band shifts were comprised of NF-κB, lysates from cells that were stimulated by TNF-α  were incubated with anti-NF-κB p65 antibody .Cells were pre-treated with NO-Cbl (300 µM) for 16 h followed by doxorubicin  or cisplatin  or 5 flurouracil  or etoposide  or paclitaxel . As a positive control for NF-κB induction, cells were treated with TNF-α . Plates were washed three times with ice-cold 1× DPBS . Cells were scraped from plates and resuspended in 1× high salt lysis buffer as above. The lysates were transferred to pre-chilled microcentrifuge tubes and incubated at 4°C using a vortex for 30 min followed by centrifugation at 20,000g for 15 min. Samples were snap frozen with liquid nitrogen and stored at −70°C until needed. Supernatants were transferred to fresh tubes and protein concentrations were assessed using the Bradford method . We used a commercially available consensus binding site for NFκB/c-Rel homodimeric and heterodimeric complexes as a probe to assess DNA binding activity. The NF-κB consensus binding sequence  was added and mixed by rotation in the cold for 30 min. Protein G immunopellets were collected by centrifugation at 800 g for 30 sec, washed 3 times with Buffer B (Buffer A plus 250 mM NaCl), and once with Buffer C (Buffer A plus 50 mM NaCl and 10 mM MgCl2). Immunopellets were re-suspended in 30 µL kinase buffer with 0.1 mM orthovanadate, 50 µM unlabeled ATP, 5 µCi γ32P-ATP, 2 mM DTT, and 2 µg of recombinant GST-IκBα1-54Whole cell extracts (200 µg) treated as above were supplemented with 150 µL of Buffer A , 10 mM 2-mercaptoethanol (BME), 0.5 mM PMSF, and protease cocktail), 2 µL normal rabbit serum (NRS), and mixed by rotation at 4°C for 1 h as previously describedt test, using a pooled estimator of variance, to determine statistical significance using Sigma Plot 10.0 . The logrank test was used to calculate the significance of the Kaplan Meir survival curve. The experimental design utilized the same set of cell lysates for Median effect analysis was used to characterize the interaction between NO-Cbl and various chemotherapeutic agents


The N-Nitrosomethylurea breast cancer model induced in rats is used for the study of carcinogenesis in mammary cancer, prostate, pancreas, etc. This model is very similar to human neoplastic disease.The present experimental study was designed to assess whether metoclopramide administration has any effect on development of MNU-induced tumours, and evaluate the treatment of goserelin acetate on PRL, TNF alpha and NO expression. NMU was administered to female Wistar rats on 2 occasions (5 mg/100 g body w/rat). PRL and TNF alpha were performed by immune-assay. Nitric Oxide by semi automated-assay and ploidy analyses by flow cytometry.The administration of metoclopramide made the induction time shorter and increased the incidence and average of tumours per rat. Tumours development was inhibited by a goserelin chronic administration. The ploidy of adenocarcinoma was polyploid-aneuploid type (average S = 60%). It was higher basal PRL plasma levels in rats with NMU induced tumours than in basal controls without tumour (p < 0.001). The goserelin "in bolus" administration showed maximal inhibition of plasma PRL at 90 min. Plasmatic TNF alpha expression was inhibited at 60 min and also remained inhibited in tissue homogenate post chronic treatment (P < 0.0125). Plasmatic NO expression is higher in rats with induced tumours than healthy controls (P < 0.001). In tissue homogenate NO values were inhibited at 90 min (P < 0.01), as well during chronically goserelin treatment (P < 0.005).The increase of blood PRL levels in NMU-induced rats may be an indicator of a poor prognosis of mammary cancer evolution. The metoclopramide administration accelerates tumour growth. However goserelin administration achieves regression in tumour development associated to inhibition PRL, TNF alpha and NO expression. N-nitroso-N-methylurea (NMU) is an effective chemical carcinogen for the induction of mammary carcinoma in rats and a very good model for human mammary carcinomas -6. Prola14H22ClN3O2), their IUPAC name: 4-amino-5-chloro-N-(2-diethylamino)ethyl-2-methoxybenzamide is a potent dopamine receptor antagonist used for its antiemetic effect and by inhibiting the action of PRL inhibiting hormone and has sometimes been used to stimulate lactation. It thus may induce hyperprolactinemia in rats.Metoclopramide  