Candida spp., are commonly found on the skin and at mucosal surfaces. Yet, they rarely cause invasive infections in immunocompetent individuals, an observation reflecting the ability of our innate immune system to control potentially invasive microbes found at biological boundaries. Antimicrobial proteins and peptides are becoming increasingly recognized as important effectors of innate immunity. This is illustrated further by the present investigation, demonstrating a novel antifungal role of histidine-rich glycoprotein (HRG), an abundant and multimodular plasma protein. HRG bound to Candida cells, and induced breaks in the cell walls of the organisms. Correspondingly, HRG preferentially lysed ergosterol-containing liposomes but not cholesterol-containing ones, indicating a specificity for fungal versus other types of eukaryotic membranes. Both antifungal and membrane-rupturing activities of HRG were enhanced at low pH, and mapped to the histidine-rich region of the protein. Ex vivo, HRG-containing plasma as well as fibrin clots exerted antifungal effects. In vivo, Hrg−/− mice were susceptible to infection by C. albicans, in contrast to wild-type mice, which were highly resistant to infection. The results demonstrate a key and previously unknown antifungal role of HRG in innate immunity.Fungi, such as  Candida is present as a commensal at mucosal surfaces and on skin. Although it may cause life-threatening infections, such as sepsis, particularly in immunocompromised individuals, it seldom causes disease in normal individuals. In order to control our microbial flora, humans as well as virtually all life forms are armoured with various proteins and peptides that comprise integral parts of our innate immune system. Here we describe a new component in this system; histidine-rich glycoprotein (HRG), an abundant plasma protein. We show, using a combination of microbiological, biochemical, and biophysical methods, that HRG exerts a potent antifungal activity, which is mediated via a histidine-rich region of the protein, and targets ergosterol-rich membrane structures such as those of Candida. HRG killed Candida both in plasma as well as when incorporated into fibrin clots. In mouse infection models, HRG was protective against systemic infection by Candida, indicating a novel antifungal role of HRG in innate immunity.It has been estimated that humans contain about 1 kg of microbes, an observation that reflects our coexistence with colonizing microbes such as bacteria and fungi. The fungal species  During recent years it has become increasingly evident that many AMPs, such as defensins and cathelicidins, are multifunctional, also mediating chemotaxis, apoptosis, and angiogenesis The innate immune system, based on antimicrobial peptides (AMP) and proteins, provides a first line of defence against invading microbes et al.2+, or by protonation of histidine residues at acidic conditions 2+ sensor, providing a mechanism for regulating the various activities of HRG in vitro, as well as antitumor in vivo. Recent studies on Hrg−/− mice furthermore suggest that HRG plays a role as both an anticoagulant and an antifibrinolytic modifier, and may regulate platelet function in vivoHistidine-rich glycoprotein (HRG) is a plasma protein which was first isolated in 1972 by Heimburger in vitro which are dependent on Zn2+and pH in vitro, the ultimate role(s) of HRG in innate immunity in vivo still remained unresolved. During the course of our studies, we observed that HRG had a significant activity against Candida. Candida, an eukaryote, is present as a commensal at mucosal surfaces and on skin. Although it may cause life-threatening sepsis in immunocompromised individuals it seldom causes invasive disease in immunologically normal individuals Candida infections. In the present study we show, using a combination of microbiological, biochemical, and biophysical methods, that HRG exerts a potent antifungal activity particularly at low pH, which is mediated via its HRR, and targets ergosterol-rich membrane structures such as those of Candida. In mouse infection models, HRG protects against systemic infection by Candida, indicating a previously undisclosed antifungal role of HRG in innate immunity.Previous work has also demonstrated that HRG exert direct antibacterial activities Candida isolates. HRG was shown to be antifungal against C. parapsilosis at normal pH , and the activity was significantly increased in low pH buffer  (in vitro− levels (0.1 M) but only at low pH  were incubated with HRG (at 3 µM) at neutral as well as low pH. C. parapsilosis, C. albicans and C. krusei were all nearly completely killed by HRG at low pH, whereas C. glabrata exhibited a partial resistance at this concentration of HRG, the latter in analogy to C. glabrata displaying some resistance against histatin 5 C. papapsilosis was incubated with HRG at low pH, washed, and analysed by immunoblotting. Since previous results indicated that the HRR of HRG, which binds heparin/heparan sulfate, mediates antibacterial effects Candida. C. parapsilosis at neutral pH, and the binding was significantly increased at pH 5.5  . It is wt low pH . The antependent . In subst pH 5.5 , resultst pH 5.5 . In summCandida cells, it was of interest to further study the possible mode of action for HRG on Candida. Electron microscopy demonstrated that HRG caused membrane breaks in Candida cells and release of cytoplasmic components  and a truncated version (rHRG1-240), lacking the HRR and C-terminal domain. In contrast to full-length rHRG, truncated rHRG (0.6 µM) displayed no activity at pH 5.5 against Candida . Taken t4C. parapsilosis and C. albicans, particularly at low pH  and in platelets, also in wound fluid from acute wounds, and chronic leg ulcers . The observation that clots devoid of HRG showed detectable, although reduced, antifungal activity  when compared with wild type mice, and the wild type mice regained their initial weight after three days. Blood samples were collected from the animals 2 days post infection, and the fungal load in blood was determined (Candida cells was detected in the blood of Hrg−/− mice when compared with wild type mice (p = 0.032), indicating that a systemic infection has developed in HRG-deficient mice. In a similar experiment, we determined the ability of the fungi to establish infection in target organs distant from the site of administration. The spleen and kidney were harvested 3 days after initiation of intraperitoneal infection and the fungal load was determined. The results showed significant differences between Hrg−/− mice and the wild type mice; one animal out of 10 in the control group showed fungal load in the spleens and kidneys compared with 8 out of 10 in the Hrg−/− group (p = 0.009)  . Histopailtrates . These rin vivo together with the characterization of possible epitopes of HRG mediating this effect, as well as mechanistic data on HRG targeting of Candida membranes. The results have implications for our understanding of novel antifungal properties of HRG, and demonstrate that HRG constitutes a previously undisclosed natural and antimicrobial defence system.The key findings in our study are the identification of an antifungal activity of HRG Candida membranes. Although the 3D structure of HRG has not yet been determined, modelling studies suggest that the HRR of HRG forms a polyproline (II) helical structure with numerous histidines. At physiological pH, HRG is net negatively charged (pI 6.45). However, due to its high content of histidine residues (∼13%), which are concentrated to the HRR, it can acquire a positive charge by protonation Candida. These results were substantiated by the finding that a region of HRG containing the motif sequence GHHPH, was antifungal, and that low pH enhanced this activity. The high conservation of this sequence among vertebrates likely reflects its importance for membrane interactions of HRG Candida, pointing to the HRR as an important, possibly the most important, effector of HRGs antifungal effects.From a structural perspective, several lines of evidence indicate that the HRR is, at least to a significant extent, responsible for the HRG interaction with in vitro of various histidine-rich peptides, both consensus motifs and peptides derived from domain 5 of HMW kininogen 2+. Others have reported that the antimicrobial activity of clavanins were substantially increased in low pH as compared with neutral pH Candida membranes, bind mitochondria, and induce cell death by non-lytic ATP-release Many histidine-rich AMPs are known, among these the clavanins per se, including bacterial permeability increasing protein, serprocodins such as proteinase 3, elastase and heparin binding protein, as well as lactoferrin Many AMPs are generated by proteolysis of larger, and non-antimicrobial holoproteins. For example, the cathelicidin LL-37 is released from hCAP18, and other AMPs are proteolytically generated from complement factor C3 and high molecular weight kininogen in vivo functions of host defence peptides is increasing, it is important to consider the direct antimicrobial and immunomodulatory properties observed. Nevertheless, several findings in this study unequivocally demonstrate that HRG, like many AMPs, acts directly on microbes. Thus, in addition to the antifungal in vitro data, the enhanced fungal growth in HRG-deficient plasma, as well as the finding that Candida was detected at higher levels in blood of Hrg−/− animals, indicates a direct antifungal action of the molecule. It is also interesting to note that these HRG deficient animals have also been shown to be more susceptible to Streptococcus pyogenes infection . However, considering both AMP and HRG multifunctionality in vitro as well as in vivo, it may be envisaged that additional actions, resulting in the observed antifungal effects, will likely be revealed. All of these effects may be dependent on binding of HRG to microbes and subsequent interactions with cells  in different compartments . In this respect, the pH dependence of HRG is particularly interesting and relevant. It is well known that infection foci, including abscesses, are characterized by low pH levels reaching as low as pH 5, due to increased anaerobic metabolism and lactate production, as well as leukocyte mediated oxidative burst and subsequent acidification Candida at these pH levels and the corresponding increase in salt-resistance at low pH suggest that HRG could target infection foci, resulting in a physiologically relevant concentration and localization of antifungal activity. As previously mentioned, HRG's opsonising activity could hypothetically lead to enhanced phagocytosis. Although it remains to be investigated, such localisation of antifungal activity to endosomal compartments, where acidification could result in enhanced HRG-mediated killing of phagocytosed fungi, could serve as an effective way of eliminating invading Candida cells at sites of tissue inflammation without releasing potentially toxic microbial components.As previously mentioned, HRG is involved in various aspects of angiogenesis, coagulation, and fibrinolysis −/− mice, which had a shorter prothrombin time CandidaAgain hypothetically, genetic deficiencies of HRG or acquired functional defects could provide interesting clues with respect to functional roles of HRG. In some patients, reduced levels of HRG are associated with a thrombophilic phenotype, indeed compatible with the phenotype observed in HrgDuring the last three decades, research on innate immune molecules has demonstrated the significance of the innate immune system for prevention of invasion by microbes at biological boundaries. Previous studies have emphasized that various molecules, such as “classic” AMPs, complement factors, and cytokines, bridge between innate and adaptive immunity. The present work adds another significant component to this family of molecules, the plasma protein HRG.http://www.ncbi.nlm.nih.gov/Genbank/index.html) accession number of human histidine-rich glycoprotein is NP_000403.The peptides GHH20 (GHHPHGHHPHGHHPHGHHPH) and histatin 5 (DSHAKRHHGYKRKFHEKHHSHRGPY) were synthesized by Innovagen AB , and were of >95% purity. The purity and molecular weight was confirmed by MALDI-TOF MS analysis . 