High-resolution copy number variation analysis of schizophrenia in Japan.

Recent schizophrenia (SCZ) studies have reported an increased burden of de novo copy number variants (CNVs) and identified specific high-risk CNVs, although with variable phenotype expressivity. However, the pathogenesis of SCZ has not been fully elucidated. Using array comparative genomic hybridization, we performed a high-resolution genome-wide CNV analysis on a mainly (92%) Japanese population (1699 SCZ cases and 824 controls) and identified 7066 rare CNVs, 70.0% of which were small (<100 kb). Clinically significant CNVs were significantly more frequent in cases than in controls (odds ratio=3.04, P=9.3 × 10-9, 9.0% of cases). We confirmed a significant association of X-chromosome aneuploidies with SCZ and identified 11 de novo CNVs (e.g., MBD5 deletion) in cases. In patients with clinically significant CNVs, 41.7% had a history of congenital/developmental phenotypes, and the rate of treatment resistance was significantly higher (odds ratio=2.79, P=0.0036). We found more severe clinical manifestations in patients with two clinically significant CNVs. Gene set analysis replicated previous findings (e.g., synapse, calcium signaling) and identified novel biological pathways including oxidative stress response, genomic integrity, kinase and small GTPase signaling. Furthermore, involvement of multiple SCZ candidate genes and biological pathways in the pathogenesis of SCZ was suggested in established SCZ-associated CNV loci. Our study shows the high genetic heterogeneity of SCZ and its clinical features and raises the possibility that genomic instability is involved in its pathogenesis, which may be related to the increased burden of de novo CNVs and variable expressivity of CNVs.

In utero gene therapy rescues microcephaly caused by Pqbp1-hypofunction in neural stem progenitor cells.

Human mutations in PQBP1, a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain. Examination of a conditional Pqbp1-knockout (cKO) mouse with microcephaly failed to reveal either abnormal centrosomes or mitotic spindles, increased neurogenesis from the neural stem progenitor cell (NSPC) pool or increased cell death in vivo. Instead, we observed an increase in the length of the cell cycle, particularly for the M phase in NSPCs. Corresponding to the developmental expression of Pqbp1, the stem cell pool in vivo was decreased at E10 and remained at a low level during neurogenesis (E15) in Pqbp1-cKO mice. The expression profiles of NSPCs derived from the cKO mouse revealed significant changes in gene groups that control the M phase, including anaphase-promoting complex genes, via aberrant transcription and RNA splicing. Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO. These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation. Finally, we demonstrated that in utero gene therapy for Pqbp1-cKO mice by intraperitoneal injection of the PQBP1-AAV vector at E10 successfully rescued microcephaly with preserved cortical structures and improved behavioral abnormalities in Pqbp1-cKO mice, opening a new strategy for treating this intractable developmental disorder.

ASKP1240, a fully human anti-CD40 monoclonal antibody, prolongs pancreatic islet allograft survival in nonhuman primates.

A strategy for inhibiting CD40 has been considered as an alternative approach for immunosuppression because of undesirable effects of anti-CD154 monoclonal antibodies (mAbs). Previously, we demonstrated that ASKP1240, which is a fully human anti-CD40 mAb, significantly prolonged kidney and liver allograft survival in cynomolgus monkeys without causing thromboembolic complications. Herein, we evaluated the effect of ASKP1240 on pancreatic islet transplantation (PITx) in cynomolgus monkeys. Diabetes was induced by total pancreatectomy, and islet allografts were transplanted into the liver. Following PITx (8201-12 438 IEQ/kg), blood glucose levels normalized promptly in all animals. Control islet allografts were rejected within 9 days (n = 3), whereas ASKP1240 (10 mg/kg) given on postoperative days 0, 4, 7, 11 and 14 (induction treatment, n = 5) significantly prolonged graft survival time (GST) to >15, >23, 210, 250 and >608 days, respectively. When ASKP1240 (5 mg/kg) was administered weekly thereafter up to post-PITx 6 months (maintenance treatment, n = 4), GST was markedly prolonged to >96, >115, 523 and >607 days. During the ASKP1240 treatment period, both anti-donor cellular responses and development of anti-donor antibodies were abolished, and no serious adverse events were noted. ASKP1240 appears to be a promising candidate for immunosuppression in clinical PITx.