is known to suppress ovarian function and plasma prolactin levels and its antitumour activity has been evaluated extensively .In chemical carcinogenesis, internal cytokines, such as interleukin (IL) 1α and tumour necrosis alpha (TNFα) contribute to tumour progression. However it has been suggested that TNF on tumour vasculature has a direct anti-tumour mechanism -14. IndeInducible nitric oxide synthase (iNOS) and nitric oxide (NO) are implicated in tumour pathology and, it is well known that TNFα induces iNOS and NO release. While low concentrations of NO alter transcription factor and act as an antioxidant factor, high concentrations of NO can damage DNA and proteins. Moreover NO-induced alteration in cellular function can lead to cell death or stimulation of growth, and stimulate tumour growth by promoting angiogenic and invasive capacities -19.The aim of the study was to assess the effect of metoclopramide on tumour development of NMU-induced tumours in Wistar rats, and at the same time to study acute and chronic administration of  goserelin on PRL, TNFα and NO expression in this experimental model.Wistar rats were used for all the experiments and were obtained at 45 days of age. Animals were housed 2/cage in a room illuminated for 12 h each day at a temperature of 25°C. The animals had free access to Purina laboratory chow and drinking water. All the experiments in this study were performed in compliance with the Royal Decree 223/1988 of March (BOE 8 18) and the Ministerial Order of 13 October 1989 (BOE 8) regarding protection of the experimental animals, as well as with the European Council Directive of 24 November 1986 (86/609/EEC).Crystalline NMU  was dissolved in 0.85% NaCl solution and acidified (acetic acid) at 5.0 pH. The concentration was then adjusted to 5 mg/100 g body wt/rat. Heparin-treated blood (0.2 ml) was obtained by cardiac puncture from rats lightly anesthetized with ether.Animals were palpated for detection of mammary tumours initialling after injection of NMU and the animals were examined by palpation for tumour masses thrice weekly post administration of carcinogen. Tumour sizes were measured with a calliper and their growth and histological parameters were tested.The diagnoses of mammary gland cancer were made by histological examination of paraffin sections stained with haematoxylin/eosin in all tumours. Autopsy was performed when the neoplasms were large. A nodule of mammary cancer is designated an active tumour, the number of active tumours is expressed per rat with mammary cancer.The tumour samples were prepared in the following manner: after sacrifice of the animals by decapitation the tumours were removed immediately. All further steps in the preparation were performed at 0–4°C. After being washed in ice-cold PBS buffer (pH 7.4) the tumours were homogenized in this buffer (10 ml/5 g of wet tumour). The homogenate was isolated by centrifugation for 30 min at 9000 × g and the supernatant were stored in aliquots at -80°C until used in each of the corresponding assays.Tumour homogenate and plasma were analyzed for prolactin concentration using a commercially available ELISA kit . The basal level of rat plasma was in accord to that indicated and obtained using standard rat plasma provided by the ELISA kit and indicating that rat stress was avoided, as confirmed by the variability of the plasma blood levels in controls.For TNF α concentration was using a commercially available kit: .An assay for Nitric Oxide concentration was performed according to Navarro-Gonzáles et al .The blood was centrifuged at 1500 g for 15 min; plasma was harvested and stored at -80°C until the hormonal concentration and biomarkers concentration were measured.Total protein concentration was determined by the Lowry assay  for tumour homogenates. In homogenate TNF α, NO concentrations were expressed relative to total protein.Histological analysis was performed on primary NMU tumours on haematoxylin-eosin stained sections. Flow cytometry  was performed of rat NMU induced tumours. Flow cytometry was performed on nuclei prepared from 40 μm thick sections from formalin fixed paraffin tissue by the modified technique of Hedley et al. and McLemore et al ,22.At 55 days of age, the female rats were divided into the following groups:1- Healthy Controls (n = 10) without NMU-induction.2- NMU controls (n = 20) received weekly an