Functional Characterization of Key Enzymes involved in l-Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

Bacillus methanolicus wild-type strain MGA3 secretes 59 g/liter⁻¹ of l-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in l-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low K_m value for ammonium (10 mM) and a high K_m value for l-glutamate (250 mM), and the V_max value is 7-fold higher for l-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar K_m values (1 to 1.4 mM) and V_max values (4 U/mg) for both l-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating l-glutamate production in this organism.     

炭疽芽胞杆菌中CRISPR位点

【目的】考察炭疽芽胞杆菌中规律成簇的间隔短回文序列(Clustered regularly interspaced short palindromic repeats,CRISPR)位点多态性情况及基于CRISPR位点多态性的分子分型方法是否在炭疽芽胞杆菌分型中适用。【方法】下载NCBI数据库中6株炭疽芽胞杆菌基因组并截取其中CRISPR位点片段序列。根据炭疽芽胞杆菌内CRISPR位点信息,设计相关引物,以193株炭疽芽胞杆菌基因组为模板PCR扩增CRISPR位点片段,测序。本地Blast比对截取序列及测序结果,查看CRISPR位点在炭疽芽胞杆菌中的多态性情况,并比较炭疽芽胞杆菌与蜡样芽胞杆菌和苏云金芽胞杆菌内CRISPR位点情况。【结果】炭疽芽胞杆菌内CRISPR位点不存在多态性。【结论】基于CRISPR位点多态性的分子分型方法不适用于炭疽芽胞杆菌分型,但可以用于区分炭疽芽胞杆菌与蜡样芽胞杆菌和苏云金芽胞杆菌。     

Envelope cross-reactivity between human immunodeficiency virus types 1 and 2 detected by different serological methods: correlation between cross-neutralization and reactivity against the main neutralizing site.

A total of 70 human immunodeficiency virus type 1 (HIV-1) and 42 HIV-2 antibody-positive serum samples, collected from groups of individuals in which only one type of HIV prevails, were tested for cross-reactivity against HIV-2 and HIV-1 proteins by Western blot (WB) (immunoblot), radioimmunoprecipitation assay (RIPA), neutralization analysis, and enzyme-linked immunosorbent assay with as antigen synthetic peptides representing selected parts of the envelope (env) glycoproteins. Cross-reactions against the env glycoproteins were observed by WB in 10% (7 of 70) and by RIPA in 40% (28 of 70) of the HIV-1 antibody-positive serum samples and by WB in 29% (12 of 42) and by RIPA in 48% (20 of 42) of the HIV-2 antibody-positive serum samples. Testing by enzyme-linked immunosorbent assay against a 36-amino-acid peptide (Cys-301-Cys-336) of the external glycoprotein of strain HTLV-IIIB of HIV-1 (HIV-1HTLV-IIIB) (known to represent a dominating, linear neutralizing site) showed type-specific reactions in 67% (38 of 57) of HIV-1 antibody-positive serum samples. Type-specific reactions against a homologous 35-amino-acid peptide of strain SBL-6669 of HIV-2 (HIV-2SBL-6669) were found in 75% (30 of 40) of HIV-2 antibody-positive serum samples, and these reactions were correlated to neutralization against HIV-2SBL-6669. Cross-reactions against these peptides were seen in 23% (13 of 57) and 33% (13 of 40) of the HIV-1 and HIV-2 antibody-positive serum samples, respectively. These cross-reactions were correlated to cross-neutralization against HIV-1HTLV-IIIB and HIV-2SBL-6669. Cross-neutralization against one heterotypic virus strain was demonstrated in 16% (9 of 57) of HIV-1 antibody-positive serum samples and in 22% (5 of 22) of HIV-2 antibody-positive serum samples, but no correlation was found between cross-neutralization and env cross-reactivity in WB or RIPA.     

Protection against lethal measles virus infection in mice by immune-stimulating complexes containing the hemagglutinin or fusion protein.