Long-term hepatic allograft acceptance based on CD40 blockade by ASKP1240 in nonhuman primates.

Blockade of the CD40-CD154 costimulatory signal is an attractive strategy for immunosuppression and tolerance induction in organ transplantation. Treatment with anti-CD154 monoclonal antibodies (mAbs) results in potent immunosuppression in nonhuman primates (NHPs). Despite plans for future clinical use, further development of these treatments was halted by complications. As an alternative approach, we have been focusing on the inhibition of the counter receptor, CD40 and have shown that a novel human anti-CD40 mAb, ASKP1240, markedly prolongs renal allograft survival in NHPs, although allografts eventually underwent chronic allograft nephropathy. On the basis of our previous findings that a CD40-CD154 costimulation blockade induces tolerance to hepatic, but not cardiac, allografts in rodents, we tested here our hypothesis that a blockade of CD40 by ASKP1240 allows acceptance of hepatic allografts in NHPs. A 2-week ASKP1240 induction treatment prolonged liver allograft survival in NHPs; however, the graft function deteriorated due to chronic rejection. In contrast, a 6-month ASKP1240 maintenance monotherapy efficiently suppressed both cellular and humoral alloimmune responses and prevented rejection on the hepatic allograft. No serious side effects, including thromboembolic complications, were noted in the ASKP1240-treated monkeys. We conclude that CD40 blockade by ASKP1240 would be a desirable immunosuppressant for clinical liver transplantation.

A human anti-CD40 monoclonal antibody, 4D11, for kidney transplantation in cynomolgus monkeys: induction and maintenance therapy.

Blockade of CD40-CD154 signaling pathway is an attractive strategy to induce potent immunosuppression and tolerance in organ transplantation. Due to its strong immunosuppressive effect shown in nonhuman primate experiments, anti-CD154 monoclonal antibodies (mAbs) have been tried in clinical settings, but it was interrupted by unexpected thromboembolic complications. Thus, inhibition of the counter molecule, CD40, has remained an alternative approach. In the previous preliminary study, we have shown that 4D11, a novel fully human anti-CD40 mAb, has a fairly potent immunosuppressive effect on kidney allograft in nonhuman primates. In this study, we aimed to confirm the efficacy and untoward events of the 2-week induction and 180-day maintenance 4D11 treatments. In both, 4D11 significantly suppressed T-cell-mediated alloimmune responses and prolonged allograft survival. Addition of weekly 4D11 administration after the induction treatment further enhanced graft survival. Complete inhibition of both donor-specific Ab and anti-4D11 Ab productions was obtained only with higher-dose maintenance therapy. No serious side effect including thromboembolic complications was noted except for a transient reduction of hematocrit in one animal, and decrease of peripheral B-cell counts in all. These results indicate that the 4D11 appears to be a promising candidate for immunosuppression in clinical organ transplantation.

Barium isotopes in allende meteorite: evidence against an extinct superheavy element.

Carbon and chromite fractions from the Allende meteorite that contain isotopically anomalous xenon-131 to xenon-136 (carbonaceous chondrite fission or CCF xenon) at up to 5 x 10(11) atoms per gram show no detectable isotopic anomalies in barium-130 to barium-138. This rules out the possibility that the CCF xenon was formed by in situ fission of an extinct superheavy element. Apparently the CCF xenon and its carbonaceous carrier are relics from stellar nucleosynthesis.

Samarium-146 in the early solar system: evidence from neodymium in the allende meteorite.

