Analysis of outer membrane vesicle protein involved in biofilm formation of Helicobacter pylori

Helicobacter pylori is one of the most common causes of bacterial infection in humans. Infection with H. pylori is closely associated with gastritis and peptic ulcers and is a risk factor for gastric cancer and mucosa-associated lymphoid tissue lymphoma. H. pylori forms biofilms on glass surfaces at the air–liquid interface in in-vitro batch cultures. We previously reported that strain TK1402 showed a strong biofilm-forming ability in vitro. We also suggested the outer membrane vesicles (OMV) produced by strain TK1402 might be related to its biofilm forming ability. In the present study, we analyzed the protein profile of the OMV produced by strain TK1402 and found a unique 22-kDa protein in TK1402 OMV cultured for 2–3 days. In addition, this protein could not be detected in the OMVs produced by other H. pylori strains. These results suggest that the 22-kDa protein is involved in effective biofilm formation by strain TK1402.     

An inter-laboratory collaborative study by the Non-Genotoxic Carcinogen Study Group in Japan, on a cell transformation assay for tumour promoters using Bhas 42 cells

The Bhas promotion assay is a cell culture transformation assay designed as a sensitive and economical method for detecting the tumour-promoting activities of chemicals. In order to validate the transferability and applicability of this assay, an inter-laboratory collaborative study was conducted with the participation of 14 laboratories. After confirmation that these laboratories could obtain positive results with two tumour promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA) and lithocholic acid (LCA), 12 coded chemicals were assayed. Each chemical was tested in four laboratories. For eight chemicals, all four laboratories obtained consistent results, and for two of the other four chemicals, only one of the four laboratories showed inconsistent results. Thus, the rate of consistency was high. During the study, several issues were raised, each of which were analysed step-by-step, leading to revision of the protocol of the original assay. Among these issues were the importance of careful maintenance of mother cultures and the adoption of test concentrations for toxic chemicals. In addition, it is suggested that three different types of chemicals show positive promoting activity in the assay. Those designated as T-type induced extreme growth enhancement, and included TPA, mezerein, PDD and insulin. LCA and okadaic acid belonged to the L-type category, in which transformed foci were induced at concentrations showing growth-inhibition. In contrast, M-type chemicals, progesterone, catechol and sodium saccharin, induced foci at concentrations with little or slight growth inhibition. The fact that different types of chemicals similarly induce transformed foci in the Bhas promotion assay may provide clues for elucidating mechanisms of tumour promotion.     

Rheb binds and regulates the mTOR kinase

BACKGROUND: The target of rapamycin (TOR), in complex with the proteins raptor and LST8 (TOR complex 1), phosphorylates the p70S6K and 4E-BP1 to promote mRNA translation. Genetic evidence establishes that TOR complex activity in vivo requires the small GTPase Rheb, and overexpression of Rheb can rescue TOR from inactivation in vivo by amino-acid withdrawal. The Tuberous Sclerosis heterodimer (TSC1/TSC2) functions as a Rheb GTPase activator and inhibits TOR signaling in vivo. RESULTS: Here, we show that Rheb binds to the TOR complex specifically, independently of its ability to bind TSC2, through separate interactions with the mTOR catalytic domain and with LST8. Rheb binding to the TOR complex in vivo and in vitro does not require Rheb guanyl nucleotide charging but is modulated by GTP and impaired by certain mutations (Ile39Lys) in the switch 1 loop. Nucleotide-deficient Rheb mutants, although capable of binding mTOR in vivo and in vitro, are inhibitory in vivo, and the mTOR polypeptides that associate with nucleotide-deficient Rheb in vivo lack kinase activity in vitro. Reciprocally, mTOR polypeptides bound to Rheb(Gln64Leu), a mutant that is nearly 90% GTP charged, exhibit substantially higher protein kinase specific activity than mTOR bound to wild-type Rheb. CONCLUSIONS: The TOR complex 1 is a direct target of Rheb-GTP, whose binding enables activation of the TOR kinase.     

Smad3 mediates TGF-beta1 induction of VEGF production in lung fibroblasts

Transforming growth factor-beta1 (TGF-beta1) is a key factor in a variety of physiological and pathological processes. Vascular endothelial growth factor (VEGF) is a key angiogenic factor, and vascular change is one of the features of airway remodeling. We examined the effect of TGF-beta1 on VEGF production by fibroblasts from mice lacking expression of Smad2 or Smad3 as well as human lung fibroblasts treated with or without Smad2 or Smad3 siRNA. TGF-beta1 stimulated VEGF production by fibroblasts from Smad2 deficient animals and wildtype animals. In contrast, TGF-beta1 did not affect VEGF production by fibroblasts from Samd3 deficient mice. Similarly, TGF-beta1 failed to stimulate VEGF production by HFL-1 cells treated with Samd3 siRNA but significantly increased VEGF production by the cells treated with Smad2 siRNA. These result suggest that TGF-beta1 stimulation of VEGF production by fibroblasts is regulated by Smad3 but not by Smad2 signaling.     