injection of NMU [a set of 2 injections during 2 weeks NMU (5 mg/100 g weight/rat)] and was observed after carcinogen administration for tumour development.3- NMU plus metoclopramide (M) (n = 20) in drinking water (0.125 mg/L), "at libitum", was observed weekly for tumour development. The elimination half-life for (M) is 5–6 hours.4- NMU plus goserelin (n = 40) and the metoclopramide administration was suspended in this group when tumour was detected and they were subdivided into 2 groups for (a) acute and (b)chronic study, respectively:a) Received goserelin administration "in bolus"  and killed 2 h after injection.b) Received goserelin (subcutaneous/daily) during 60 days (0.25 mg/ml/day).2 analysis and Yates correction. Inter-group comparisons were performed by Student t test. Values of p < 0.05 were considered to be significantly different.Differences in tumour incidence were demonstrated using xPost administration of NMU in control group, mammary cancer was detected in 9 of 20 (45%) rats on days 90–120. Metoclopramide treatment had effect on NMU-induced tumours, where the incidence was 85%. Latency to the appearance of first palpable tumour tended to be shorter in all rats that received metoclopramide. Indeed the average of tumours was higher than in controls, Table th day, compared with the administered goserelin, where the tumours stop in their development or diminish in size  the size of tumours observed an increase in growth at 400% on 20The histology revealed that all the tumours were adenocarcinoma.Analyses by flow cytometry of nuclear DNA patterns revealed 100% polyploidy in NMU induced tumours. According to this study all tumour samples were polyploidy-aneuploid and none of them was diploid type, percentage of S = 60%, Mitosis (ratio) = 1.99. DNA Index = 1.50 – 2.00, and with a CV (G0/G1) = 9%. Fig plasma the mean value of PRL levels was lower in healthy controls [C]  than in rats with NMU tumours [T] , P < 0.01. During metoclopramide (M) administration the PRL basal mean levels were 125.6 ± 8.5 ng/ml, n = 20 in healthy controls, Fig In The PRL plasma mean levels of NMU tumour rats [T], were higher than in tumour homogenates [TH] (corresponding to the same group of rats and extraction time) 5.8 ± 1.2 ng/ml (n = 20); these results were statistically significant (P < 0.0005).The PRL plasma levels decreased in NMU rats when they were treated with goserelin. During acute administration the time course was, basal: 51.3 ± 2.2 ng/ml (n = 20); 30 min 26.2 ± 6.5 ng/ml (n = 20), P < 0.001; 60 min 17.5 ± 13.5 ng/ml (n = 20), P < 0.0125; and at 90 min: 12.2 ± 4.2 ng/ml (n = 20), P < 0.0005.During chronic administration of goserelin the mean PRL levels were similar to those in healthy control rats: 49.0 ± 23.4 ng/ml (n = 20) P = 0.40, Fig tumour homogenate [TH] there was no statistically significant variations of PRL mean levels during acute administration of goserelin neither post chronically treated: Basal 5.8 ± 1.2 ng/ml (n = 20); 30 min 6.9 ± 0.1 ng/ml (n = 20), P = 0.20; 60 min 5.6 ± 0.3 ng/ml (n = 20); at 90 min 6.8 ± 0.2 ng/ml (n = 20), P = 0.35. At 60 days treatment the mean value was 5.9 ± 0.1 ng/ml (P = 0.35).In plasma. The mean values were: Basal 178.7 ± 11.6 pg/ml (n = 20); at 30 min 150.0 ± 15.0 pg/ml (n = 20), P = 0.10; at 60 min 145.2 ± 2.5 pg/ml (n = 20), P < 0.05; and at 90 min. 135.0 ± 5.0 (n = 20), P < 0.05; Fig TNFα expression during time course of acute administration of goserelin observed inhibition in The inhibition of TNF α expression was observed during post-chronic treatment with goserelin: 132.1 ± 52.2 pg/ml, n = 20 (P < 0.0125).tumour homogenates mean basal values of TNFα were: 255.0 ± 30.0 pg/mg of protein (n = 20), at 30 min 210.0 ± 12.5 pg/mg of protein (n = 18), P = 0.20; at 60 min 200.0 ± 15.0 pg/mg of protein (n = 18), P < 0.05; and at 90 min 210.0 ± 15.0 pg/mg of protein (n = 18), P = 0.10.In At 60 days of goserelin treatment, the TNFα expression observed a statistically significant inhibition when compared with the basal values, 255.0 ± 30.5 vs. 159.0 ± 5.2 pg/mg of protein (n = 18), P < 0.0125, Fig plasma inhibition of NO expression during time course of acute administration of goserelin has been observed but these differences were not statistically significant. Basal mean values were: 59.6 ± 5.5 μg/μl (n = 20), 30 min 56.7 ± 17.8 μg/μl (n = 20), P = 0.10; at 60 min 64.0 ± 17.9 μg/μl (n = 20) P = 0.15 and at 90 min 52.2 ± 7.5 μg/μl (n = 20) P = 0.25. Lack of inhibition of NO expression by goserelin was observed during chronic treatment 46.0 ± 19.9 μg/μl, n = 20 (P = 0.25) when compared with NMU induced basal values, Fig In female Wistar rats without NMU induced tumour, the basal mean NO concentration in plasma was 31.5 ± 9.5 μg/μl, n = 20. The mean NO concentrations were higher in plasma of female Wistar rats with NMU induced tumours than basal healthy controls . In tumour homogenate. The mean basal concentration was: 205.9 ± 42.3 μg/μg of protein (n = 20), at 30 min 134.0 ± 17.9 μg/μg of protein (n = 20) P = 0.05, at 60 min: 118.0 ± 22.9 μg/μg of protein (n = 20), P < 0.05, and at 90 min. 82.3 ± 9.9 μg/μg of protein (n = 20), P < 0.01.The NO concentration was inhibited by goserelin acute treatment in In chronically administered goserelin (at 60 day) the NO values lowered to 72.4 ± 9.9 μg/μg of protein and when compared with basal NO values statistically significant differences was observed between them (P < 0.005), Fig The MNU breast cancer model induced in rats is used for the study of carcinogenesis in mammary cancer, prostate, pancreas, etc. This model is very similar to human neoplastic disease . The devA great variability of various mouse and rat strains for sensitivity on mammary tumours with chemical initiating agents was studied and considered that their results support the need to use more then one rat strain for the initiation of mammary carcinogenesis, due to "very low"-sensitivity for the NMU tumour development in Wistar-Han rats . NeverthOur study demonstrated that metoclopramide (as dopaminergic antagonist) has a very high capacity to induce hyperprolactinemia in rat, indeed the tumour growth accelerated during the observation period and this effect could be influenced or stimulated by PRL release. These results are in agreement with different previous experimental and clinical studies on PRL-induced proliferation. ,27-33. TThe histological study of our tumours revealed adenocarcinoma type, similar to observed in human breast cancer. Flow cytometric S-phase fraction contributes to diagnosis of tumours. At present to our knowledge, only flow cytometric analyses were performed in NMU-induced solid tumours in rat prostate . MonakovInitially an acute administration of LH-RH agonist induces a release of LH and FSH. However, continuous and chronic administration produces an inhibition of the hypophyseal-gonadal-axis though the process of "down regulation" of pituitary receptors for LH-RH. Furthermore the expression on LH-RH receptor gene was established in tumours . In our It is well known that NO is a free radical that mediates a variety of cell functions. The plasmatic NO expression of our study showed a statistically significant increase of NO concentration in rats with NMU-induced tumours when compared to controls, which was observed in basal conditions. These results suggest that NO can stimulate tumour growth. Additionally, TNFα induces iNOS and NO production and could mediate tumour promoter-induced transformation.In this study we have examined PRL, TNF alpha and NO levels in plasma and in tumour homogenates during goserelin administration and, similar behaviour was observed. PRL levels in homogenate presented no statistical significant differences. It could be due to the initial low values in homogenate or because the sensitivity of the assay was insufficient.It is well known that NO levels in cancer cells contribute to tumour angiogenesis and during acute administration of goserelin it did not completely inhibit this increase. At present, the practical importance of these values or relevance with respect to predicting the disease course is not clear.This study demonstrates firstly the role "in vivo" of metoclopramide on tumour growth and secondly for goserelin in NMU-induced mammary tumours in which inhibitory effects were observed on growth as well as with significant tumour regression, besides it also exerted inhibitory effects on plasmatic and tissular PRL, TNFα and ON expression. In summary, these findings could reveal a possible new inhibitory pathway in the tumour development regulation by LH-RH analogues.IV designed and carried out the experiments and wrote the manuscript and REdeS participated in study analysis and drafted the manuscript. All authors read and approved the final manuscript.