A carbon-chromite fraction from the Allende C3V chondrite shows strikingly large isotopic enrichments of neodymium-142 (0.47 percent) and neodymium- 143 (36 percent). Both apparently formed by alpha decay of samarium-146 and samarium-147 (half-lives 1.03 x 10(8) and 1.06 x 10(11) years), but the isotopic enrichment was greatly magnified by recoil of residual nuclei into a carbon film surrounding the samarium-bearing grains. These data provide an improved estimate of the original abundance of extinct samarium-146 in the early solar system [(146)Sm/(144)Sm = (4.5 +/- 0.5) x 10(-3)], higher than predicted by some models of pprocess nucleosynthesis. It may be possible to use this isotopic pair as a chronometer of the early solar system.

Examination of in vivo gelatinolytic activity in rheumatoid arthritis synovial tissue using newly developed in situ zymography and image analyzer.

OBJECTIVE: The aim of this study was to examine in vivo gelatinolytic activity of rheumatoid arthritis (RA) synovium using a newly developed in situ zymography (ISZ) method and pathological image analyzer, and to evaluate the relationship between this activity and several features on RA. METHODS: A total of 8 samples of synovium were obtained from RA patients during surgery, and 8 samples from osteoarthritis (OA) patients were examined as controls. Furthermore, total 14 samples of syovium were obtained for comparison among radiographical classifications as Larsen grade (4 cases of grade III, 5 cases of grade IV and 5 cases of grade V). These specimens were frozen with OCT compound immediately after surgery. Frozen sections were applied to a newly developed gelatin-coated FIZ film (Fuji Film Co.Tokyo.Japan) designed for use ISZ, and incubated at 37 degrees C for 6 hours. Using an image analyzer (image processor for analytical pathology; IPAP), two variables were measured as indicators of in vivo gelatynolytic activity: optical density of gelatinolyzed area (ODG), and ratio of gelatinolyzed area (RGA). Also, we investigated the relationship between these indicators and the following variables: radiographic changes (Larsen grades), clinical data (C-reactive protein concentration), histological score of synovial tissue (modified Rooney's score), and expression of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 (assessed by immunohistochemistry). RESULTS: RA synovium had significantly higher RGA and lower ODG than OA, indicating higher gelatinolytic activity in RA. Synovium from cases with Larsen grade IV or V had significantly lower ODG than cases with grade III, but there was no significant difference in RGA between grades. There was no significant correlation between gelatinolytic activity (ODG or RGA) and either CRP or modified Rooney's Histological Score. The results of ISZ indicate that the gelatinolyzed areas were mainly localized in the lining area, with a small amount scattered throughout the stroma. The results of immunohistochemistry indicate that MMP-2, MMP-9, TIMP-1 and TIMP-2 were expressed in areas of gelatinolysis. CONCLUSIONS: The present results indicate that in vivo gelatinolytic activity of synovium is stronger in RA than in OA. They also indicate that gelatinolytic activity of RA synovial cells is stronger in cases with Larsen grade IV or V than in cases with grade III, although the gelatinolyzed area is similar. Gelatinolytic activity, as indicated by optical density and the gelatinolyzed area, differed between regions, even within the same specimen, suggesting an imbalance between production of proteinases and their inhibitors. We believe that the present zymography method can contribute to the elucidation of biological enzymatic activity of RA synovium.

The major lung cancer-derived mutants of ERBB2 are oncogenic and are associated with sensitivity to the irreversible EGFR/ERBB2 inhibitor HKI-272.