20-mer synthetic peptides (PEP-screen) spanning the sequence of HRG (Table 1) were obtained from Sigma-Genosys . Polyclonal rabbit antibodies against GHH20 and TAMRA-labeled GHH20 were from Innovagen AB . HRG was FITC-labeled using the FluoroTag FITC Conjugation Kit . Human serum and plasma were collected from healthy volunteers. Sterile wound fluids were obtained from surgical drainages after mastectomy. The use of human wound fluid was approved by the Ethics Committee at Lund University (LU 708-01). Seminal plasma was collected at the Fertility Center at Malmö University Hospital, Sweden. The GenBank .The fungi Serum HRG was purified using nickel-nitrilotriacetic acid (Ni-NTA) agarose as described before Recombinant His-tagged HRGP and truncated version of HRG (HRG1-240), containing amino acids 1-240 was produced and purified as previously described 3 cells, disrupted by freeze thawing) were electrophoresed on 8% SDS-polyacrylamide (SDS-PAGE) gel or an 16.5% Tris-tricine gel and transferred to a nitrocellulose membrane  Plasma, serum, wound fluids, seminal plasma (1 µl), and platelets (fluid from 1×10−/− and C57BL/6 (wild type) mice 2+ in eppendorf tubes at 37°C over night. Clots were washed three times and then stored in 10 mM 2-Morpholinoethanesulfonic acid (MES), pH 5.5. Clots (∼0.04g) were used in viable count experiments. To investigate the localization of HRG in fibrin clots, human plasma and HRG-deficient plasma were incubated with 10 µl FITC-labeled HRG (0.4 mg/ml) and then processed as before in the presence of 10 mM Ca2+ over night. The clots were then washed in distilled water and mounted on slides using Dako mounting media (Dako).Human plasma was subjected to a Ni-NTA agarose gel. The eluent (plasma completely depleted of HRG) was collected and used to form clots. HrgC. parapsilosis, C. albicans, C. glabrata and C. krusei were grown to mid-logarithmic phase in Todd-Hewitt (TH) medium  at 27°C and washed in 10 mM Tris, pH 7.4 or 10 mM MES, pH 5.5. For dose-response experiments, purified HRG or GHH20 (0.03–6 µM) were incubated with 1×105C. parapsilosis ATCC 90018 or C. albicans ATCC 90028 for 2 h at 37°C in 10 mM, Tris, pH 7.4 or in 10 mM MES-buffer, pH 5.5, plated on Sabouraud dextrose broth (Becton and Dickinson) agar, and incubated 48 hours at 27°C, whereafter the number of cfu was determined. In order to investigate the antifungal activity of HRG in presence of salt, 6 µM HRG were incubated with 1×105C. parapsilosis ATCC 90018 for 2 h at 37°C in 10 mM, MES, pH 5.5 containing 0, 25, 50, 100 or 150 mM NaCl, plated and the number of cfu was determined. In kinetic experiments, 0.3 and 3 µM HRG were incubated with C. parapsilosis ATCC 90018 for 5, 15, 30, 60, or 120 minutes in 10 mM MES, pH 5.5, plated and the number of cfu was determined. For determination of the effect of HRG on various Candida strains, HRG (3 µM) was incubated with C. parapsilosis ATCC 90018 or BD 17837, C. albicans ATCC 90028 or BD 1060, C. glabrata ATCC 90030 or C. krusei ATCC 6258 in 10 mM Tris, pH 7.4 or 10 mM MES, pH 5.5, plated and number of cfu determined. Truncated and full length recombinant HRG, 0.6 µM rHRG, or rHRG1-240 were incubated with C. parapsilosis (1×105) for two hours and then plated and number of cfu determined. To investigate the in vitro antifungal activity of HRG, normal or HRG-deficient fibrin clots (∼0.04g) were incubated with C. parapsilosis ATCC 90018 for 2 h in 10 mM MES, pH 5.5, plated and number of cfu were determined. For inhibition studies, 0.3 µM HRGP were incubated with C. parapsilosis (1×105) in 10 mM MES, pH 5.5, in presence or absence of heparin (50 µg) for two hours and then plated and number of cfu was determined. In all experiments, 100% survival was defined as total survival of fungi in the same buffer and under the same conditions in absence of peptide, protein, or clots. The p-values were determined using Kruskall-Wallis one-way ANOVA analysis.C. parapsilosis ATCC 90018 and C. albicans ATCC 90028 were grown to midlogarithmic phase in TH-medium, and then washed with distilled water. 4×106 colony forming units was added to 5 ml of the underlay agarose gel (0.03% (w/v) trypticase soy broth (TSB), 1% (w/v) low electroendosmosis type agarose (Sigma), 0.02% (v/v) Tween 20 (Sigma). The buffers used in the underlay gels were 10 mM Tris, pH 7.4 or 10 mM MES, pH 5.5. The underlay gel was poured into an 85-mm Petri dish. After agarose solidification, wells of 4 mm in diameter were punched, and 6 µl of peptide solution was added to each well. Buffers were used as a negative control. Plates were incubated at 28°C for 3 h to allow diffusion of the peptides. The underlay gel was then covered with 5 ml of molten overlay. Antimicrobial activity of a peptide is visualized as a zone of clearance around each well after 18–24 h of incubation at 28°C. Peptides were tested in concentrations of 100 µM.Radial diffusion assay (RDA) was performed essentially as described earlier C. parapsilosis (1×105 cfu) were incubated with 0.6 µM HRG in 50 µl 10 mM MES, pH 5.5, with or without heparin (50 µg/ml) for 2 h at 37°C, centrifuged and the pellet was washed three times in 10 mM MES, pH 5.5. The pellet and the supernatant were resuspended in SDS sample buffer, electrophoresed (8% SDS-PAGE), and then transferred to a nitrocellulose membrane. Western blotting was performed as above.C. parapsilosis ATCC 90018 fungi were grown in TH medium at 27°C to mid-logarithmic phase. The fungi were washed in 10 mM Tris, pH 7.4, and resuspended in the same buffer. C. parapsilosis (2×106/ ml) were incubated with 1 µl of TAMRA-labeled GHH20 (2 mg/ml) in 10 mM MES, pH 5.5, with or without heparin (50 µg/ml), left standing for 5 minutes on ice, and then washed twice in 10 mM Tris, pH 7.4. Fungi were fixed with 4% paraformaldehyde by incubation on ice for 15 minutes and in room temperature for 45 minutes. The fungi were then applied onto Poly-L-lysine coated cover glass and after an incubation time of 30 minutes, finally mounted on slides using Dako mounting media . In order to assess permeabilisation, C. albicans ATCC 90028 (2×106 cfu) were incubated with HRG or LL-37 (both at 10 µM) in 10 mM Tris, pH 7.4 or 10 mM MES, pH 5.5 for 30 minutes at 37°C. Samples were transferred to Poly-L-lysine coated cover glass and incubated for 45 minutes at 37°C, washed and 2 µg of FITC were added in a volume of 200 µl, and incubated for 30 minutes at 30°C, washed and then fixed as above. Samples were visualized using a Nikon Eclipse TE300 inverted fluorescence microscope equipped with a Hamamatsu C4742-95 cooled CCD camera, a Plan Apochromat 100X objective and a high N.A. oil condenser.C. parapsilosis ATCC 90018 were grown in TH medium at 37°C to mid-logarithmic phase. The fungi were washed in 10 mM Tris, pH 7.4 or 10 mM MES, pH 5.5, and resuspended in the same buffer. HRG or LL-37 (10 µM) was incubated with C. parapsilosis (20×106 cfu) for two hours in a total volume of 10 µl in Tris buffer, pH 7.4 or in MES buffer, pH 5.5. Samples of C. parapsilosis fungi suspensions were adsorbed onto carbon-coated copper grids for 1 min, washed briefly on two drops of water, and negatively stained on two drops of 0.75 % uranyl formate. The grids were rendered hydrophilic by glow discharge at low pressure in air. Specimens were observed in a Jeol JEM 1230 electron microscope operated at 60 kV accelerating voltage. Images were recorded with a Gatan Multiscan 791 CCD camera.C. parapsilosis ATCC 90018 were grown in TH medium at 27°C to mid-logarithmic phase. The fungi were washed in 10 mM Tris, pH 7.4 or 10 mM MES, pH 5.5 and resuspended in the same buffer. C. parapsilosis  were incubated with 10 µl of FITC-labeled HRG (0.4 mg/ml) or 10 µl TAMRA-labeled GHH20 (2 mg/ml) in 10 mM Tris, pH 7.4 or in 10 mM MES, pH 5.5, let stand for 5 minutes on ice and then washed in 10 mM Tris, pH 7.4. The cells were fixed with 4% paraformaldehyde by incubation on ice for 15 minutes and in room temperature for 45 minutes. Flow cytometry analysis was performed using a FACS-Calibur flow cytometry equipped with a 15 mW argon laser turned a 488 mm . The fungal population was selected by gating with appropriate settings of forward scatter (FSC) and sideward scatter (SSC). The FL1 fluorescence channel (λem = 530 nm) was used to record the emitted fluorescence of FITC, and the FL3 fluorescence channel (λem = 585 nm) was used to record the emitted fluorescence of Texas red.Dry lipid films were prepared by dissolving dioleoylphosphatidylcholine  (60 mol%) and either ergosterol or cholesterol  (40 mol%), and then removing the solvent by evaporation under vacuum overnight. Subsequently, buffer  was added together with 0.1 M carboxyfluorescein (CF) . After hydration, the lipid mixture was subjected to eight freeze-thaw cycles consisting of freezing in liquid nitrogen and heating to 60°C. Unilamellar liposomes, of about Ø140 nm were generated by multiple extrusions through polycarbonate filters (pore size 100 nm) mounted in a LipoFast miniextruder  at 22°C. Untrapped CF was then removed by two gel filtrations (Sephadex G-50) at 22°C, with Tris buffer as eluent. CF release was determined by monitoring the emitted fluorescence at 520 nm from liposome dispersions (10 mM lipid in 10 mM Tris). An absolute leakage scale was obtained by disrupting the liposomes at the end of the experiment through addition of 0.8 mM Triton X100 , causing 100% release and dequenching of CF. Although calcein is frequently used for pH-dependent leakage studies, the high charge of this dye has been noted to influence its leakage behaviour in the presence of highly cationic peptides Saccharomyces cerevisiae .The CD spectra of the peptides in solution were measured on a Jasco J-810 Spectropolarimeter . Measurements were performed at 37°C in a 10 mm quartz cuvet under stirring and the effect on protein/peptide secondary structure monitored in the range 200–260 nm. The background value, detected at 250 nm, was subtracted, and signals from the bulk solution were corrected for. The secondary structure was monitored at a concentration of 0.25 µM of HRG in buffer, in the presence of liposomes (lipid concentration 100 µM), and in the presence of mannan from C. parapsilosis ATCC 90018 were grown in TH medium at 27°C to mid-logarithmic phase. The fungi were washed in 10 mM MES, pH 5.5 and resuspended in the same buffer. C. parapsilosis (2×107) cfu in a total volume of 10 µl was added to 50 µl of human normal plasma or HRG-depleted plasma (eluent from Ni-NTA agarose gel), and incubated for 0, 4, 8 or 18 hours at 27°C and then plated and number of cfu determined.HRGtm1wja1 were crossed with C57BL/6 mice (Taconic) for 14 generations to obtain uniform genetic background. These HRG-deficient mouse strain was called B6-HRGtm1wja1 following ILAR  rules. Wildtype C57BL/6 control mice and C57BL/6 Hrg−/− mice  were bred in the animal facility at Lund University. C57BL/6 Hrg−/−, lacks the translation start point of exon 1 of the Hrg gene Candida dissemination, C. albicans ATCC 90018 were grown to midlogarithmic phase, washed and diluted in PBS, pH 7.4. Two hundred and fifty µl containing 1×109 cfu was injected intraperitoneally into C57BL/6 or C57BL/6 Hrg−/− mice, divided into weight and sex matched groups. The animals were sacrificed 48 hours post infection, and blood was collected by cardiac puncture. The number of cfu was determined by viable count. In order to study fungal dissemination to target organs, the mice were infected as previously described and three days later the spleen and kidney were harvested on ice.The original knockout mice 129/B6-Representative animals were sacrificed three days post infection and the kidneys were removed into 4% formalin. The tissues were embedded in paraffin, sectioned and stained with Hematoxylin and eosin (H&E) and with Periodic acid-Schiff (PAS).Table S1C. albicans and C. parapsilosis, content (%) of the basic amino acids K, R, H, and the acidic D, E.Synthetic 20-mer peptides spanning the whole sequence of HRG, used in the screening of antifungal activity in (0.07 MB DOC)Click here for additional data file.Figure S1C. albicans ATCC 90028 in 10 mM Tris, pH 7.4 (•) or in 10 mM MES, pH 5.5 (○). A 4 mm diameter well was loaded with 6 µl of 100 µM peptide. The clearance zones (mm) were measured after an overnight incubation at 27°C. The equation for the line of regression is y = 0.605x + 1.222 for peptides in pH 7.4 and y = 0.543x + 0.662 for pH 5.5.Correlation between net charge and antifungal activity. 20-mer peptides spanning the whole sequence of HRG (for sequences see (31.73 MB TIF)Click here for additional data file.Figure S25 cfu C. parapsilosis ATCC 90018 in 10 mM MES, pH 5.5 with or without 50 µg heparin, plated and the number of cfu determined (n = 6).Inhibition of antifungal activity by heparin. 0.3 µM HRG were incubated with 1×10(1.16 MB PSD)Click here for additional data file.