Antiangiogenic photodynamic therapy (PDT) by using long-circulating liposomes modified with peptide specific to angiogenic vessels

For the improvement of therapeutic efficacy in photodynamic therapy (PDT) by using a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), we previously prepared polyethylene glycol (PEG)-modified liposomes encapsulating BPD-MA (PEG-Lip BPD-MA). PEGylation of liposomes enhanced the accumulation of BPD-MA in tumor tissue at 3 h after injection of it into Meth-A-sarcoma-bearing mice, but, unexpectedly, decreased the suitability of the drug for PDT when laser irradiation was performed at 3 h after the injection of the liposomal photosensitizer. To improve the bioavailability of PEG-Lip BPD-MA, we endowed the liposomes with active-targeting characteristics by using Ala-Pro-Arg-Pro-Gly (APRPG) pentapeptide, which had earlier been isolated as a peptide specific to angiogenic endothelial cells. APRPG-PEG-modified liposomal BPD-MA (APRPG-PEG-Lip BPD-MA) accumulated in tumor tissue similarly as PEG-Lip BPD-MA and to an approx. 4-fold higher degree than BPD-MA delivered with non-modified liposomes at 3 h after the injection of the drugs into tumor-bearing mice. On the contrary, unlike the treatment with PEG-Lip BPD-MA, APRPG-PEG-Lip BPD-MA treatment strongly suppressed tumor growth after laser irradiation at 3 h after injection. Finally, we observed vasculature damage in the dorsal air sac angiogenesis model by APRPG-PEG-Lip BPD-MA-mediated PDT. The present results suggest that antiangiogenic PDT is an efficient modality for tumor treatment and that tumor neovessel-targeted, long-circulating liposomes are a useful carrier for delivering photosensitizer to angiogenic endothelial cells.     

The effective size of mixed sexually and asexually reproducing populations

Using the transition matrix of inbreeding and coancestry coefficients, the inbreeding (N(eI)), variance (N(eV)), and asymptotic (N(e lambda)) effective sizes of mixed sexual and asexual populations are formulated in terms of asexuality rate (delta), variance of asexual (C) and sexual (K) reproductive contributions of individuals, correlation between asexual and sexual contributions (rho(ck)), selfing rate (beta), and census population size (N). The trajectory of N(eI) toward N(e lambda) changes crucially depending on delta, N, and beta, whereas that of N(eV) is rather consistent. With increasing asexuality, N(e lambda) either increases or decreases depending on C, K, and rho(ck). The parameter space in which a partially asexual population has a larger N(e lambda) than a fully sexual population is delineated. This structure is destroyed when N(1 - delta) < 1 or delta > 1 - 1/N. With such a high asexuality, tremendously many generations are required for the asymptotic size N(e lambda) to be established, and N(e lambda) is extremely large with any value of C, K, and rho(ck) because the population is dominated eventually by individuals of the same genotype and the allelic diversity within the individuals decays quite slowly. In reality, the asymptotic state would occur only occasionally, and instantaneous rather than asymptotic effective sizes should be practical when predicting evolutionary dynamics of highly asexual populations.     

Simultaneous imaging of initiator/effector caspase activity and mitochondrial membrane potential during cell death in living HeLa cells

A family of cystein proteases, the caspases, plays a central role in mediating cell death. In this study, we measured the activation of the initiator and effector caspase in real time, and studied the relationship between caspase activity and mitochondrial membrane potential in living cells by means of bioimaging. We also designed and developed a fluorescence resonance energy transfer (FRET)-based genetically encoded fluorescent indicator, which consisted of yellow fluorescent protein (YFP), a peptide sequence which can be cleaved by specific caspases, and cyan fluorescent protein (CFP). Two peptide sequences which could be cleaved by initiator caspases and effector caspases, respectively, were used. Simultaneous real-time measurements of the caspase activity and mitochondrial membrane potential in the cells treated with TNF-alpha and staurosporine revealed that dying cells showed caspase activation and mitochondrial depolarization, and that these events, however, were not firmly linked. Although it takes anywhere from 1 to over 10 h after the addition of the cell death inducer for the caspases to begin to be activated, initiator caspases and effector caspases are activated within a short period of time at the last stage in the entire process leading to cell death.     

Abolished interaction of NUB1 with mutant AIPL1 involved in Leber congenital amaurosis

Constructs based on the pSUPER vector [Science 296 (2002) 550] and encoding small interfering RNAs specific for the Type I, Type II, or Type III isozymes of mammalian (rat) hexokinase were prepared. Transfection of Chinese hamster ovary and HeLa cells with these vectors resulted in selective depletion of the respective isozymes. A Zeocin marker was incorporated into the modified pSUPER vector, permitting isolation of stably transfected cell lines selectively depleted of the respective isozyme.     

Maintenance of DNA methylation during the Arabidopsis life cycle is essential for parental imprinting

Imprinted genes are expressed predominantly from either their paternal or their maternal allele. To date, all imprinted genes identified in plants are expressed in the endosperm. In Arabidopsis thaliana, maternal imprinting has been clearly demonstrated for the Polycomb group gene MEDEA (MEA) and for FWA. Direct repeats upstream of FWA are subject to DNA methylation. However, it is still not clear to what extent similar cis-acting elements may be part of a conserved molecular mechanism controlling maternally imprinted genes. In this work, we show that the Polycomb group gene FERTILIZATION-INDEPENDENT SEED2 (FIS2) is imprinted. Maintenance of FIS2 imprinting depends on DNA methylation, whereas loss of DNA methylation does not affect MEA imprinting. DNA methylation targets a small region upstream of FIS2 distinct from the target of DNA methylation associated with FWA. We show that FWA and FIS2 imprinting requires the maintenance of DNA methylation throughout the plant life cycle, including male gametogenesis and endosperm development. Our data thus demonstrate that parental genomic imprinting in plants depends on diverse cis-elements and mechanisms dependent or independent of DNA methylation. We propose that imprinting has evolved under constraints linked to the evolution of plant reproduction and not by the selection of a specific molecular mechanism.