Mutations in the ERBB2 gene were recently found in approximately 2% of primary non-small cell lung cancer (NSCLC) specimens; however, little is known about the functional consequences and the relevance to responsiveness to targeted drugs for most of these mutations. Here, we show that the major lung cancer-derived ERBB2 mutants, including the most frequent mutation, A775insYVMA, lead to oncogenic transformation in a cellular assay. Murine cells transformed with these mutants were relatively resistant to the reversible epidermal growth factor receptor (EGFR) inhibitor erlotinib, resembling the resistant phenotype found in cells carrying the homologous mutations in exon 20 of EGFR. However, the same cells were highly sensitive to the irreversible dual-specificity EGFR/ERBB2 kinase inhibitor HKI-272, as were those overexpressing wild-type ERBB2. Finally, the NSCLC cell line, Calu-3, overexpressing wild-type ERBB2 owing to a high-level amplification of the ERBB2 gene were highly sensitive to HKI-272. These results provide a rationale for treatment of patients with ERBB2-mutant or ERBB2-amplified lung tumors with HKI-272.

Does the permanent portacaval shunt for a small-for-size graft in a living donor liver transplantation do more harm than good?

In order to obviate a small-for-size graft syndrome (SFSGS), a portacaval (PC) shunt had been considered in a case of adult-to-adult living donor liver transplantation (AA-LDLT). In a recent AA-LDLT case, we adopted the PC shunt to resolve SFSGS; however, graft atrophy was observed in the late period of LDLT, thereby resulting in liver dysfunction. Due to the surgical closure of the PC shunt at 11 months post-LDLT, the graft regenerated gradually and resulted in the recovery of the liver function. This experience indicates that the portacaval shunt would overcome SFSGS in the early period of LDLT, while it would cause the graft atrophy and the graft dysfunction in the late period of LDLT.

Tuberculosis-related technical enquiries received by a national level institution in Japan, 2014-2016.

Background: Japan had a tuberculosis (TB) notification rate of 13.9 per 100 000 population in 2016. Objectives: To characterise TB-related enquiries received by the Research Institute of Tuberculosis, Tokyo, Japan, between January 2014 and December 2016. Design: A descriptive study of the time, place and other attributes of the enquiries. Results: A total of 1864 enquiries were listed for analysis. On average, 51.8 enquiries (range 30-77) were received per month. The enquiry rates were highest for Yamanashi (5.87/100 000) and Kochi (5.77) Prefectures, and lowest in Miyazaki (0.45) and Saga (0.48) Prefectures. Enquirers belonged mostly to local governments (n = 1212, 65%) and health care facilities (n = 386, 21%), and included medical doctors (n = 412, 22%), nurses (n = 926, 50%) and the general public (n = 150, 8.0%). Most enquiries concerned TB diagnosis and treatment (n = 475, 25%), including diagnosis in general (n = 38, 2.0%), laboratory diagnosis (n = 83, 4.5%), anti-tuberculosis treatment in general (n = 62, 3.3%) and the management of comorbidities or adverse events (n = 60, 3.2%), followed by contact investigations (n = 371, 20%). Conclusions: As most enquiries concerned the diagnosis and treatment of TB, the Ministry of Health of Japan should maintain a number of specialised TB institutions with TB clinicians to provide technical assistance.

Contexte : Le Japon avait un taux de notification de la tuberculose (TB) de 13,9 par 100 000 habitants en 2016.Objectif : Caractériser les demandes liées à la TB reçues par le Research Institute of Tuberculosis, Tokyo, Japon, de janvier 2014 à décembre 2016.Schéma : Une étude descriptive en termes de date, lieu et autres caractéristiques des demandes.Résultats : Un total de 1864 demandes ont été listées pour l'analyse. En moyenne, 51,8 demandes (fourchette 30­77) par mois ont été reçues. Les taux de demande ont été les plus élevés pour les préfectures de Yamanashi (5,87/100 000 habitants) et de Kochi (5,77) et les plus bas dans les préfectures de Miyazaki (0,45) et de Saga (0,48). Les organisations principales auxquelles appartenaient les demandeurs ont été les gouvernements locaux (n = 1212 ; 65%) et les structures de soins de santé (n = 386 ; 21%). Les demandeurs ont été des médecins (n = 412 22%), des infirmiers (n = 926 ; 50%) et le public général (n = 150 ; 8,0%). Les demandes les plus fréquentes ont été liées au diagnostic et au traitement de la TB (n = 475 ; 25%), incluant le diagnostic en général (n = 38 ; 2.0%), le diagnostic de laboratoire (n = 83 ; 4,5%), le traitement de la TB en général (n = 62 ; 3,3%) et la prise en charge des comorbidités ou des effets secondaires (n = 60 ; 3,2%), suivies par la recherche des contacts (n = 371 ; 20%).Conclusion : Comme les demandes les plus fréquentes ont été relatives au diagnostic et au traitement de la TB, le Ministère de la santé du Japon devrait maintenir quelques institutions spécialisées dans la TB avec des cliniciens qui pourraient fournir une assistance technique.