By contrast, the CB1R antagonist/inverse agonist LY320135 (500 nM), increased GABAergic synaptic activity and beta/gamma oscillatory activity in superficial mEC, was suppressed, whilst that in deep mEC was enhanced. These data indicate that cannabinoid-mediated effects on inhibitory synaptic activity may be constitutively active in vitro, and that modulation of CB1R activation using inverse agonists unmasks complex effects of CBR function on network activity.Cannabinoids modulate inhibitory GABAergic neurotransmission in many brain regions. Within the temporal lobe, cannabinoid receptors are highly expressed, and are located presynaptically at inhibitory terminals. Here, we have explored the role of type-1 cannabinoid receptors (CB1Rs) at the level of inhibitory synaptic currents and field-recorded network oscillations. We report that arachidonylcyclopropylamide (ACPA; 10  In the cerebrum, the hippocampal formation and the entorhinalcortex (EC) show the highest density of staining for CB1R.Cannabinoid receptors are afamily of G-protein coupled, presynaptic receptors , 2. AutoA receptor mediated synaptic transmissionoccurs through the inhibition of voltage-dependent calcium channels . In the . The mE  chilled to 4°C. Slices (450 μm) were cut using a vibrating microtome , and stored in ACSF continuously bubbled with 95% O2/5% CO2,at room temperature. Following a recovery period of at least 1 hour, individualslices were transferred to a recording chamber mounted on the stage of anOlympus (BX50WI) upright microscope. The chamber was continuously perfused withoxygenated ACSF at 30–32°C, at a flow rate of approximately 2 mL/min. The ACSFcontained the following (in mM): NaCl (126), KCl (3.25), NaH2PO4 (1.25), NaHCO3 (24), MgSO4 (2), CaCl2 (2.5),and D-glucose (10). The solution was continuously bubbled with 95% O2/5%CO2 to maintain a pH of 7.4. Neurones were visualized using differentialinterference contrast optics and an infrared video camera.Combined EC-hippocampal slices were prepared from young maleWistar rats (50–110 g) as previously described . All exp2 (5.0),TEA-Cl (10), phosophocreatine (7) ATP (4), GTP (0.4). The solution was adjustedto 290 mOsmol with sucrose and to pH 7.4 with CsOH.Whole-cell voltage clamp recordings were made from neurones in layers II and Vof the medial division of the EC, using an Axopatch 700 series amplifier.The holding potential in all cases was –70 mV. Underthese experimental conditions, layer II/V neurones exhibited sIPSCs, mediatedby GABA acting primarily at GABAA receptors.Patch-clamp electrodeswere pulled from borosilicate glass  andhad open tip resistances of 4-5 MΩ. They were filled with a solution containingthe following (in mM): CsCl (90), HEPES (33), QX-314 (5), EGTA (0.6), MgClData were recordeddirectly to computer hard disk using AxoScope software . Mini Analysis  was usedfor analysis of sIPSCs offline. sIPSCs were detected automatically using athreshold-crossing algorithm, and their frequency and amplitude are analysed.200 sIPSCs were sampled during a continuous recording period for each neuroneunder each condition. The nonparametric Kolmogorov-Smirnoff (KS) test was usedto assess the significance of shifts in cumulative probability distributions ofinterevent interval (IEI). Differences between drug and control situations instudies of sIPSCs were assessed by means of a one-way ANOVA. All error valuesstated in the text refer to the S.E.M.All saltsused in preparation of ACSF were Analar grade and purchased from Merck/BDH (UK). LY320135 and ACPA were obtained from Tocris Cookson(UK).t-tests were carried out to determine statisticalsignificance.For fieldrecordings of oscillatory activity, slices were placed into an interface chamber and the chamber wascontinuously perfused with oxygenated ACSF at 30–32°C, at a flow rate ofapproximately 1-2 mL/min. Extracellularpopulation recordings were made with glass microelectrodes filled with aCSF, ofresistance 1–3 MΩ. Signals were amplified 1000-fold and recordedunfiltered. Low amplitude 50 Hz interference was removed using a HumBug . Signals were digitized and recorded at 10 kHz using an NPI EXT-02F amplifier and pClamp 10 software . Following 30–90 minutes control period of stableoscillatory activity, drugs were applied to the bath in known concentration. Pharmacological oscillatory activity wasanalysed using the fast Fourier transform , cross-correlationanalysis and Morlet-wavelet time-frequency spectrogram analysis . Student We analysed oscillations at beta(15–29 Hz) and gamma (30–90 Hz) bands, using bandpass filters (Clampfit 10.1)and measurement of the area under the power spectrum curve in Sigmaplot 8.0.μM, onto slices from which stable gammaactivity had been induced by 300–400 nM kainate. As γ-band was 561 ± 179 μV2 and mean controlgamma frequency was 40.7 ± 2.4 Hz. Following 40–60 minutes bath application of ACPA, we applied theCB1R-specific inverse agonist LY320135 . In pooled data, ACPA didsignificantly reduce mean peak gamma frequency to 35.6 ± 1.8 Hz , although thiseffect was variable, and some recordings showed multiple peaks.Whilstrecording from layers II and V of the mEC, we applied the CBR agonistarachidonylcyclopropylamide (ACPA) at 10 . FigureP ≤ .0006, n = 9). In addition, mean peakfrequency returned to 41.2 ± 1.8 Hz .Following perfusion of the CB1R-specificinverse agonist LY320135, there was a marked reduction in normalised gammapower to 39.4 ± 10.1% of control, and this was highly significant ,and ACPA had no significant effect on mean peak beta frequency . However, when we added the CB1R-specific inverseagonist LY320135, there was a reduction in normalised beta power to 57 ± 13%of control, and this was highly significant . LY320135 had no effect on mean peak frequency .Whenbeta power in layer II was measured, we noted a similar pattern of drugresponses to that observed for gamma activity. Mean area powerin the beta band was lower than that of gamma activity at 26 ± 6 μV2 and mean peak frequencywas similar to layer II at 39.19 ± 3.1 Hz.During the above experiments, wesimultaneously recorded oscillatory activity in deep entorhinal cortex (layerV). Oscillatory activity in layer V was lower in power in layer V compared tolayer II, with mean area gamma power just 60 ± 10 P ≤ .03, n = 9), however, baseline gamma power was verylow in this layer, and the absolute change in gamma power was difficult todiscern. Peak frequency was again slightly reduced to 36.0 ± 2.4 Hz, butthis was not significant . On subsequent additionof the CB1R-specific inverseagonist LY320135, there was a strong increase in normalised gamma power to108.4 ± 58% of control, and this reached significance . Again, LY320135 did not significantly alter meanpeak gamma frequency .When we applied ACPA whilst recording in layer V we observed asignificant increase in mean gamma power . ACPA had no significant effect on mean peak beta frequency . When we nextapplied the inverse agonist LY320135, we noted an increase in normalised betapower by 142.4 ± 88% of control, and this just failed to reach significance . LY320135  had no effect on peak frequency.When beta power in layer V was analysed,we noted a similar pattern of drug responses to that observed for gammaactivity. Mean area power in the beta band was lower than that ofgamma activity at 9.6 ± 0.6 P ≤ .01, n = 5). When beta activity was measured, itwas apparent that in LY320135, there was a significant reduction in meannormalised beta power .Wehypothesised that the lack of effects of ACPA in layer II might reflectconstitutive or tonic activation of CBR, perhaps due to persistentkainate-induced activation of pyramidal neurones. To test this hypothesis, we appliedLY320135 in the absence of ACPA. Application of LY320135 suppressed gamma band activity to 19.5 ± 11% of control, and this was highly significant  had subtleeffects on sIPSCs in layer II, decreasing their frequency without affecting meanamplitude. Cumulative probability plots for sIPSC amplitude in the presence ofACPA . When we analysed amplitude distribution using thenonparametric Kolmogorov-Smirnov test, the shift towards higher amplitudesIPSCs was just significant (P ≤ .021 KS test). In the case of interevent interval, we noted a shift to the right in thecumulative probability plot , and theincrease in IEI time distribution towards higher values was highly significant .When we performed similar experiments using LY320135 (500 nM), we noted effectswhich tended towards the opposite of those seen with ACPA, that is, increasedsIPSC amplitude and frequency. P ≤ .013, ANOVA, n = 6). Similarly, theshift in distribution towards larger amplitudes was highly significant . The mean median IEIshowed a slight decrease from 86.6 ± 15.8 milliseconds in control to 80.6 ± 16.1 milliseconds LY320135, but this decrease in IEI time was not significant . ofACPA  indicateity plot ,indicatThecumulative probability plots for sIPSC amplitude  and IEI P ≥ .23)). Mean amplitude rose slightly from59.41 ± 7.33 pA in control to 70.49 ± 8.89 pA in ACPA, but this increase was notsignificant . Whenwe analysed IEI, the change in distribution towards larger IEI values was significant(P ≤ .0001 KS test), and mean median IEI was foundto increase very significantly from 792 ± 41 milliseconds in control to 1317 ± 75 milliseconds in ACPA . This effect of ACPA on IEIin layer V was consistent in all recordings. Cumulativeprobability plots for sIPSC amplitude . Cumulative probability plots for sIPSC amplitude in the presence of LY320135. In the case of IEI, we noteda shift to the left in the cumulative probability plot , as was the change in distribution .When we performed similarexperiments using LY320135 (500 nM), we again noted robust effects, which opposedthose seen with ACPA, that is, increased sIPSC amplitude and frequency. LY320135 show theity plot , indicatWefound that the cannabinoid receptor agonist ACPA had little effect on eitheroscillatory activity or synaptic inhibition in superficial layers of the mEC,and that effects in deep layers were more robust, especially in the case ofsIPSC frequency. However, the inverse agonist, LY320135, strongly suppressedoscillatory activity in superficial mEC even while its effects on sIPSCfrequency and amplitude were not great. We also observed that suppression ofoscillatory activity in layer II by LY3201235 was accompanied by augmentationof oscillatory power in layer V, and that the suppression of sIPSCs by ACPA andsubsequent enhancement by LY320135 in this layer were marked.2+ dependent release of GABA and have no effect on mIPSCs([Wehave reported previously  that spo mIPSCs(; it seemSince,compared to layer II, both ACPA andLY320135 had more profound effects on synaptic inhibition in layer V, it seemslikely that the relative dominance of mIPSCs in layer II may mask CB1R effectson the minority of AP-dependent sIPSCs to some degree. The lack of a robusteffect of ACPA on oscillatory activity in layer V suggests, however, that CB1Rmay already be activated by ongoing network activity, and that further attemptsat activation using an agonist did not increase any effect that CB1R might haveon oscillatory power. This appears to be supported by the effects of theinverse agonist LY320135 in layer II. Here, we observed a robust reduction inboth beta and gamma power in layer II, suggesting that CB1R do contribute tomaintaining oscillatory activity in this layer. The apparently contradictoryresult of enhanced oscillatory activity in layer V in response to LY320125 mayrelate to effects that are secondary to activity in layer II, for example, Bragin , workingCannabinoidreceptors exert powerful control over GABA release from presynaptic terminals,with CB1 receptors having been shown to suppress both IPSPs and IPSCs inpyramidal neurones (IPSPs, ; IPSCs, Itseems possible that PV−/CCK+/CB1R+ inhibitory interneurones might similarly selectpopulations of pyramidal cells involved in rhythmogenesis in the mEC, whichcontains both PV+ and PV− neurones , 32 and increased gamma and beta power and thisappeared to be correlated with decreased superficial beta and gamma power. Atfirst, this appears paradoxical, however, oscillatory activity in specificlaminae does not exist in isolation, and we might expect interactions between,as well as within, networks of neurones. Bragin et al. [Whenwe measured oscillatory activity in layer V, inverse agonists at CBR n et al.  have dem