N,N,N-trimethyl chitosan (TMC) with 93% degree of trimethylation was synthesized. TMC and the chitosan starting material were subjected to acidic hydrolysis to produce 49 different samples with a reduced average molecular weight (Mw) ranging from 2 to 144 kDa. This was done to allow the investigation of the relationship between antibacterial activity and Mw over a wide Mw range. NMR investigation showed that hydrolysis did not affect the degree of trimethylation (DSTRI) or the structure of the polymer backbone. The activity of TMC against Staphylococcus aureus (S. aureus) increased sharply with Mw until a certain Mw value  was reached. After the CMW, the activity was not affected by a further increase in the Mw. A similar pattern of activity was observed for chitosan. The CMW was determined to be 20 kDa for TMC and 50 kDa for chitosan.  Several studies reported so far produced varying Mw ranges for chitosan and chitooligosaccharides   were purchased from Oxoid .Chitosan TM 4138 , followed by addition of NaOH  under cooling (10 °C). Methyl iodide  was then added dropwise to the reaction mixture. After completion of addition, the reaction mixture was allowed to reach room temperature and then stirred for 48 h. The reaction mixture was then precipitated with acetone (150 mL) and the solid obtained was collected by filtration, followed by washing with fresh acetone (2 × 150 mL). The product was then subjected to the same reaction conditions three more times. The final product obtained was first purified by ion-exchange with 10% NaCl solution followed by dialysis for 2 days and freeze-drying. The pure product was obtained as a white solid. Yield: 4.7 g (80.0%). Chitosan  was stirred in DMF-H−1. 1H NMR : δ 2.01 (NAc), 3.28 [N(CH3)3], 3.68–4.07 , 4.30–4.42 , 5.42 ppm (H-1).FT-IR (KBr): 3239 (O–H), 2927 (C–H), 1636 (C=O amide I), 1473 (C–H), 1030 (C–O–C), 880 (C–H) cmTMC  was stirred in (a) concentrated HCl solution (7.5 mL) at 30 °C and samples (0.5 mL) were collected at time intervals of 0.15 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, 120 h, 216 h, 384 h, and 528 h and (b) in 1 M HCl solution (7.5 mL) at 60 °C and samples (0.5 mL) were collected at time intervals of 0.15 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, 120 h, and 216 h. Chitosan  was stirred in (a) concentrated HCl (7.5 mL) at 30 °C and samples (0.5 mL) were collected at time intervals of 0.15 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, 120 h, 216 h, 384 h, 528 h, 884 h, and 1388 h and (b) in 1 M HCl solution (7.5 mL) at 60 °C and samples (0.5 mL) were collected at time intervals of 0.15 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, 120 h, and 216 h. The samples were precipitated in acetone (60 mL), filtered and dried in vacuum for 3 h.1H-NMR, COSY-NMR and IR spectroscopy. 1H-NMR spectra were recorded using a Bruker Avance 400 instrument operating at 400.13 MHz at 300 K. NMR samples were prepared in D2O at concentrations of 10–15 mg/mL. The N-acetyl peak (2.08 ppm) was used as the internal reference in all spectra. FT-IR measurements were performed with a Nicolet iZ10 FT-IR instrument . Samples were placed over a diamond crystal and the spectrum was recorded using OMNIC 7 software. Each spectrum was collected in transmittance using 32 scans, resolution of 4 and data spacing of 0.482 cm−1. Characterization of the compounds was performed using N,N,N-trimethyl chitosan derivative was evaluated on the basis of the integral values in 1H NMR. The following equations were used for calculating the degree of acetylation (DA) and the degree of substitution (DS) for the chitosan derivatives:(1)Degree of acetylation, (2)N,N,N-trimethylation, DS for where H2 and H6 are the protons at position 2 and 6 respectively in the glucosamine unit; HAc is the protons in the N-acetyl group; N–CH3 are the protons in the N,N,N-trimethyl group.Equivalent quantities of reagents were calculated using one glucosamine unit. The degree of substitution (DS) for the GPC measurements were done using the Polymer Standards Service (PSS) , Dionex Ultimate 3000 HPLC system , Dionex Ultimate 3000 HPLC pump and Dionex Ultimate 3000 autosampler , Shodex RI-101 refractive index detector  and PSS’s ETA-2010 viscometer. WINGPC Unity 7.4 software  was used for data collection and processing. The eluent used was 0.1 M NaCl/0.1% TFA solution. Each sample was dissolved in the same eluent as mentioned above at a concentration of 1 mg/mL, filtered through a 0.45 μL filter  before measurement at 25 °C using a flow rate of 1 mL/min. Each sample had an injection volume of 100 µL and a retention time of 25 min and all the measurements were done in triplicates.S. aureus (ATCC 29213). The broth microdilution method was used to determine the MIC values using Mueller–Hinton broth at pH 7.2 for TMC and 5.5 for chitosan. Samples for TMC were prepared in sterile water (pH = 7.2) while chitosan samples were prepared at pH = 5.5 at