The importance of each of the two surface glycoproteins of measles virus in active and passive immunization was examined in mice. Infected-cell lysates were depleted of either the hemagglutinin (H) or fusion (F) glycoprotein by using multiple cycles of immunoaffinity chromatography. The products were used to prepare immune-stimulating complexes (iscoms) containing either F or H glycoprotein. Such complexes are highly immunogenic, possibly as a result of effective presentation of viral proteins to the immune system [B. Morein, B. Sundquist, S. H?glund, K. Dalsgaard, and A. Osterhaus, Nature (London) 308:457-460, 1984]. Groups of 3-week-old BALB/c mice were inoculated with the iscom preparations. All animals developed hemolysis-inhibiting antibodies, whereas only sera of animals immunized with the iscoms containing the H glycoprotein had hemagglutination-inhibiting antibodies. Sera from animals immunized with the H or F preparation only precipitated the homologous glycoprotein in radioimmune precipitation assays. The immunized animals were challenged with a lethal dose of the hamster neurotropic variant of measles virus. Of the 7-week-old animals in the nonimmunized control group, 50% died within 10 days after challenge. No animals in the immunized groups showed symptoms of disease throughout the observation period of 3 months. Passive administration of anti-H monoclonal antibodies gave full protection against the 100% lethal acute infection with the hamster neurotropic variant of measles virus in newborn mice, whereas anti-F monoclonal antibodies failed to protect the animals. This study emphasizes that both H and F glycoproteins need to be considered in the development of measles virus subunit vaccines.     

Measles virus hemagglutinin. Removal of the initiator methionine in the mature protein, and evidence for further processing to produce a 'ragged' end

Measles virus hemagglutinin has been isolated by immunoadsorption. The total composition of the protein and its N-terminal amino acid sequence give data matching the structure indirectly deduced from cDNA. However, direct analysis of the hemagglutinin also shows that the mature protein is proteolytically processed and has a partly heterogeneous N-terminus. The initiator Met is removed, and non-stoichiometrically also the second residue.     

LOCAL MITOGENIC EFFECT OF TISSUE MAST CELL SECRETION

The effect of drug-induced mast cell secretion on proliferation was studied in fibroblast-like and mesothelial-like cells in organ-cultured rat mesentery. Mast cell degranulation achieved by Compound 48/80 was followed by a marked mitogenic reaction in the surrounding tissue cells. The drug itself lacked mitogenic effect on cultured guinea-pig mesentery, the mast cells of which are unresponsive to the drug, and on a human normal fibroblast-like cell line. In contrast, histamine at about 10^?10 M, a major mast cell component, induced marked mitogenesis in guinea-pig mesentery without causing degranulation of mast cells. It is concluded that secreting rat-tissue mast cells release a mitogenic factor or factors acting locally on nearby tissue cells.     

Mechanical stresses in carotid plaques using MRI-based fluid-structure interaction models

Risk assessment in patients with carotid atherosclerosis relies on the degree of luminal stenosis. Incorporating morphological information on plaque composition obtained noninvasively through the use of magnetic resonance imaging (MRI) could include other variables besides the degree of stenosis into carotid plaque risk assessment. Knowledge of the morphologic composition of the plaque allows determination of mechanic stresses exerted on the protective fibrous cap, which may be of importance in the assessment of plaque vulnerability. Based on image processing of transverse MRI scans, longitudinal 2D fluid-structure interaction (FSI) simulations of carotid atherosclerotic plaques were performed facilitating in-vivo estimation of longitudinal internal fibrous cap stresses. The FSI simulation combined finite element analysis (FEA) with computational fluid dynamics (CFD) simulations of blood-flow variables. Preliminary results from two symptomatic patients revealed longitudinal stress levels (max. 254.1 and 143.2 kPa) approaching established criteria for plaque rupture at known predilection sites of plaque rupture. Determination of longitudinal fibrous cap stresses may prove useful in assessing plaque vulnerability and improve risk stratification in patients with carotid atherosclerosis.