Background. Sepsis is an important cause of mortality in newborns. However, a single reliable marker is not available for the diagnosis of neonatal late-onset sepsis (NLS). The aim of this study is to evaluate the value of serum amyloid A (SAA) and procalcitonin (PCT) in the diagnosis and follow-up of NLS. Methods. 36 septic and healthy newborns were included in the study. However, SAA, PCT, TNF-α, IL-1β, and CRP were serially measured on days 0, 4, and 8 in the patients and once in the controls. Töllner's sepsis score (TSS) was calculated for each patient. Results. CRP, PCT, and TNF-α levels in septic neonates at each study day were significantly higher than in the controls (P = .001). SAA and IL-1β levels did not differ from healthy neonates. The sensitivity and specificity were 86.8% and 97.2% for PCT, 83.3% and 80.6% for TNF-α, 75% and 44.4% for SAA on day 0. Conclusion. Present study suggests that CRP seems to be the most helpful indicator and PCT and TNF-α may be useful markers for the early diagnosis of NLS. However, SAA, IL-1β, and TSS are not reliable markers for the diagnosis and follow-up of NLS. Despite advances in medicine, diagnosis of neonatalsepsis remains as a major challenge. Early clinical signs arenonspecific and the laboratory criteria are also not fully reliable.Warning signs and symptoms are often subtle and can easily beconfused with noninfective causes such as apnea, hypothermia, andacute exacerbation of chroniclung disease. So that hematologicaland biochemical markers such as immature/total neutrophil ratio,platelet count, C-reactive protein (CRP), various cytokines,procalcitonin (PCT), and tumornecrosis factor- infants –4. Recen infants , 3–6. BuThirty six newborn infants who were diagnosed ashaving clinical suspected NLS in the neonatal intensive care unit at the medicalfaculty of Eskisehir Osmangazi Universitybetween June 2003 and June 2004 were included in this prospective study.Thirty six healthy newborns who had normal clinical and laboratory findingswere included as a control group. They were selected from the neonatal unit orwell-baby outpatient clinic. Diagnosis of sepsis was done according to the 2001International Sepsis Definitions Conference criteria. According to thisconference, diagnostic criteria for sepsis in the pediatric population are signs and symptoms ofinflammation including arterial hypotension-decreased capillary refill ormottling cardiac index >5.5, significant edema or positive fluid balance,ileus plus infection (documented or suspected) with hyper or hypothermia,tachycardia, and at least one of the following indications of altered organfunction: altered mental status, acute oliguria, hypoxemia, increased serumlactate level, or bounding pulses . Laboratα, IL-1β, PCT, and SAA were serially measured. The initial (2 mL) blood samples wereobtained from patients on day 0 (at the time of sepsis diagnosis). Two furthersamples were obtained from each patient on days 4 and 8 for follow-up in thepatient group. However, only one blood sample (2 mL) was obtained from each healthycontrol subject.Töllner's sepsis score (TSS) was calculated for eachpatient . This scα (cut-off level: 1 pg/mL), IL-1β, and SAA (cut-off level: 6610 ng/mL) levels were measured by ELISA .PCT (cut-off level: 0.8 ng/mL) levels were analyzed by immunoluminometricmethod .Blood specimens for TNF-α, IL-1β, PCT, and SAA wereimmediately immersed in ice and transported to the laboratory for processing.Serum was separated by centrifugation at 4°C and stored in 200 μL aliquots at −72°C until analysis.Serum CRP levels were measured by radio-immuno-assaymethod (cut-off level: 0.8 mg/dL). TNF-α, PCT, and SAA.The receiver operator characteristic (ROC) method wasused to determine the best cut-off point of CRP, TNF-The study was approved by the Research Ethics Committee of the Facultyof Medicine, Eskisehir Osmangazi University.Informed consents were obtained from the parents or guardians for all studypatients and control subjects.χ2, post hoc and Mann Whitney-U tests were used to compare categorical predictors.Also, multivariate analyses were performed for logistic regressionanalysis. The sensitivityand specificity were calculated for days 0, 4, and 8 to construct the receiveroperating characteristic curves for each biochemical marker.Statistical analyses were performed using SPSS10.0. 3 versus 215000 ± 47600/mm3; P < .001 and 0.2 ± 0.1 versus 0.002 ± 0.004; P < .001; resp., but leukocyte counts (Mean ± SEM) were similar in patientsand controls 19294.4 ± 8598/mm3 versus 46777.8 ± 10720.6/mm3; P > .05).The demographic characteristics of the study and control groups aresummarized in During the study period, 28 (77.8%) patientswere treated with mechanical ventilation. Eight out of these 28 (28.6%) patients hadrespiratory dystress syndrome and were treated with surfactant. Thirty of 36 (83.3%)patients were given oxygen therapy. None of the patients had been treated with pre-/postnatalantibiotics or steroids and none of them had been operated prior to theinclusion to the study. Also none of the patients had a central line.The ratio of blood culture-proven septic patients among the studygroup was 72.2%. Klebsiella pneumonia was the most prevalent microorganism in theseptic patients.α, and IL-1β in the whole patient group and control group on days 0, 4, and 8 are shown inα in the whole patient group on day 0 were found to be significantlygreater than the controls (P = .001). Also, serum levels of CRP, PCT, and TNF-αin the patients with proven sepsis on day 0 were found to be significantlyhigher than in controls. These high levels were continuedon days 4 and 8. On the other hand, serum levels of IL-1β were not higher inthe patient group on days 0, 4, and 8 than the control group. The mean levelsof SAA of the study group were found to be greater than that of the controlgroup but the difference was not statistically significant (P > .05).Individual serum levels of PCT, SAA, CRP, TNF-P < .05), there were no significantdifferences between PCT levels on day 4 and day 8 or day 0 and day 8 (P > .05).TNF-α and SAA levels did not change statistically during the study period (P > .05). CRP levels on day 0 were significantly higher than on day 4 and day 8 . However, CRP levels on day 4 and day 8 were similar (P > .05).While PCT levels on day 0 weresignificantly higher than on day 4 . Also, the serum levels of PCT, SAA,CRP, TNF-α, or IL-1β did not correlate with platelet counts (P > .05).TSS did not correlate with the serum levels of PCT,SAA, CRP, TNF-α on day 0 and with the levels of SAA on day 4 . In addition, the levels of SAA on day 0 positively correlated with the ratio of I/T .The serum levels of CRP negatively correlated with the levels of SAA and positively correlatedwith TNF-α level . The sensitivity and specificity of PCT, SAA, CRP, and TNF-α for determining sepsis are summarized in α , SAA . Based on these analyses, the most sensitiveparameter was CRP on day 0. Other sensitive parameters were PCT, TNF-α, andSAA, respectively, on day 0.Multiple regression analysis showed that the most important factor which influenced thelevels of CRP on days 0, 4, and 8 was TNF-α are seen in The ROC curves of PCT, SAA, CRP, and TNF-α and SAA in this patients in comparison to thosewho survived, but high levels of PCT were found in these patients on days 0, 4,and 8 than the control group .Three patients (8.3%) died due to systemic inflammatory response syndrome. One of them had hydrops fetalis and two others had respiratory dystress syndrome and also all these three patients had disseminated intravascular coagulation (DIC). Interestingly, we did not find any significantincrease in the levels of TNF-P > .05). These results may explaindefective IL-1β production in newborn. In fact, it has been reported that SAA genetranscription is induced mainly by glucocorticoids, IL-1, IL-6, and TNF-α [α could induce SAAprotein synthesis in human hepatocytes and that IL-6 was the most effectiveinducer but synergy with either IL-1 or TNF-α was not seen [α seems not to influence the production of SAA in septic newborns. Therefore, theproduction of SAA may not be adequately stimulated in newborn. Like IL-1, we donot propose the use of SAA as an acute phase reactant in NLS.Culture-provenbacterial infections in newborn infants are associated withsubstantial morbidity and mortality. However, a single reliable biochemicalmarker is not available for the diagnosis of neonatal sepsis. SAAhas been proposed for early diagnosis of NLS , 9, 10. nd TNF-α . Howevernd TNF-α  reportednot seen . OurexpAlthough, in recent years, several new markers of infection have beeninvestigated, some studies suggested that CRP remains to be the best diagnostictest for neonatal sepsis. Especially serial measurements of CRP are highlyspecific and sensitive in newborn sepsis –16. Alsoβin infected infants were remarkably low and IL-1β was a less satisfactory marker. It seems that the monocytes of newborninfants may be unable to secrete adequate IL-1β and prostaglandin E2  and IL-6 may suppress IL-1β and TNF-α production in infections [β as an acute phase reactant in NLS.Our dataindicated that the plasma concentrations of IL-1fections –20. Therα is one of the primary agents which sets inmotion the exaggerated cellular, metabolic, and vascular responses of sepsis[αwere 83.3% and 80.6%, respectively, on day 0. We found that thelevels of TNF-α positively correlated with the serum levels of CRP on day 0.So that TNF-α is also a useful marker withhigh sensitivity and specificity for the detection of NLS.TNF-f sepsis–23. Itsf sepsis, 17, 24.α > SAA at the time of diagnosis. Therefore, we found that PCTwas the second useful diagnostic marker in sepsis after CRP.A few new of markers including PCT seem to be promising as adiagnostic test for sepsis , 25–29. Our study confirmed previous findings that neonateswith bacterial sepsis have reduced thrombocytecount, high I/T ratio and presence of toxic granulation in granulocytes , 29, butIn conclusion, our data indicate that CRP is the bestreliable marker of inflammation for the diagnosis of NLS. PCT may also be usedas a sensitive and specific diagnostic marker in NLS. The SAA did not increasein early phase in our patients, therefore it cannot be used alone for thediagnosis and followup of NLS. Also, TSS is not beneficial in late-neonatalsepsis.