an initial concentration of 32,768 µg/mL. This was then serially diluted by twofold dilutions in a 96-well plate using Muller–Hinton broth, to get final concentrations varying from 16,384 to 4 µg/mL. Gentamicin was used as the performance control during the test. A standard 0.5 McFarland suspension (1–2 × 108 colony-forming units (CFU)/mL) was prepared by direct colony suspension in Mueller–Hinton broth. This suspension was further diluted to achieve a final test concentration of 5 × 105 CFU/mL in the wells of the microtiter plate. Mueller–Hinton broth without any addition was used as a sterility control and Mueller–Hinton broth with bacterial suspension as a growth control. The microtiter plates were then incubated at 35 °C for 18 h under moistened conditions. The MIC values were determined as the lowest concentration of the antibacterial agent that completely inhibited the visible growth of the microorganism in the microtiter plate. The antibacterial activity was tested against the Gram-positive bacteria N,N,N-trimethyl chitosan (TMC) with a very high degree of N,N,N-trimethylation and low O-methylation was synthesized from chitosan starting material with DA of 8%. w for studying the relationship between antibacterial activity and Mw. The polymers were treated at different temperatures with concentrated (12 M) or diluted (1 M) HCl. Rapid hydrolysis of the polymer was observed in the first 24 h with more than 50% reduction in the Mw. Continued incubation up to 1388 h resulted in slow further degradation. NMR analysis confirmed the hydrolysis of the glycosidic bond. N,N,N-Trimethylation (DSTRI) was not affected and no other change in the structure was observed with extensive acid treatment. Acidic hydrolysis was performed on TMC and chitosan material and to produce samples of different Mw ranging from 133 to 2 kDa and 24 samples of TMC with Mw ranging from 144 to 2 kDa. The hydrolysis of chitosan gave 26 samples with MS. Aureus with all samples, as well as glucosamine.The antimicrobial assay was performed to determine the MIC for activity against S. aureus decreased with Mw until a certain Mw value (CMW) was reached. After this, no change in activity with a further increase in Mw was observed. The CMW was determined to be 20 kDa for TMC. The activity of chitosan also increased with Mw up to a CMW value of 50 kDa after which the Mw had no effect on activityThe MIC values for chitosan and TMC against w chitosan against different microorganism and the relationship for other chitosan derivatives may also be similar. This is the first study of molecular weight activity relationship for TMC. A number of studies have reported a similar pattern of activity for different MThese results on the Mw activity relationship may have implication for the possible mechanism of action for TMC and chitosan. This will be investigated in further studies.


Pinelliae Rhizoma (RPR) is a representative toxic herb that is widely used for eliminating phlegm or treating cough and vomiting. Given its irritant toxicity, its processed products, including Pinelliae Rhizoma Praeparatum (PRP) and Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine (PRPZA), are more commonly applied and administered concomitantly with other chemical drugs, such as cough medications. This study aimed to investigate the effects of RPR, PRP, and PRPZA on CYP3A activity. Testosterone (Tes) and buspirone (BP) were used as specific probe substrates ex vivo and in vivo, respectively. CYP3A activity was determined by the metabolite formation ratios from the substrates. Ex vivo results show that the metabolite formation ratios from Tes significantly decreased, indicating that RPR, PRP, and PRPZA could inhibit CYP3A activity in rats. CYP3A protein and mRNA levels were determined to explore the underlying mechanism. These levels showed marked and consistent down-regulation with CYP3A activity. A significant decrease in metabolite formation ratios from BP was also found in PRPZA group in vivo, implying that PRPZA could inhibit CYP3A activity. Conclusively, co-administration of PR with other CYP3A-metabolizing drugs may cause drug–drug interactions. Clinical use of PR-related formulae should be monitored carefully to avoid adverse interactions.Raw  Pinelliae Rhizoma (PR), the tuber of Pinellia ternate (Thunb.) Breit, is a commonly used Chinese herb with high bioactivity against cough and vomiting, or for eliminating the stagnation of phlegm . CY. CYex vi-OH-Tes)  35]. Th. Thex vi−1·day−1, p.o.) or saline. Compared with that in the control group, the formation rates of 6β-OH-Tes from 4, 10, and 30 μmol·L−1 Tes in the 0.45 g·kg−1·day−1 dose group decreased by 14% (p < 0.05), 12% (p < 0.05), and 4% (p > 0.05), respectively; those in the 0.9 g·kg−1·day−1 dose group decreased by 24% (p < 0.05), 19% (p < 0.05), and 11% (p < 0.05), respectively; those in the 1.8 g·kg−1·day−1 group decreased by 45% (p < 0.05), 36% (p < 0.05), and 32% (p < 0.05), respectively.