Marco de referencia: En el Japón, la tasa de notificación de tuberculosis (TB) fue 13,9 por 100 000 habitantes en el 2016.Objetivos: Caracterizar las consultas relacionadas con la TB que se recibieron en el Instituto de Investigación en Tuberculosis, Tokyo, Japón, de enero del 2014 a finales de diciembre del 2016.Método: Se llevó a cabo un estudio descriptivo de las consultas con respecto al tiempo, el lugar y otros atributos de las mismas.Resultados: Se registraron 1864 consultas para análisis. En promedio, se recibieron 51,8 consultas por mes (entre 30 y 77). La tasa de consultas más alta correspondió a las prefecturas de Yamanashi (5,87/100 000 habitantes) y Kochi (5,77) y la más baja a las prefecturas de Miyazaki (0,45) y Saga (0,48). Las principales organizaciones de los solicitantes pertenecían a los gobiernos locales (n = 1212; 65%) y a los establecimientos de salud (n = 386; 21%). Los solicitantes fueron médicos (n = 412; 22%), enfermeros (n = 926; 50%) y el público en general (n = 150; 8,0%). Las consultas más frecuentes se relacionaban con el diagnóstico y el tratamiento de la TB (n = 475; 25%), entre otros, el diagnóstico en general (n = 38; 2,0%), el diagnóstico en el laboratorio (n = 83; 4,5%), el tratamiento antituberculoso en general (n = 62; 3,3%) y el manejo de las enfermedades intercurrentes o las reacciones adversas (n = 60; 3,2%), seguidos de la investigación de contactos (n = 371; 20%).Conclusiones: Dado que las consultas más frecuentes se referían al diagnóstico y el tratamiento de la TB, el Ministerio de Salud del Japón debe conservar algunas instituciones especializadas en TB con médicos experimentados que presten asistencia técnica en esta esfera.

Real-time monitoring of nitric oxide (NO) and pO2 levels under ischemic conditions associated with small bowel ischemia/reperfusion injury using selective electrodes for NO and oxygen molecules.

The present study demonstrated the feasibility of monitoring nitric oxide (NO) and pO2 levels under ischemic conditions associated with small bowel ischemia/reperfusion (I/R) injury through the use of selective electrodes for NO and oxygen molecules. NO levels gradually increased during ischemia. When reperfusion was started, the NO level decreased suddenly and returned to pre-ischemia values within 10 minutes. After clamping, pO2 decreased rapidly. When reperfusion was started, pO2 increased suddenly, returning to pre-ischemia values within 10 minutes. We concluded that it is feasible to monitor NO and pO2 levels under ischemic conditions of small bowel I/R injury through the use of electrodes selective for NO and oxygen molecules.

Protective effects of MnM2Py4P and Mn-salen against small bowel ischemia/reperfusion injury in rats using an in vivo and an ex vivo electron paramagnetic resonance technique with a spin probe.