CERKL) to determine CERKL function and contribution to pathogenesis.Retinitis pigmentosa (RP), a retinal neurodegenerative disorder characterized by apoptosis of photoreceptor cells, is caused by mutations in many different genes. We analyzed the RP gene ceramide kinase-like -polymerase (PARP) caspase-dependent cleavage.RT–PCR was performed to characterize CERKL transcripts underwent alternative splicing. In the human retina, four different CERKL isoforms of 532, 558, 419, and 463 amino acids were expressed. CERKL proteins were mainly localized in the endoplasmic reticulum and Golgi compartments, but they also shifted localization to nuclei and nucleoli. We also found that CERKL prevented cells from entering apoptosis induced by oxidative-stress conditions.CERKL remains a unique orphan lipid kinase in that no candidate substrate has been identified after intense research. The dynamic localization of CERKL suggests multiple sites of action. Remarkably, CERKL (but not the RP R257X mutant) exerts a protective role in cells against oxidative stress, consistent with RP mutations impairing the normal protein function in photoreceptors and thus tilting the balance toward apoptosis. These results provide valuable insights into the molecular mechanisms causing retinal degeneration. CERKL) , as described elsewhere [Human embryonic kidney HEK293T) or African green monkey kidney (COS-7) cells were seeded at 3×103T or Afr2, with the corresponding lipid and protein sources. Reactions were started by adding MgCl2 at 0.5 mM final concentration plus γ-32P-ATP (2–5 µCi/reaction), incubated at 30 °C for 30 min, 60 min, or overnight, and stopped with 250 µl of chloroform, 250 µl of methanol and 125 µl HCl 2.4 M. After vortexing, the phases were separated by centrifugation, and the organic phase removed and vacuum-dried. Lipids were resuspended in 25 µl chloroform:methanol , spotted onto silica gel plates and separated by thin-layer chromatography (TLC). For the enzymatic assays in vivo, we seeded 1.5×106 661W cells on 10 cm diameter plates and transfected using either the two pEGP-CERKL long isoforms (a and b) or the pEGFP empty-vector (negative control). Transfected cells were enriched by using Fluorescence Activated Cell Sorting (FACS) to select green fluorescent protein (GFP) positive cells. These cells were reseeded on smaller plates, and 4 μl of radioactive 32P-orthophosphate was added to the medium. Cells were collected after 4 h, and lysed directly in a solution of chloroform:methanol:chlorhydric acid (100:100:1). After vigorous vortexing, cellular debris was sedimented by centrifugation in a microfuge (100x g) and the liquid phase was vacuum-dried on completion. The lipid pellet was resuspended in a solution of 1:1 chloroform:methanol and loaded onto silica gel 60 TLC plates . All TLCs (assays in vivo and in vitro) were run in chloroform:acetone:methanol:acetic acid:water . Radiolabeled lipids were visualized by autoradiography.DAG and ceramide kinase activities were assayed in vitro as described . BrieflyE.coli cells (Stratagene) to avoid premature protein truncation due to the strongly biased codon usage of the CERKL human gene. Recombinant proteins were produced in 500 ml cultures induced by the addition of 0.5 mM Isopropyl-β-D-thiogalactopyranoside (IPTG). Purified GST-recombinant proteins were obtained after elution with reduced glutathione of the protein bound to the glutathione-sepharose bead batch, following the manufacturer’s instructions (Amersham Biosciences).To express the four CERKL isoforms as glutathione-S-transferase (GST) fusion proteins, we subcloned each variant in-frame into pGEX-4T-1 , between the XhoI and NotI sites. The constructs were transformed into BL21 Codon Plus Binding of CERKL isoforms to sphingolipids was assessed using SphingoStrips™ and PIP Strips™ , which consist of several nitrocellulose-immobilized sphingolipids and phosphoinositides (100 pmol/spot) respectively. The membranes were equilibrated for 5 min in 10 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.1% v/v Tween-20 (TBST). They were then blocked with 3% BSA in TBST (blocking solution) for 1 h at room temperature. Subsequently, the membrane was then incubated overnight at 4 °C in blocking solution containing 0.5 µg/ml of the GST-fusion protein on a rocking platform. The membranes were washed twice for 15 min in TBST with gentle agitation, and then incubated for 1 h with 1:1,000 anti-GST monoclonal antibody . After washing as before, membranes were incubated for 1 h with 1:3,000 horseradish peroxidase-conjugated anti-mouse IgG antibody (Sigma). Finally, the membranes were washed 6 times in an hour in TBST, and developed with the ECL western blotting detection system (Amersham Biosciences).2PO4, 8 mM Na2HPO4, pH 7.4) and fixed in 3% paraformaldehyde and 2% sucrose in 0.1 M phosphate buffer at 4 °C for 30 min. Cells were then washed and permeabilized for 10 min with a solution that contained 0.1% Triton X-100, 20 mM glycine, and 10 mM PBS. After permeabilization, cells were rinsed and blocked in a solution composed of 1% BSA, 20 mM glycine, and 10 mM PBS. Then, cells were incubated with 1:250 anti-HA polyclonal antibody and either specific 1:25 anti-calnexin, 1:250 anti-GM130, or 1:1,000 anti-EEA1 monoclonal primary antibodies  at 37 °C for 1 h. Mitochondria were labeled by adding reduced MitoTracker Orange  to the cell culture medium at a final concentration of 500 nM for 45 min before the fixation procedure and subsequent immunocytochemistry. Upon washing, cells were incubated with 1:300 AlexaFluor 488-conjugated anti-rabbit and 1:300 AlexaFluor 546-conjugated anti-mouse (Molecular Probes) secondary antibodies. When required, slides were counter-stained with 1:200 DAPI (Sigma) nuclear blue dye in PBS for 15 min. All preparations were mounted in Vectashield medium for fluorescence  and analyzed by confocal laser scanning microscopy with Olympus Fluoview 500  and Leica TSC NT and TSC SPII  laser scanning microscopes. In silico predictions for CERKL subcellular localization were performed using PSORT, ProSLP v2.0, ESLPred, and SubLoc v1.0.COS-7 cells were grown in Dubecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 4 mM L-glutamine, 100 U/ml of penicillin, and 100 µg/ml streptomycin (Invitrogen Life Technologies). Cells were grown on coverslips in 12 well plates. Transient transfections were performed using Lipofectamine™2000 (Invitrogen Life Technologies) according to the manufacturer’s protocol. Next, 24 h or 48 h post-transfection cells were rinsed with PBS 1X  in 10 cm plates and transfected with the CERKLa isoform tagged with the HA epitope. At 48 h post-transfection, cells were washed twice with 1X PBS, and then incubated 20 min at 4 °C in a solution that contained 10 mM Tris HCl, pH 7.4, 3% sucrose w/v, and 1% protease inhibitors (Roche). After this incubation, the concentration of sucrose was increased up to 0.25 M, and cells were broken by 15 strokes in an iced-chilled Dounce homogenizer. Cellular debris, nuclei, and unbroken cells were separated by centrifugation at 1,300x g for 5 min at 4 °C, and the supernatant was further centrifuged overnight at 100,000x g at 4 °C. The pellet containing cell organelles was resuspended in 1.5 M sucrose and laid at the bottom of a 1.8 ml swing-out  centrifuge tube. Subsequent layers of equal volumes of 1.15, 0.9, 0.6, and 0.25 M sucrose were added, and the final sucrose gradient was centrifuged for 2 h at 100,000x g at 4 °C. Next, 11 fractions of approximately 150 μl were collected from bottom to top. Samples were loaded onto 10% polyacrylamide SDS–PAGE gels, transferred to 0.45 µm polyvinylidene fluoride PDVP membranes  and underwent immunodetection with the corresponding antibodies.HeLa cells  were seeded , membranes were incubated overnight at 4 °C or 1 h at room temperature, separately, with any of the following primary antibodies, all diluted at 1:1000, anti-GM130 (BD Biosciences), anti-PDI (BD Biosciences), anti human TGN38  or anti-HA . After extensive washes in MTP, membranes were incubated for 1 h with 1:3,000 of the corresponding peroxidase-conjugated secondary antibody (Sigma), and the bands were visualized using the ECL western blotting detection system (Amersham Biosciences).2O2  and 3 h later propidium iodide was added at a final concentration of 7 µg/ml. After 1 h, cells were rinsed twice with PBS and fixed in 3% paraformaldehyde and 2% sucrose in 0.1 M phosphate buffer at 4 °C for 30 min. They were then washed 2 times in 0.1 M PBS and mounted in Vectashield medium (Vector Laboratories). Several fields were analyzed under the visible and green light channels in a fluorescence microscope (Leica DM IL) coupled to a Leica DFC camera . A minimum of 500 cells were counted and compared to detect pignotic nuclei in the total cell count. Three independent replicates were performed for each construct.COS-7 cells were grown on coverslips in 24 well plates. Transient transfections were performed on the following: the constructs encoding each of the four isoforms (pcDNA3-CERKL), the truncated variant (pcDNA3-R257X), empty pcDNA3.1 (negative control), or pcDNA6.2-DEST-CERK . At 44 h post-transfection, cells were treated with 200 µM H2O2, 400 µM H2O2, or 0.3 M sodium nitroprusside (Fluka), or serum-deprived medium. After 24 h, cells were rinsed with PBS, harvested, and lysed directly in protein electrophoresis loading buffer. Samples were then boiled for 5 min and centrifuged to sediment cellular debris. Protein preparations were loaded onto 7.5% SDS–PAGE gels. Either 1:1,000 monoclonal anti-PARP (Invitrogen Life Technologies) or 1:8,000 monoclonal anti-tubulin (Sigma) were used as primary antibodies. Anti-mouse secondary antibody was conjugated to 1:3,000 horseradish peroxidase. Immunodetection was performed