Isolation and immunological characterization of the four non-identical subunits of the soluble NAD-linked hydrogenase from Alcaligenes eutrophus H16

The soluble NAD-linked hydrogenase of Alcaligenes eutrophus H16 is a tetramer consisting of 4 non-identical subunits with molecular weights of 63,000, 56,000, 30,000 and 26,000. Conditions have been elaborated to separate and isolate each of these subunits as a single polypeptide by a preparative scale of polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS). Against each of the 4 subunits, polyclonal antibodies were produced. From the crude sera isolated from rabbits, the antibodies (IgG fractions) were purified by Protein A-Sepharose chromatography. By the double immunodiffusion method, comparison of the 4 types of subunits revealed that they are in fact different polypeptides. Subunit 1 (Mr = 63,000) and subunit 2 (Mr = 56,000) only reacted with their own specific antibodies and showed no cross-reaction whatsoever with the antibodies raised against the other subunits. The only immunological relationship among the different subunits was observed with subunit 3 (Mr = 30,000) and subunit 4 (Mr = 26,000); the type of cross-reaction indicated that they are partially identical. A. eutrophus H16 contains, in addition to the soluble hydrogenase, a membrane-bound hydrogenase which is a dimer composed of 2 subunits with Mr of 61,000 and 30,000. Whereas the 2 native enzymes did not show any immunological cross-reaction with the respective antibodies, it was demonstrated by double immunofluorescence labeling on nitrocellulose filters that the larger subunit of the membrane-bound hydrogenase cross-reacted significantly with the antibodies raised against subunit 2 of the soluble hydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct Observation of Surface Plasmon Polariton Propagation and Interference by Time-Resolved Imaging in Normal-Incidence Two Photon Photoemission Microscopy

Plasmonics - Time-resolved imaging of the propagation and interference of isolated ultrashort surface plasmon polariton wave packets is demonstrated using two photon photoemission microscopy. The...     

A Link between Arabinose Utilization and Oxalotrophy in Bradyrhizobium japonicum

Rhizobia have a versatile catabolism that allows them to compete successfully with other microorganisms for nutrients in the soil and in the rhizosphere of their respective host plants. In this study, Bradyrhizobium japonicum USDA 110 was found to be able to utilize oxalate as the sole carbon source. A proteome analysis of cells grown in minimal medium containing arabinose suggested that oxalate oxidation extends the arabinose degradation branch via glycolaldehyde. A mutant of the key pathway genes oxc (for oxalyl-coenzyme A decarboxylase) and frc (for formyl-coenzyme A transferase) was constructed and shown to be (i) impaired in growth on arabinose and (ii) unable to grow on oxalate. Oxalate was detected in roots and, at elevated levels, in root nodules of four different B. japonicum host plants. Mixed-inoculation experiments with wild-type and oxc-frc mutant cells revealed that oxalotrophy might be a beneficial trait of B. japonicum at some stage during legume root nodule colonization.     

Identification of blapsins A and B as potent small-molecule 14-3-3 inhibitors from the insect Blaps japanensis

In this study, we report three novel naturally occurring compounds, blapsins A (1) and B (2), and blapsamide (3) from the ethanol extract of the stink beetle, Blaps japanensis. The structures of these compounds were determined using spectroscopic methods. Compound 3 is a phenolic compound bearing a formamido group in the structure. Functional studies revealed that compounds 1 and 2 potently inhibited 14-3-3 protein-protein interactions (PPIs) with IC(50) values of 9.2 and 10.0 μM as determined by an ELISA assay, and 2.0 and 2.5 μM in an FP assay, respectively. These compounds represent the first example of natural small-molecule 14-3-3 inhibitors.     

Draft Genome Sequence of Corynebacterium bovis DSM 20582, Which Causes Clinical Mastitis in Dairy Cows

Bovine mastitis represents the most economically important disease in dairy cows and can be caused by Corynebacterium bovis, a commensal in the bovine udder. The draft genome sequence provides insights into the adaptation of this bacterium to the bovine habitat and its lipolytic capabilities to utilize components of cow's milk.     