BACKGROUND: Reperfusion of ischemic tissues results in the formation of toxic reactive oxygen species (ROS), such as superoxide anion, hydroxyl radicals, hydroperoxide, and peroxynitrite. ROS are potent oxidizing agents, capable of damaging cellular membranes by lipid peroxidation. In the present study, we applied an in vivo electron paramagnetic resonance (EPR)/spin probe and an ex vivo EPR technique to provide direct evidence of ROS following experimentally induced small bowel ischemia/reperfusion (I/R) injury. MATERIALS AND METHODS: We used a rat model of small bowel I/R injury to explore the possibility that MnM2Py4P or Mn-salen can prevent the accumulation of ROS species following experimentally induced I/R injury. We examined the ability of MnM2Py4P and Mn-salen to scavenge radicals in living Sprague-Dawley (SD) rats using an in vivo and an ex vivo EPR technique with a spin probe. RESULTS: The CP decay rates in the MnM2Py4P- and Mn-salen-treated rats were significantly higher than those in the untreated rats and almost equal to those in sham group rats. There were no significant differences between the MnM2Py4P-treated group and the Mn-salen-treated group. Superoxide scavenging activities (SSA) in the MnM2Py4P- and EUK-8-treated group were higher than those in the untreated group and almost equal to the sham group. CONCLUSION: The present study suggested that the protective effects of MnM2Py4P and Mn-salen against small bowel IR injury were mediated by the inhibition of O2, H2O2, and NO production.

Addiction to the IGF2-ID1-IGF2 circuit for maintenance of the breast cancer stem-like cells.

The transcription factor nuclear factor-κB (NF-κB) has important roles for tumorigenesis, but how it regulates cancer stem cells (CSCs) remains largely unclear. We identified insulin-like growth factor 2 (IGF2) is a key target of NF-κB activated by HER2/HER3 signaling to form tumor spheres in breast cancer cells. The IGF2 receptor, IGF1 R, was expressed at high levels in CSC-enriched populations in primary breast cancer cells. Moreover, IGF2-PI3K (IGF2-phosphatidyl inositol 3 kinase) signaling induced expression of a stemness transcription factor, inhibitor of DNA-binding 1 (ID1), and IGF2 itself. ID1 knockdown greatly reduced IGF2 expression, and tumor sphere formation. Finally, treatment with anti-IGF1/2 antibodies blocked tumorigenesis derived from the IGF1Rhigh CSC-enriched population in a patient-derived xenograft model. Thus, NF-κB may trigger IGF2-ID1-IGF2-positive feedback circuits that allow cancer stem-like cells to appear. Then, they may become addicted to the circuits. As the circuits are the Achilles' heels of CSCs, it will be critical to break them for eradication of CSCs.

Effects of hepatic CYP3A4 activity on disposition of micafungin in liver transplant recipients with markedly small-for-size grafts.

Micafungin, the first candin antifungal drug developed in Japan, has a significant therapeutic effect against deep-seated mycoses caused by Candida or Aspergillus. Little is known, however, about the optimal dosage or disposition of micafungin in patients with severe hepatic impairment. Nine liver transplant recipients (5 males and 4 females) were enrolled in this study. In 1 recipient with a markedly small-for-size graft (ratio of graft volume to standard liver volume at the time of transplantation: 25.9%), the areas under the plasma concentration-time curves up to 12 hours postdose (AUC(0-12 h)) at doses of 50 and 100 mg/d were 79.38 and 601.17 mug.h/mL, respectively. The corresponding elimination half-life (T(1/2)) values were 16.01 and 75.75 hours, and saturated elimination was observed only at the dose of 100 mg/d. The mean urinary ratio of 6beta-hydroxycortisol to cortisol (6beta-OHF/F) in the small-for-size graft recipient was significantly (P < .05) lower than that in the other recipients. In conclusion, graft size was an important factor affecting disposition of micafungin. For liver transplant recipients with markedly small-for-size grafts, the optimal dosage of micafungin to reach and maintain therapeutic plasma levels is estimated to be 50 mg/d.

14-3-3ζ-Mediated Stimulation of Oxidative Phosphorylation Exacerbates Oxidative Damage Under Hypothermic Oxygenated Conditions in Human Renal Tubular Cells (HK-2).