using chemiluminescent ECL reagents , and the intensity of each band was quantified using the Quantity-One 1-D Analysis Software .HeLa cells were seeded in 24 well plates and transfected with either empty pcDNA3.1 (negative control) or pcDNA3-CERKLa (containing the lipid kinase domain). At 40 h post-transfection, the medium was replaced by fresh solution containing one of the following: 300 µM HCERKL gene spanned 13 exons in chromosome 2q31.2–31.3 (CERKL on retina cDNA using primers spanning the ATG (forward) and the STOP (reverse) codons, we observed several faint bands, which were indicative of alternatively spliced variants. Therefore, we devised a two-step strategy of RT–PCR and cloning (see Methods) to characterize the full-length isoforms based on the structure of the gene. The RT–PCR analysis of human retina mRNA using primers from exons 2 and 13 allowed us to identify several spliced transcripts (AY357073); 2) CERKLb, a 14 exon isoform, which includes an additional exon (4b) between exons 4 and 5 and encodes a 558 amino acid protein (accession number AY690329); 3) CERKLc, a splicing variant devoid of exons 3-4-4b-5, which encodes a 419 amino acid protein (accession number AY690330); and 4) CERKLd, a splicing variant that skips exons 4-4b-5 and encodes a 463 amino acid protein (accession number AY690331). Two very faint bands corresponded to not in-frame splicing variants (accession numbers AY690332 and AY690333) that would generate prematurely truncated proteins. These were considered aberrant transcripts, and given the mRNA quality control regulation, if they were produced in cells, they would be most probably cleared by the nonsense mediated decay mechanism.The initially characterized 1.2–31.3 . While anscripts  that wounscripts : 1) CERKThe longest isoforms (CERKLa and CERKLb) contained the complete DAG domain and a putative N-terminal pleckstrin homology (PH) region. In contrast, the short forms (CERKLc and CERKLd) skipped most of the DAG kinase domain, although the putative PH domain (encoded in exons 1 and 2) was preserved .Cerkl gene, which shows overall strong conservation in exon and intron structure, has no equivalent for exon 4b, thus suggesting that there are only 3 CERKL isoforms in the mouse. Again, the donor site of exon 5 in rodents is GC.Noticeably, exons 4b and 5, which are alternatively spliced, show the rare GC donor splice site instead of the more common GT . No kinase activity was detected for any isoform at any combination of the lipid and protein lysate sources  or as total fresh protein lysate from HEK293T cultured cells transiently transfected with the CERKLa HA-tagged construct. Again, no positive binding signals were produced (data not shown).The absence of kinase activity prompted us to test the ability of CERKL isoforms to bind sphingolipids by protein-lipid overlay. As PH domains have been reported to have an affinity for phosphoinositides, these substrates were also included. We used the CERKLa isoform  either as a pure preparation of recombinant GST-CERKL fusion protein , as well as the fusion CERKL-GFP constructs, in these transient transfections.Interestingly, neither a unique localization pattern nor a restricted subcellular localization could be ascribed to any CERKL isoform, as all variants showed similar patterns that appeared to be cell-specific. Two main protein localizations emerged consistently in all preparations, as seen for the isoform CERKLa  or distinctive organelle markers. The results  clearly CERKL cause RP is based on its cellular protective role through the regulation of the ceramide/ceramide-1-phosphate ratio [CERKL) would make them more susceptible to damage, tilting the survival/death balance and triggering apoptosis. To test whether CERKL has a protective role, we assayed the effect of CERKL overexpression in transiently transfected cells under normal or oxidative stress conditions. As most neuronal cell lines, retina derived cells are very poorly transfected, so we had to resort to transfections on COS-7 and HeLa cell lines to obtain statistically significant results.One of the working hypotheses to explain why mutations in te ratio . Photore2O2 for 4 h. The results were compared to those obtained with cells transfected with either empty vector or a construct expressing the known ceramide kinase, CERK (this construct was a gift from F. Bornancin). Dead cells were stained with propidium iodide and quantified (as a percentage) against total cell number  deprivation and 0.3 mM sodium nitroprusside (SNP). The analysis was performed at different window times to determine the best range of conditions for quantification of any potential protective effect. CERKL did protect against apoptosis induced by oxidative stress , than at 400 μM H2O2 (no significant protection). It was also dependent on the type of inducer, as cells overexpressing CERKLa did not behave differently from empty-vector transfected cells after FBS deprivation or SNP addition.Propidium iodide labels both apoptotic and necrotic cell nuclei. We attempted then to determine whether this protective effect of CERKL overexpression was on preventing cells from entering apoptosis. Activation of caspases is a well established event in apoptosis, and detection of caspase-cleaved substrates, such as PARP-1 (poly-ADP-ribose-polymerase)-1, is one of the most widespread hallmarks to quantify cell apoptosis ,16. PARPe stress  in cellsThis protective effect was also dependent on time, and presented an effective window frame. It was already detectable at 12 h  with an CERKL, which is responsible for an autosomal recessive form of retinitis pigmentosa (RP26). In our case, the isoform multiplicity increased the complexity of the functional analysis of a yet to be characterized lipid kinase. For instance, comparison of isoforms CERKLa and CERKLb revealed an additional exon (E4b), which introduces 26 additional amino acids in-frame within the DAG kinase catalytic domain and immediately upstream of the phosphate-donor binding site (GGDGS). The position of this exon in the presumptive catalytic domain makes it a good candidate for modulating the catalytic, substrate- or partner-binding abilities of the protein, as has been postulated for alternatively-spliced exons within encoded functional domains after an in silico genomic survey [CERKL splicing is probably highly regulated.There is mounting evidence that most human genes show alternatively spliced isoforms, many of them regulated by tissue-specific factors. Indeed, alteration of alternative splicing is frequently linked to severe genetic diseases . We repoc survey . Interesc survey , making c survey , and thaCERKL may be exclusive to the vertebrate lineage. Highly homologous sequences can be found in chick and fish, but no evidence of CERKL is found in the lancelet, the closest chordate ancestor to the vertebrates, which instead shows an unambiguous hit to CERK (data not shown). Further support to its vertebrate confinement comes from the fact that other invertebrate genomes, such as Drosophila and C. elegans, are also devoid of any CERKL homologs. In this evolutionary context, the more complex vertebrate retina may have required genes with novel enzymatic functions to protect cells against stress, adding further refinement to the metabolism of bioactive lipids as second messengers.From sequence comparisons and phylogenetic analysis, CERKL unambiguously clusters within the ceramide kinase subfamily of lipid kinases . HoweverThe versatile subcellular localization of CERKL in the COS-7 and HEK293T cell lines is common to all isoforms. This complex and highly dynamic pattern may reflect changes in the intracellular sphingolipid pools, as it occurs with other proteins in the ceramide metabolism –24. CERK2O2 was no longer detectable after 24 h treatment. This RP mutation lies within the CERKL exon 5, which is skipped in the variants that do not contain the DAG kinase domain (isoforms CERKLc and CERKLd). Thus, these two variants probably are not compromised in the RP26 patients. In this context, the physiologic relevance of each CERKL isoform and their particular contribution to retinal disorders is yet to be determined. Conceivably, the RP pathology could be caused by the absence of the longer isoforms or alternatively, the toxicity of the 2 truncated proteins. Our results, and the fact that most transcripts with premature STOP codons are degraded by nonsense mediated decay, support the first scenario.RP is characterized by progressive depletion of photoreceptor cells through apoptosis. Many different genetic defects converge in retinal cell malfunction and trigger programmed cell death. The retina consists of several ordered layers of differentiated neuronal cells that are under constant oxidative stress conditions due to light exposure and a high metabolic rate. Therefore, photoreceptor survival depends on the action of anti-apoptotic mechanisms to prevent premature death. Our data support that CERKL plays a crucial protective role as its overexpression protects cells from entering apoptosis. This protective effect is dependent on time and the severity of the insult suggesting that CERKL overexpression is effective only under a range of stress conditions, but probably not after massive cell damage. Remarkably, the R257X protein causing RP cannot protect cells after sustained injury. The weak protective effect observed at 4 h treatment with HTo our knowledge this is the first time CERKL has been shown to be directly involved in protection against apoptosis. Thus, we link RP-causative mutations to a regulatory mechanism for cell survival, which highlights the feasibility of an RP-gene therapy approach based on protection factors to prevent photoreceptor neurodegeneration and promote cell survival.