Proteomic analysis of maternal serum in down syndrome: identification of novel protein biomarkers

Down syndrome (DS) is the most prevalent chromosomal disorder, accounting for significant morbidity and mortality. Definitive diagnosis requires invasive amniocentesis, and current maternal serum-based testing requires a false-positive rate of about 5% to detect 85% of affected pregnancies. We have performed a comprehensive proteomic analysis to identify potential serum biomarkers to detect DS. First- and second-trimester maternal serum samples of DS and gestational age-matched controls were analyzed using multiple, complementary proteomic approaches, including fluorescence 2-dimensional gel electrophoresis (2D-DIGE), 2-dimensional liquid chromatography-chromatofocusing (2D-CF), multidimensional protein identification technology (MudPIT; LC/LC-MS/MS), and MALDI-TOF-MS peptide profiling. In total, 28 and 26 proteins were differentially present in first- and second-trimester samples, respectively. Of these, 19 were specific for the first trimester and 16 for the second trimester, and 10 were differentially present in both trimesters. Analysis of MALDI-TOF-MS peptide profiles with pattern-recognition software also discriminated between DS and controls in both trimesters, with an average recognition capability approaching 96%. A majority of the biomarkers identified are serum glycoproteins that may play a role in cellular differentiation and growth of fetus. Further characterization and quantification of these markers in a larger cohort of subjects may provide the basis for new tests for improved DS screening.     

Cytoplasmic thioredoxin reductase is essential for embryogenesis but dispensable for cardiac development

Two distinct thioredoxin/thioredoxin reductase systems are present in the cytosol and the mitochondria of mammalian cells. Thioredoxins (Txn), the main substrates of thioredoxin reductases (Txnrd), are involved in numerous physiological processes, including cell-cell communication, redox metabolism, proliferation, and apoptosis. To investigate the individual contribution of mitochondrial (Txnrd2) and cytoplasmic (Txnrd1) thioredoxin reductases in vivo, we generated a mouse strain with a conditionally targeted deletion of Txnrd1. We show here that the ubiquitous Cre-mediated inactivation of Txnrd1 leads to early embryonic lethality. Homozygous mutant embryos display severe growth retardation and fail to turn. In accordance with the observed growth impairment in vivo, Txnrd1-deficient embryonic fibroblasts do not proliferate in vitro. In contrast, ex vivo-cultured embryonic Txnrd1-deficient cardiomyocytes are not affected, and mice with a heart-specific inactivation of Txnrd1 develop normally and appear healthy. Our results indicate that Txnrd1 plays an essential role during embryogenesis in most developing tissues except the heart.     

Structural basis of the differential stability and receptor specificity of H-2Db in complex with murine versus human beta2-microglobulin

beta(2)-Microglobulin (beta(2)m) is non-covalently linked to the major histocompatibility complex (MHC) class I heavy chain and interacts with CD8 and Ly49 receptors. Murine MHC class I heavy chains can bind human beta(2)m (hbeta(2)m) and peptide, and such hybrid molecules are often used in structural and functional studies. The replacement of mouse beta(2)m (mbeta(2)m) with hbeta(2)m has several functional consequences for MHC class I complex stability and specificity, but the structural basis for this is presently unknown. To investigate the impact of species-specific beta(2)m subunits on MHC class I conformation, we provide a crystallographic comparison of H-2D(b) in complex with LCMV-derived gp33 peptide and either hbeta(2)m or mbeta(2)m. The conformation of the gp33 peptide is not affected by the beta(2)m species. Comparison of the interface between beta(2)m and the alpha(1)alpha(2) domains of the heavy chain in these two crystal structures reveals a marked increase in both polarity and number of hydrogen bonds between hbeta(2)m and the alpha(1)alpha(2) domains of H-2D(b). We propose that the positioning of two hydrogen bond rich regions at the hbeta(2)m/alpha(1)alpha(2) interface plays a central role in the increased overall stability and peptide exchange capacity in the H-2D(b)/hbeta(2)m complex. These two regions act as bridges, holding and stabilizing the underside of the alpha(1) and alpha(2) helices, enabling a prolonged peptide-receptive conformation of the peptide binding cleft. Furthermore, analysis of H-2D(b) in complex with either mbeta(2)m or hbeta(2)m provides a structural explanation for the differential binding of H-2D(b)/hbeta(2)m to both Ly49A and Ly49C. Our comparative structural study emphasizes the importance of beta(2)m residues at positions 3, 6 and 29 for binding to Ly49A and suggests that sterical hindrance by residue K6 on hbeta(2)m impairs the recognition of Ly49C by H-2D(b)/gp33/hbeta(2)m. Finally, comparison of the two H-2D(b) crystal structures implies that the beta(2)m species may affect the strength of TCR recognition by affecting CD8 binding.