Spontaneous transformation and its use for genetic mapping in Bacillus subtilis

Using a simple semi-synthetic competence and sporulation medium (CSM), we found evidence that Bacillus subtilis cells transformed in the competence phase can sporulate, indicating that genetic information acquired during the competence phase is inherited by the next generation after germination of the transformed spores. Moreover, the results from mixed cell culture experiments suggest that spontaneous genetic transformation can occur between competent cells and DNA released from lysed cells in the natural environment. We also found evidence that the spontaneous transformation system can be used for genetic mapping in B. subtilis.     

Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology

We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications.     

p38alpha mitogen-activated protein kinase plays a critical role in cardiomyocyte survival but not in cardiac hypertrophic growth in response to pressure overload

The molecular mechanism for the transition from cardiac hypertrophy, an adaptive response to biomechanical stress, to heart failure is poorly understood. The mitogen-activated protein kinase p38alpha is a key component of stress response pathways in various types of cells. In this study, we attempted to explore the in vivo physiological functions of p38alpha in hearts. First, we generated mice with floxed p38alpha alleles and crossbred them with mice expressing the Cre recombinase under the control of the alpha-myosin heavy-chain promoter to obtain cardiac-specific p38alpha knockout mice. These cardiac-specific p38alpha knockout mice were born normally, developed to adulthood, were fertile, exhibited a normal life span, and displayed normal global cardiac structure and function. In response to pressure overload to the left ventricle, they developed significant levels of cardiac hypertrophy, as seen in controls, but also developed cardiac dysfunction and heart dilatation. This abnormal response to pressure overload was accompanied by massive cardiac fibrosis and the appearance of apoptotic cardiomyocytes. These results demonstrate that p38alpha plays a critical role in the cardiomyocyte survival pathway in response to pressure overload, while cardiac hypertrophic growth is unaffected despite its dramatic down-regulation.     

Detection of biosphere frontier by using phosphatase activity.

A wide variety of methods have been proposed to detect microbial activities, but most of them can be applied to limited categories of terrestrial organisms. We propose here to use phosphatase activity, which seems to be an essential catalytic activity for all the terrestrial organisms, and possibly for extraterrestrial organisms. We determined phosphatase activity in core samples, chimney samples, and sea water samples obtained in submarine hydrothermal systems located at Suiyo Seamount, Izu-Bonin Arc, and South Mariana. It was shown that phosphatase activity is one of possible biomarkers for extant life.     

Molecular and structural basis for redox regulation of beta-actin

An essential consequence of growth factor-mediated signal transduction is the generation of intracellular H₂O₂. It operates as a second messenger in the control of actin microfilament dynamics, causing rapid and dramatic changes in the morphology and motile activity of stimulated cells. Little is understood about the molecular mechanisms causing these changes in the actin system. Here, it is shown that H₂O₂ acts directly upon several levels of this system, and some of the mechanistic effects are detailed. We describe the impact of oxidation on the polymerizability of non-muscle β/γ-actin and compare with that of muscle α-actin. Oxidation of β/γ-actin can cause a complete loss of polymerizability, crucially, reversible by the thioredoxin system. Further, oxidation of the actin impedes its interaction with profilin and causes depolymerization of filamentous actin. The effects of oxidation are critically dependent on the nucleotide state and the concentration of Ca²⁺. We have determined the crystal structure of oxidized β-actin to a resolution of 2.6 Å. The arrangement in the crystal implies an antiparallel homodimer connected by an intermolecular disulfide bond involving cysteine 374. Our data indicate that this dimer forms under non-polymerizing and oxidizing conditions. We identify oxidation of cysteine 272 in the crystallized actin dimer, likely to a cysteine sulfinic acid. In β/γ-actin, this is the cysteine residue most reactive towards H₂O₂ in solution, and we suggest plausible structural determinants for its reactivity. No other oxidative modification was obvious in the structure, highlighting the specificity of the oxidation by H₂O₂. Possible consequences of the observed effects in a cellular context and their potential relevance are discussed.     