−1·day−1, p.o.) or saline. Compared with that in the control group, the formation rates of 6β-OH-Tes from 4, 10, and 30 μmol·L−1 Tes in the 0.45 g·kg−1·day−1 dose group decreased by 29% (p < 0.05), 29% (p < 0.05), and 31% (p < 0.05), respectively; those in the 0.9 g·kg−1·day−1 dose group decreased by 32% (p < 0.05), 27% (p < 0.05), and 23% (p < 0.05), respectively; those in the 1.8 g·kg−1·day−1 dose group decreased by 24% (p < 0.05), 28% (p < 0.05), and 18% (p < 0.05), respectively.−1·day−1, p.o.) or saline. Compared with that in the control group, the formation rates of 6β-OH-Tes from 4, 10, and 40 μmol·L−1 Tes in the 0.45 g·kg−1·day−1 dose group decreased by 16% (p < 0.05), 1% (p > 0.05), and 1% (p > 0.05), respectively; those in the 0.9 g·kg−1·day−1 dose group decreased by 21% (p < 0.05), 16% (p < 0.05), and 9% (p < 0.05), respectively; those in the 1.8 g·kg−1·day−1 dose group decreased by 23% (p < 0.05), 9% (p > 0.05), and 10% (p < 0.05), respectively.−1·day−1 had the strongest inhibitory effects on CYP3A activity ex vivo. The processed products, especially PRPZA, only slightly inhibited CYP3A activity. Furthermore, some treated groups showed dosage dependency of the inhibitory effects. The inhibitory effects on CYP3A activity increased gradually with increasing dose of RPR. The PRP-treated groups showed an opposite tendency at 30 μmol·L−1 Tes that the inhibitory effects decreased gradually with rising dose. The PRPZA groups did not show marked dose-dependent inhibition on CYP3A activity. Therefore, the different processing methods of PR and different administration doses produced various but regular inhibitory effects on CYP3A. These findings may provide useful guidance for the clinical application of PR.As shown in −1·day−1), or saline. The ratio of CYP3A to β-actin concentration was used as the relative protein expression level. Compared with the control group, the CYP3A protein expression levels in the RPR group at 0.45, 0.9 and 1.8 g·kg−1·day−1 doses decreased by 42% (p < 0.01), 52% (p < 0.01) and 60% (p < 0.001), respectively; those in the PRP group decreased by 43% (p < 0.01), 41% (p < 0.01) and 31% (p < 0.01), respectively; those in the PRPZA group decreased by 34% (p < 0.01), 31% (p < 0.01) and 44% (p < 0.01), respectively.−1·day−1 doses decreased by 61% (p < 0.001), 69% (p < 0.001) and 53% (p < 0.001), respectively; those in the PRP group decreased by 22% (p < 0.01), 27% (p < 0.001) and 42% (p < 0.001), respectively; those in the PRPZA group decreased by 47% (p < 0.001), 57% (p < 0.001) and 66% (p < 0.001), respectively.−1·day−1 doses decreased by 37% (p < 0.001), 32% (p < 0.001) and 42% (p < 0.001), respectively; those in the PRP group decreased by −0.2% (p > 0.05), 6% (p > 0.05) and 23% (p < 0.01), respectively; those in the PRPZA group decreased by 36% (p < 0.001), 37% (p < 0.001) and 37% (p < 0.001), respectively.CYP3A protein and mRNA levels were determined to explore the underlying mechanism of changes in CYP3A activity. The CYP3A isoforms in rat include CYP3A1, CYP3A2, and CYP3A23. CYP3A1 and CYP3A2 are the most versatile isoforms in rat liver, which is involved in the metabolism of currently used drugs. Thus, in this research, we focused on CYP3A1/2 in rats. However, given the high homology of CYP3A1 and CYP3A2 , we inveex vivo, we chose the medium dose (0.9 g·kg−1·day−1) as the treated dose to further study the influence of PR on CYP3A activity via BP metabolism in vivo. −1 BP·HCl. As shown in 0-t of 6'-OH-BP /AUC0-t of BP) in the control group was 0.24 ± 0.06 [0-t of 1-PP/AUC0-t of BP) in the control group was 0.30 ± 0.11 [p < 0.01) in the PRPZA group; other formation ratios of 6'-OH-BP or 1-PP from BP showed no evident change (p > 0.05).Based on the results 4 ± 0.06 , whereas0 ± 0.11 , whereasin vivo. Conclusively, the inhibitory effects of RPR and PRP ex vivo were more evident than those in vivo. We presume that the inconsistent effects may be mainly attributed to the complex environment of rat in vivo, which will be explored urgently in the next research.The BP pharmacokinetic behaviors showed that the formation ratio of 1-PP from BP significantly decreased in the PRPZA group, but other formation ratios of 6'-OH-BP or 1-PP from BP did not markedly change. This finding demonstrates that PRPZA could inhibit CYP3A activity, but RPR and PRP had no significant effects on CYP3A activity Buspirone HCl, testosterone (purity > 98%), 6β-hydroxytestosterone (purity > 98%), and Gemfibrozil  were purchased from Sigma–Aldrich . 1-(2-pyrimidinyl) piperazine, 6'-hydroxybuspirone and SKF-525A HCl (used as a positive control drug) were obtained from Toronto Research Chemicals, Inc. . An NADPH regenerating system was purchased from BD Gentest Corp. . Acetonitrile, dichloromethane, and formic acid were high-performance liquid chromatography grade. All other chemicals used were of analytical reagent grade or better.P. ternate (Thunb.) Breit.  