Cellular survival and death are at least partially regulated by the phosphorylation of proteins. A chaperon protein, 14-3-3ζ, regulates the activity of many proteins by covering the phosphorylation site within a 14-3-3 binding motif. Therefore, regulation of 14-3-3ζ activity may affect the fate of cells subjected to cold preservation and/or hypothermic oxygenated conditions. The present study assessed whether 14-3-3ζ protects cells from hypothermic oxygenation-induced injury and clarified its role in mitochondrial functions. Human renal tubular cell line HK-2 or 14-3-3ζ-overexpressed HK-2 (ζHK-2) cells were subjected to 72 hours of normoxic cold preservation in UW solution with or without antioxidants and hydroperoxides. Cellular death, adenosine triphosphate (ATP) content, and MTT catabolism were evaluated. Deferoxamine treatment reduced cellular death and augmented ATP content in both cell types. These indices were higher in ζHK-2, regardless of deferoxamine treatment. Exposure to hydroperoxides did not affect cellular death in either cell type, whereas hydroperoxide supplementation significantly reduced ATP content, except for low-dose hydrogen peroxide in HK-2 cells. MTT assay at normal state showed higher values in ζHK-2 cells, whereas it was impaired by hydroperoxides in both cell types. These results suggest that accumulation of hydroperoxides as a byproduct of the augmented oxidative phosphorylation by 14-3-3ζ overexpression causes mitochondrial dysfunction. In conclusion, despite possessing many potentially protective functions, 14-3-3ζ exacerbates cellular injury under hypothermic oxygenated conditions. 14-3-3ζ accelerates mitochondrial functions together with iron-dependent oxidative damage. Although further investigations are necessary, upregulation of 14-3-3ζ could be a method to maintain mitochondrial function under hypothermic oxygenated conditions, as shown in hypothermic machine preservation of renal grafts, when appropriate antioxidant treatment is administered.

Bactericidal catechins damage the lipid bilayer.

The mode of antibacterial action of, the green tea (Camellia sinensis) extracts, (-)-epigallocatechin gallate (EGCg) and (-)-epicatechin (EC) was investigated. Strong bactericidal EGCg caused leakage of 5,6-carboxyfluorescein from phosphatidylcholine liposomes (PC), but EC with very weak bactericidal activity caused little damage to the membrane. Phosphatidylserine and dicetyl phosphate partially protected the membrane from EGCg-mediated damage when reconstituted into the liposome membrane with PC. EGCg, but not EC, caused strong aggregation and NPN-fluorescence quenching of PC-liposomes and these actions were markedly lowered in the presence of negatively charged lipids. These results show that bactericidal catechins primarily act on and damage bacterial membranes. The observation that Gram-negative bacteria are more resistant to bactericidal catechins than Gram-positive bacteria can be explained to some extent by the presence of negatively charged lipopolysaccharide.

Two forms of Vibrio cholerae O1 El Tor hemolysin derived from identical precursor protein.

Vibrio cholerae O1 grown in heart infusion broth produces two forms of El Tor hemolysin (ETH) monomers of 65 and 50 kDa. These monomers form several different sizes of mixed oligomers ranging from 180 to 280 kDa in the liposomal membranes. We found that the N-terminal amino acid sequences, NH2-Trp-Pro-Ala-Pro-Ala-Asn-Ser-Glu, of both the 65- and 50-kDa toxins were identical. We assumed, therefore, that the 65- and 50-kDa toxins were derivatives of the identical precursor protein and the 50-kDa protein was a truncated derivative of 65-kDa ETH. To substantiate this assumption, we treated the 260-kDa oligomer with trypsin and obtained a 190-kDa oligomer. This 190-kDa oligomer consisted of only the 50-kDa subunits. Both 260- and 190-kDa oligomers formed ion channels indistinguishable from each other in planar lipid bilayers. These results suggest that the essential part of the ETH in forming the membrane-damaging aggregate is a 50-kDa protein.