Reduced sensitivity of Niemann-Pick C1-deficient cells to θ-toxin (perfringolysin O): sequestration of toxin to raft-enriched membrane vesicles

-Toxin (perfringolysin O) binds to cell surface cholesterol and forms oligomeric pores that cause membrane damage. Both in cytotoxicity and cell survival assays, a mutant Chinese hamster ovary cell line NPC1(–) that lacked Niemann-Pick C1 showed reduced sensitivity to -toxin, compared with wild-type (wt) cells. BC is a derivative of -toxin that retains cholesterol-binding activity but lacks cytotoxicity. Confocal and electron microscopy revealed the presence of multiple vesicles which bound BC, both on the cell surface and in the extracellular space of these cells. BC binding to raft microdomains was verified by its resistance to 1% Triton X-100 at 4°C and recovery of bound BC in floating low-density fractions on sucrose density gradient fractionation. BC-labeled vesicles were abolished when NPC1(–) cells were depleted of lipoproteins and also when treated with a Rho-associated kinase inhibitor Y-27632. In addition, similar vesicles were observed in wt cells treated with progesterone. In parallel with these results, -toxin sensitivity of NPC1(–) cells was increased when cells were depleted of lipoproteins or treated with Y-27632, whereas that of wt cells was decreased by progesterone. Our findings suggest that sequestration of toxin to raft-enriched cell surface vesicles may underlie reduced sensitivity of NPC1-deficient cells to -toxin.     

iNKT cells in microbial immunity: recognition of microbial glycolipids

Natural killer T cells expressing an invariant T cell antigen receptor (iNKT cells) are cells of the innate immune system. After recognizing glycolipid antigens presented by CD1d molecules on antigen presenting cells (APCs), iNKT cells rapidly produce large quantities of cytokines, thereby stimulating many types of cells. Recent studies have described several mechanisms of iNKT cell activation and the contribution of these cells to antimicrobial responses. iNKT cells can be activated by endogenous antigens and/or inflammatory cytokines from APCs. However, iNKT cells also recognize certain microbial glycolipids by their invariant T cell antigen receptor (TCR), and they contribute to pathogen clearance in certain microbial infections. These findings indicate that the iNKT TCR is useful for detecting certain microbial pathogens. Moreover, recent studies suggest that iNKT cell glycolipid antigens may be useful in antimicrobial therapy and vaccines.     

Biochemical characterization of rab proteins from Bombyx mori

The small GTPases known as Rab proteins are key regulators of membrane trafficking. We used RT-PCR to isolate cDNA clones of insect-specific Rab proteins (BRabN1 and BRabN2) showing low homology with known Rab proteins from other animals, from mRNA of Bombyx mori. These 2 Rabs were produced in Escherichia coli and purified. BRabN1 bound [(3)H]-GDP and [(35)S]-GTPgammaS with dissociation constants of 0.087 x 10(-6) M and 1.02 x 10(-6) M, respectively, whereas those of BRabN2 were 0.546 x 10(-6) M and 1.02 x 10(-6) M, respectively. Binding of [(35)S]-GTPgammaS to BRabN1 and N2 was inhibited by GDP and GTP. The GTP-hydrolysis activities of BRabN1 and N2 were 154 and 35.5 mmol/min/mole, respectively, and bound [(35)S]-GTPgammaS was exchanged efficiently with GTP. BRabN1 also showed ATPase activity and exchange of [(35)S]-GTPgammaS with ATP. Monoclonal antibodies against BRabN1 and N2 did not recognize any other Rab proteins, and Western blotting using the anti-BRabN1 antibody revealed a single band in the testis of B. mori. These results suggest that BRabN1 and N2 of B. mori bind GTP, convert from the GTP-bound state to the GDP-bound state by intrinsic GTP hydrolysis activity, and return to the GTP-bound state with the exchange, and that BRabN1 is specifically expressed in testis. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc.     

The E3 ubiquitin ligase activity of Trip12 is essential for mouse embryogenesis

Protein ubiquitination is a post-translational protein modification that regulates many biological conditions. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12(mt/mt)) that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12(mt/mt) embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16. In contrast, Trip12(mt/mt) ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12(mt/mt) ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex) and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.