In the diabetic animals, the plaque area was 8887 ± 5386 μm2 and 23220 ± 10400 μm2, respectively for treated and nontreated mice (P = .001). Plasma lipids were increased by omapatrilat: Meanplasma cholesterol in treated mice, diabetic and nondiabetic combined, was 39.31 ± 6.00 mmol/L, versus 33.12 ± 7.64 mmol/L in the nontreated animals (P = .008). The corresponding combined mean values of triglycerides were 4.83 ± 1.93 versus 3.00 ± 1.26 mmol/L (P = .02). Omapatrilat treatment did not affect weight or plasma glucose levels. Treatment with omapatrilat inhibits atherogenesis in diabetic as well as nondiabetic LDL receptor–deficient mice despite an increase in plasma lipids, suggesting a direct effect on the arterial wall.Omapatrilat inhibits both angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP). ACE inhibitors have been shown to inhibit atherosclerosis in apoE-deficient mice and in several other animal models but failed in low-density lipoprotein (LDL) receptor– deficient mice despite effective inhibition of the reninangiotensin- aldosterone system. The aim of the present study was to examine the effect of omapatrilat on atherogenesis in diabetic and nondiabetic LDL receptor–deficient mice. LDL receptor–deficient male mice were randomly divided into 4 groups (n = 11 each). Diabetes was induced in 2 groups by low-dose STZ, the other 2 groups served as nondiabetic controls. Omapatrilat (70 mg/kg/day) was administered to one of the diabetic and to one of the nondiabetic groups. The diabetic and the nondiabetic mice were sacrificed after 3 and 5 weeks, respectively. The aortae were examined and the atherosclerotic plaque area was measured. The atherosclerotic plaque area was significantly smaller in the omapatrilat-treated mice, both diabetic and nondiabetic, as compared to nontreated controls. The mean plaque area of omapatrilattreated nondiabetic mice was 9357 ± 7293


Legionella pneumophila, the intracellular bacterial pathogen that causes Legionnaires' disease, exhibit characteristic transmission traits such as elevated stress tolerance, shortened length and virulence during the transition from the replication phase to the transmission phase. ClpP, the catalytic core of the Clp proteolytic complex, is widely involved in many cellular processes via the regulation of intracellular protein quality.L. pneumophila at high temperatures and under several other stress conditions. We also observed that cells devoid of clpP exhibited cell elongation, incomplete cell division and compromised colony formation. Furthermore, we found that the clpP-deleted mutant was more resistant to sodium stress and failed to proliferate in the amoebae host Acanthamoeba castellanii.In this study, we showed that ClpP was required for optimal growth of L. pneumophila, and further suggest a putative role of ClpP in virulence regulation.The data present in this study illustrate that the ClpP protease homologue plays an important role in the expression of transmission traits and cell division of  Legionella pneumophila, a Gram-negative, intracellular bacterial pathogen, is the opportunistic agent responsible for a severe form of pneumonia named Legionnaires' disease and the less severe flu-like Pontiac fever , then diluted into new flasks to similar optical densities at approximate OD600 0.2. These new cultures were then incubated at 25°C, 30°C, 37°C, and 42°C, respectively. OD600 was determined by Beckman Du-530 at various time points.The growth experiments were conducted using three Resistance to stresses was measured as previously described ,65, withIn heat resistance assays, cells from 1 ml broth cultures were centrifuged at 5, 000 g for 5 min and then resuspended in AYE. Samples for heat-shock were placed in a 57°C water bath for 20 min, with the control in a 37°C water bath. Cells were washed and serially diluted in AYE, and spread on BCYE for CFU counting.-1)/(control sample CFU ml-1)] × 100.Stress resistance was calculated as [(stressed sample CFU ml-1)/(BCYE CFU ml-1)] × 100.Sodium sensitivity assay was performed as previously described . BrieflyL. pneumophila cells in exponential or stationary phase were collected by centrifugation at 5,000 g for 2 minutes, and then washed 3 times with 1×PBS. After being fixed by 2% glutaraldehyde (pH 7.4) and 1% osmium tetroxide followed by dehydration in a graded ethanol series and isoamyl acetate embedding, the cells were dried by using a critical point drying method, and mounted on aluminum stubs and shadowed with gold. For visualization, a scanning electron microscope (Hitachi/Oxford S-520/INCA 300) was used at 10 kV.For scanning electron microscopy (SEM), L. pneumophila cells were collected and washed using the same method as above. The cells were then resuspended in 1×PBS and 4 μl sample aliquots were directly applied to a holey carbon film grid , followed by blotting with filter paper (Whatman #1) for about 3 seconds. The grid was then immediately flash frozen by plunging into pre-cooled liquid ethane. The cryo-grid was held in a Gatan 626 Cryo-Holder  and transferred into TEM (JEOL JEM-2010 with 200 kv LaB6 filament) at -172°C. The sample was scanned and observed under minimal dose condition at -172°C. The micrographs were recorded by a Gatan 832 CCD camera at a nominal magnification of 10,000~ 50,000× and at the defocus of 3-5.46 μm.For Cryo-transmisson electron microscopy, A. castellanii cells were cultured in PYG medium for 3 days prior to the test. A medium change was carried out one day before the test. The amoebae cells were washed off from the tissue culture flask, collected by centrifugation at 2,000 rpm for 5 min and resuspended in PYG to a density of 2 × 106 ml-1. 2 × 106 A. castellanii cells were spread on BCYE agar plates, and incubated at room temperature overnight. Series of tenfold dilutions of stationary-phase bacterial cultures at a starting density of 1 OD600 (=109 cells ml-1) were prepared. 10 μl of each dilution were spotted onto the amoebae-CYET agar plates, and incubated at 37°C for 5 days.APT was performed as previously described . Briefly5 amoebae cells were infected by bacteria at a multiplicity of infection (MOI) of 100. 24 h post infection, propidium iodide (PI) was added to 3 mg ml-1. A. castellanii cells were detached from the wells and 2.5 × 104 infected amoebae per sample were analyzed using a FACSCalibur flow cytometer (Becton Dickinson) with a scatter gate adjusted for A. castellanii [To determine cytotoxicity, 2.5 × 10tellanii . ExcitatA. castellanii were washed with Ac (A. castellanii) buffer, resuspended in HL5 medium, seeded onto a 24-well plate (2.5 × 105 per well) and were allowed to adhere for 1-2 h. L. pneumophila was grown for 21 h in AYE broth, diluted in HL5 and used to infect amoebae at an MOI of 10. The infection was synchronized by centrifugation at 440 g for 10 min, and the infected amoebae were incubated at 30°C. Thirty minutes post infection, extracellular bacteria were removed by washing 3 times with warm HL5 medium [For intracellular growth assays, exponentially growing 5 medium . At the 5 medium .http://www.ebi.ac.uk/Tools/clustalw2.Basic statistical analyses were performed using Excel, and one-way ANOVA was performed using SPSS followed by a post hoc Student-Newman-Keul's test. The alignment of amino acid sequences was performed using the online ClustalW2 dot: defect in organelle trafficking; icm: intracellular multiplication; mip: macrophage infectivity potentiator; MIF: mature intracellular form; MOI: multiplicity of infection; APT: amoebae plate testXHL and YJL designed the experiments and drafted the manuscript. XHL performed the experiments. YLZ and YG participated in the design of the study and performed the amoebae infection analysis. XCZ carried out part of molecular cloning work. QFZ carried out the cyro- electron microscope observation. SNZ participated in designing the study and helped to draft the manuscript. All authors read and approved the final manuscript.


Fabp7 , a gene with functional links to the N-methyl-D-aspartic acid (NMDA) receptor and expression in astrocytes. Fabp7-deficient mice showed decreased PPI and a shortened startle response latency, typical of the QTL's proposed effects. A quantitative complementation test supported Fabp7 as a potential PPI-QTL gene, particularly in male mice. Disruption of Fabp7 attenuated neurogenesis in vivo. Human FABP7 showed altered expression in schizophrenic brains and genetic association with schizophrenia, which were both evident in males when samples were divided by sex. These results suggest that FABP7 plays a novel and crucial role, linking the NMDA, neurodevelopmental, and glial theories of schizophrenia pathology and the PPI endophenotype, with larger or overt effects in males. We also discuss the results from the perspective of fetal programming.Deficits in prepulse inhibition (PPI) are a biological marker for schizophrenia. To unravel the mechanisms that control PPI, we performed quantitative trait loci (QTL) analysis on 1,010 F2 mice derived by crossing C57BL/6 (B6) animals that show high PPI with C3H/He (C3) animals that show low PPI. We detected six major loci for PPI, six for the acoustic startle response, and four for latency to response peak, some of which were sex-dependent. A promising candidate on the Chromosome 10-QTL was  Fabp7 , a chaperon for the essential fatty acid docosahexaenoic acid. Human studies showed that the FABP7 gene is modestly associated with schizophrenia and that transcript expression levels are up-regulated in schizophrenic brains. From these results, we propose that a FABP7 protein-mediated disturbance of essential lipid metabolism in developing brains may be one risk factor in the development of schizophrenia, with a greater effect in males.A startle response to an unexpected, strong startling stimulus can be suppressed by an immediately preceding low-intensity stimulus, thereby eliciting little behavioral response. This phenomenon, called prepulse inhibition (PPI), has been observed in all mammals tested and is thought to reflect sensory-motor gating functions in organisms. PPI is diminished in human schizophrenia, raising the possibility that PPI might serve as a potential biological marker for the disease. Once the genes regulating PPI in lower animals are identified, it is expected that the human orthologs will be strong candidate genes for schizophrenia. In this study, we first performed a genetic dissection of mouse PPI using quantitative trait loci analysis, which detects chromosomal regions harboring causative genes. Further analyses including those of knockout mice, allowed us to identify one potential causative gene,  The search for responsible genes for prepulse inhibition, a measure deemed to be a biological trait in schizophrenia, has exposed a gene encoding essential fatty acid-binding protein. Prepulse inhibition (PPI) is the normal suppression of a startle response when a low-intensity stimulus, eliciting little or no behavioral response, immediately precedes an unexpected stronger startling stimulus . The resFabp7 as a highly promising causative gene, especially in males. Functional analysis of the gene and human studies provided fresh insight into schizophrenia mechanisms and the PPI endophenotype.To map the genetic loci determining PPI and its related behavioral profiles, we performed large-scale quantitative trait loci (QTL) analysis using selected inbred mouse strains. QTL analysis is a method of localizing chromosomal regions harboring genetic variants that affect a continuously distributed, polygenic phenotype . MoleculIn a preliminary study, we examined the supply of inbred mice for variability in the PPI response between individual animals from the same strain and consistency of PPI scores from animals with different delivery times. These longitudinal tests spanned more than 1 y. −4 for prepulse intensity of 86 dB[A]), almost conforming to a normal distribution (kurtosis: −0.358 for prepulse intensity of 86 dB[A]) (hB2) of these traits by computing the phenotypic variances (Vp) in the F1 [Vp(F1)] and F2 [Vp(F2)] population [hB2 = {Vp(F2) − Vp(F1)}/Vp(F2)] (rA)  : in gene71–0.81) , whereas04–0.16)  and betw = 0.13) . Phenoty = 0.34) . We also = 0.01) .All 1,010 F2 generation mice were genotyped using 80 microsatellite markers covering the whole genome (19 autosomes and X chromosome) with an average intermarker distance of 20 cM  as the fThe mean phenotypic value for each QTL and test results for additive and dominant models are shown in http://www.r-project.org/), which can suitably handle the X chromosome in QTL analysis ) paired stimulus or a no prepulse–no pulse pair were administered  with package nlme [ijy is the PPI phenotypes of the ith individual at the jth prepulse level denoted by jx equal to 72, 74, 78, 82, and 86 dB for j = 1, 2, 3, 4, and 5, respectively. a0 is the population average intercept, a1 is the population average slope, which is the regression coefficient of jx. iS and iL are the fixed effects of strain and sex of the ith individual, respectively, for which the B6 female mouse was regarded as control with iL = 0 for B6 strain and iS = 0 for females. ui0 and ui1 are the random effects of the intercept and slope associated with ith individual, and ije is the within-subject error term. It is assumed that  are independently and identically distributed with a bivariate normal distribution N, where D is a 2 × 2 positive definite matrix, and the ije are independently and identically distributed with a normal distribution N, independent of . The analysis of variance showed a significant difference between B6 and the other strains as well as a marginally significant sex effect  were measured for the four inbred strains , where the numbers of mice measured were 30, 30, 29, and 30 for males in B6, C3, BALB, and DBA, respectively, and were 32, 32, 30, and 32 for females in B6, C3, BALB, and DBA, respectively. This experiment tested three factors: strain, sex, and prepulse level. The third factor (prepulse level) is ordinal and further was nested, since each mouse had multiple measurements. Thus, we applied the linear mixed model to the test of the difference between strains and between sexes. Statistical analysis for the linear mixed model was performed using statistical software R2.4.1  after PCR amplification using fluorescently labeled primers. For SNP genotyping, we used TaqMan probes and an ABI 7900 sequence detection system (Applied Biosystems). Detailed information on the markers used is available on request.Microsatellite markers were typed on a 3730 iy is the phenotype of the ith individual, μ is the intercept of the model, is is the sex effect, iu and iv are dummy variables indicating the genotype of a putative QTL on the position of interest with  = ,  and  for QQ, Qq, and qq, respectively. a and d are additive and dominance effects of a QTL, respectively. iju and ijv are the variables indicating the genotype of QTL near by the jth marker included in the model to absorb the effect of a background QTL in another region. ja and jd are the additive and dominance effects of the QTL, and ie is the residual error.We used two-stage QTL analyses, the first stage using CIM  on the wN is the number of QTLs involved, klaa is the additive by additive epistatic interaction between the kth QTL and the lth QTL, and klad , klda, and kldd are interaction of additive by dominance, dominance by additive, and dominance by dominance interaction, respectively, between the kth QTL and the lth QTL, with other parameters and variables being as described above in the model of CIM. In MIM, we evaluated the quality of model-fitting with BIC [k is the number of parameters fitted in the model and n is the sample size. The model that minimized BIC specifies the genetic architecture, namely the QTL number, N, the position and effect of each of N QTLs and epistatic interaction between some QTLs.In the second QTL genome scan, we used MIM to analyze the more detailed genetic structure of the phenotypes, where male and female mice were analyzed separately. MIM uses multiple marker intervals to simultaneously construct multiple putative QTLs in the model for QTL mapping. Therefore, compared with CIM, MIM tends to be more powerful and precise in detecting QTLs. The analysis with MIM attempts to fit the following model including effects from multiple QTLs and the epistasis between QTLs to the observations:with BIC , which iWe determined the levels of mRNA from mouse and human brain tissue by real-time quantitative RT-PCR (TaqMan system and ABI 7900) and the standard curve method described elsewhere .t-test (two-tailed) (two groups) or Bonferroni test (more than two groups). The gene expression levels in human brains were evaluated using the two-tailed Mann-Whitney U-test.The differences of the means of mRNA expression levels in mouse brains were evaluated by either Student's Fabp7-disrupted mice is described in detail elsewhere [Fabp7-null mice.The generation of lsewhere . BrieflyFabp7 knockout mice (age of 8–9 wk) were used for the locomotor responses to (+)-MK-801 . MK-801 was dissolved in physiological saline and injected intraperitoneally at a dose of 0.3 mg/kg. In chronic experiments, MK-801 was administered six times at 3-d intervals. Locomotor activities were recorded from 90 min before to 180 min after single injections (acute experiments) or after the sixth injection (chronic experiments), using Supermex (Muromati Kikai) [Adult male wild-type and i Kikai) .Locomotor activity on the challenge day was analyzed using a repeated measures ANOVA (genotype × time bin) with time bin as the repeated measure, followed by Bonferroni post hoc tests.y = μ + S + L + G + R + (S × L) + (S × G) + (S × R) + (L × G)+ (L × R) + (G × R) + (S × L × G) + (S × L × R) + (S × G × R) + (L × G × R) + (S × L × G × R) + e, for data from both males and females. Here, y is the phenotype; μ is the intercept; S is the sex effect; L is the difference of effects at QTL between two strains—i.e., high (B6) and low (C3);G is the difference of effects at a gene of interest (m and +); R is the effect of runs; (S × L), (S × G), (S × R), (L × G), (L × R), and (G × R) are the two-way interactions; (S × L × G), (S × L × R), (S × G × R), and (L × G × R) are the three-way interactions; (S × L × G × R) is the four-way interaction; and e is the residual. Moreover, we performed analyses separately for male and female mice using a model: y = μ + L + G + R + (L × G) + (L × R) + (G × R) + (L × G × R) + e. L × G interaction. The detailed data are available upon request. Failure to complement is indicated by a significant interaction of (L × G) in the analysis of variance.In the quantitative complementation test, a total of 311 mice  were measured for PPI and latency phenotypes at two different times, where 159 mice  and 152 mice , respectively, were phenotyped in two independent runs. We analyzed quantitative complementation results with the statistical software R 2.4.1 using a linear model: t-test (two-tailed).Immunostaining for NeuN (neuron specific nuclear protein), GFAP, PSA-NCAM, and BrdU was performed as previously described . BrdU wan = 570) and age/sex frequency matched controls (n = 570), both of which were free of population stratification, have been described elsewhere [FABP7 interval was constructed using the Haploview program (http://www.broad.mit.edu/mpg/haploview/), using the genotype data from both case and control samples . Haplotype distributions were evaluated using the expectation-maximization algorithm implemented in the COCAPHASE of UNPHASED software package v2.403 (http://www.litbio.org). Empirical significance levels were simulated from 10,000 Monte Carlo permutations using the COCAPHASE program. The present study was approved by the ethics committee of RIKEN.Demographic data on brain tissues , and genomic DNA from patients with schizophrenia . (B) The values represent mean ± s.e. (n = 20 for each group).(A) The values represent mean ± standard error (s.e.) ((839 KB EPS)Click here for additional data file.Figure S2(38 KB PPT)Click here for additional data file.Figure S3(A) Frequency histogram of PPI (%) at prepulse levels of 82 dB for each generation. The numbers of mice used for behavioral scoring were 62 for B6, 61 for C3, 210 for F1, and 1,010 for F2.(B) PPI (%) at five different prepulse levels in each generation. The values represent mean ± s.e.(C) Acoustic startle amplitude in each generation. The values represent mean ± s.e.(D) Latency to startle reflex peak in each generation. The values represent mean ± s.e.(468 KB EPS)Click here for additional data file.Figure S4r) are shown.Pearson's correlation coefficients ((144 KB PPT)Click here for additional data file.Figure S5r) are shown.Pearson's correlation coefficients ((63 KB JPG)Click here for additional data file.Figure S6p < 0.05 and 0.01  , ASR (startle stimulus was 120 dB), and latency are shown. Analysis was performed using the CIM method. Genome-wise significant threshold values were computed by permutation testing  of 5,000and 0.01 . Peak lo.05, see ) were fo < 0.01) . Shared (87 KB JPG)Click here for additional data file.Figure S7Each vertical bar represents a mouse chromosome, with the centromere denoted by a black circle. Loci to the left of each chromosome are those identified in this study, and loci to the right are flanking microsatellite and SNP markers.(382 KB EPS)Click here for additional data file.Figure S8Fabp7 shows abundant expression in E16 (A) and P0 (B) mouse brains. At both stages, Fabp7 is strongly expressed in the ventricular zone and radial glia. The procedure for immunohistochemistry is described elsewhere ,32.(42 KB JPG)Click here for additional data file.Figure S9Fabp7 was complementary to murine cDNA corresponding to nucleotides 6809–6853 [Fabp7 was abundant in the brain at E15 and gradually decreased at P0 and P70.Total RNA was extracted from whole brains of C57BL/6 mice at embryonic day 15 (E15), postnatal day 0 (P0), and postnatal day 70 (P70) by guanidine thiocyanate/phenol/chloroform extraction (triplicated for each stage). The specific 45-mer oligonucleotide probe for 809–6853 . Note th(11 KB JPG)Click here for additional data file.Figure S10p-Values were calculated using the two-tailed Mann-Whitney U-test. Cont, control; SZ, schizophrenia(A) Male samples. (B) Female samples.(493 KB EPS)Click here for additional data file.Figure S11n = 12) in drinking water at concentration of 20 mg/l water for 3 wk. This dose corresponded to 1–5 mg/kg weight/day haloperidol intake, and gave serum haloperidol concentrations of 5.2–15.6 ng/ml at day 21. Control C3 male mice (n = 12) were given drinking water without haloperidol. On day 21, the mice were decapitated, and two brain regions  were quickly dissected. Relative mRNA expression levels were evaluated by Student's t-test (two-tailed).Haloperidol  was administered to C3 male mice ((29 KB PPT)Click here for additional data file.Figure S12n = 285 each for males and females) and age/sex-matched controls . Single-locus allelic association was tested using Fisher's exact test. Haplotype distributions were evaluated using the expectation-maximization algorithm implemented in COCAPHASE [The samples consist of schizophrenia (OCAPHASE . Empiric(382 KB EPS)Click here for additional data file.Figure S13The values represent mean ± s.e. The number of mice used for behavioral scoring were 62 for B6 and 61 for C3.(480 KB EPS)Click here for additional data file.Figure S14Cdh23 genotypes are Cdh23753A/Cdh23753A for B6/B6, Cdh23753A/Cdh23753G for B6/C3, and Cdh23753G/Cdh23753G for C3/C3.The values represent mean ± s.e. The numbers of mice used for behavioral scoring were 156 for B6/B6, 476 for B6/C3, and 177 for C3/C3. The (507 KB EPS)Click here for additional data file.Table S1(27 KB XLS)Click here for additional data file.Table S2(35 KB XLS)Click here for additional data file.Table S3(36 KB XLS)Click here for additional data file.Table S4(45 KB XLS)Click here for additional data file.Table S5(98 KB XLS)Click here for additional data file.Table S6(34 KB XLS)Click here for additional data file.Text S1(38 KB DOC)Click here for additional data file.