were purchased from Huamiao Traditional Chinese Medicine Engineering Technology Development Center  and confirmed by Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences.Male Sprague–Dawley rats (180–220 g) were obtained by the Laboratory Animal Center of Guangzhou University of Chinese Medicine  2008-0020), and housed four per cage in a unidirectional airflow room under control  with free access to standard rat food and water. All animal treatments followed guidelines for the care and use of laboratory animals and were approved by the ethics committee of Southern Medicine University .g for 30 min, and the supernatant was subjected to Ultra High Pressure Liquid Chromatography-mass spectrometry-Quadrupole-time of flight  for analysis. The detailed information of analysis conditions, the chromatograms and possible ingredients in RPR, PRP and PRPZA water extracts were shown in the RPR, PRP or PRPZA (90 g) was immersed in 1800 mL of water for 30 min and then extracted with boiling water for 30 min. The residue was again extracted with 900 mL of boiling water for 30 min. The supernatant was combined and condensed to 500 mL. The prepared water extracts (10 µL) were diluted 100 times with methanol. The mixture was subsequently vortexed and then centrifuged at 18,000 −1·day−1 as the high dose, medium dose and low dose, respectively), saline, or SKF-525A HCl (0.1 g·kg−1) for 7 day, the rats were anaesthetized with ethyl carbamate  on the eighth day, and the liver was excised. The livers of ten rats per group were combined to prepare RLMs. The detailed RLMs preparation procedures could be found in previous literature [Rats were randomly assigned to eleven groups with ten animals in each group. After oral pretreatment with RPR, PRP or PRPZA water extracts  and RLMs protein (0.02–0.05 mg·mL−1), was created to evaluate the formation rate of metabolite 6β-OH-Tes from Tes in RLMs with 7 d RPR, PRP or PRPZA pretreatment. The samples of Tes and 6β-OH-Tes were analyzed by a validated UPLC method described in our published paper [ex vivo was expressed as nanomoles of 6β-OH-Tes obtained per milligram of protein per minute. All experiments were run in triplicate.The detailed information about determination of CYP3A activity by Tes metabolism was described in our previous study . In brieed paper . CYP3A aEighty micrograms of total protein was separated by SDS-PAGE  and transferred from the gel to the PVDF membrane. Other detailed procedures were described in previous literature . ECL cheAfter pretreatment, rat hepatic total RNA was isolated using the TRIzol extraction method . cDNA was synthesized from total RNA using a reverse transcription kit . SYBR Green real-time PCR amplification and detection were then performed using an ABI 7500 system . The forward and reverse primers of CYP3A1, CYP3A2 and GAPDH were displayed in previous research , which a−1·day−1, p.o.) or saline for 7 days, the rats were given BP·HCl . Serial blood samples were collected at 0, 2, 7, 15, 30, 60, 90, 120, 180, 300, 480, and 720 min after BP·HCl administration. The plasma samples were processed and analyzed by a validated UPLC/MS/MS method as described in our previous studies [in vivo was determined by measuring the formation ratios of 6'-OH-BP and 1-PP from BP .Rats were randomly assigned to five groups with five animals in each group. After pretreatment with RPR, PRP or PRPZA water extracts  or one-way ANOVA followed by LSD test (for more than two groups) by SPSS 19.0. Statistical differences were considered significant at p < 0.05.Pharmacokinetic parameters were calculated using the standard non-compartmental method by Practical Pharmacokinetic Program Version 97 (3P97). All results were presented as mean ± standard deviation. Significant differences were analyzed using Student’s ex vivo and in vivo, which assessed the safety of co-administration of PR with other CYP3A-metabolizing drugs from the perspective of CYPs. Raw product RPR, as well as its processed products PRP and PRPZA can significantly inhibit CYP3A activity ex vivo. Such inhibition is due to the down-regulation of CYP3A protein and mRNA expression levels. Besides, the inhibitory effects caused by RPR and PRP ex vivo were more evident than that in vivo. Results of this study revealed that drug interaction between PR and the CYP3A-metabolizing drugs might be probable, suggesting that careful monitoring is essential for the concomitant use of PR with other drugs so as to avoid the adverse interactions. DDIs caused by PR-related co-therapy may be another factor that results in its toxicity. Considering no study has reported the index component in PR, so water extracts of PR were analyzed in this study based on clinical practices in China. Further research should be continued on the effects of bioactive ingredients in PR on CYP3A.This study is the first to investigate the influence of RPR, as well as its processed products PRP and PRPZA, on CYP3A