Analysis of creatinine in mouse and rat serum by ion exchange high performance liquid chromatography for in vivo studies of renal function

An ion exchange high performance liquid chromatography method was developed for determining creatinine levels in both mouse and rat serum samples. Separation of creatinine from other serum components was achieved in 10 min using a 100 x 4.1-mm, 10 microm strong cation exchange column following acetonitrile precipitation of serum proteins. Incorporation of a guard cartridge placed in-line prior to the analytical column was employed to prevent interference from compounds used in renal disease animal trials. Creatinine levels in normal and diseased animals were accurately determined in the 0.01-10 mg/dL range, and average recovery of the method was approximately 85% for both mouse and rat serum. Addition of 0.5-1.0% acetic acid to the acetonitrile used for protein precipitation significantly improved creatinine recovery to above 97% in mouse serum. The method was used for routine preclinical diagnosis of rat and mouse model renal function, and for the evaluation of renal disease treatment efficacy.     

Nonstructural proteins of respiratory syncytial virus suppress premature apoptosis by an NF-kappaB-dependent, interferon-independent mechanism and facilitate virus growth

The two nonstructural (NS) proteins NS1 and NS2 of respiratory syncytial virus (RSV) are abundantly expressed in the infected cell but are not packaged in mature progeny virions. We found that both proteins were expressed early in infection, whereas the infected cells underwent apoptosis much later. Coincident with NS protein expression, a number of cellular antiapoptotic factors were expressed or activated at early stages, which included NF-kappaB and phosphorylated forms of protein kinases AKT, phosphoinositide-dependent protein kinase, and glycogen synthase kinase. Using specific short interfering RNAs (siRNAs), we achieved significant knockdown of one or both NS proteins in the infected cell, which resulted in abrogation of the antiapoptotic functions and led to early apoptosis. NS-dependent suppression of apoptosis was observed in Vero cells that are naturally devoid of type I interferons (IFN). The siRNA-based results were confirmed by the use of NS-deleted RSV mutants. Early activation of epidermal growth factor receptor (EGFR) in the RSV-infected cell did not require NS proteins. Premature apoptosis triggered by the loss of NS or by apoptosis-promoting drugs caused a severe reduction of RSV growth. Finally, recombinantly expressed NS1 and NS2, individually and together, reduced apoptosis by tumor necrosis factor alpha, suggesting an intrinsic antiapoptotic property of both. We conclude that the early-expressed nonstructural proteins of RSV boost viral replication by delaying the apoptosis of the infected cell via a novel IFN- and EGFR-independent pathway.     

Glucans from fruit bodies of cultivated mushrooms Pleurotus ostreatus and Pleurotus eryngii: Structure and potential prebiotic activity

Cultivated oyster mushrooms (genus Pleurotus) are interesting as a source of biologically active glucans. Partially, β-glucan from Pleurotus sp. (pleuran) has been used as food supplements due to its immunosuppressive activity. Like other dietary fibre components, oyster mushroom polysaccharides can stimulate the growth of colon microorganisms (probiotics), i.e. act as prebiotics. Specific glucans were isolated from stems of Pleurotus ostreatus and Pleurotus eryngii by subsequent boiling water and alkali extraction. Obtained water soluble (L1), alkali soluble (L2) and insoluble (S) fractions were characterised by various analytical methods. Spectroscopic analysis detected glucans in all the fractions: branched 1,3-1,6-β-d-glucan predominated in L1 and S, while linear 1,3-α-d-glucan in L2. Fractions L1 also contained marked amount of proteins partially in complex with glucans; protein content in L2 was insignificant. Effective deproteinisation of L1 and separation of α- and β-glucans in L2 was achieved by the treatment with phenolic reagent. Small amount of chitin was found in S as a component of cell wall chitin–glucan complex. Potential prebiotic activity of extracts L1 and L2 was testing using nine probiotic strains of Lactobacillus, Bifidobacterium and Enterococcus. These probiotics showed different growth characteristics dependently on used extract and strain specificity due to the presence of structurally diverse compounds. The extracts L1 and L2 can be applied to synbiotic construction only for carefully selected probiotic strains. This exploitation of fruit body extracts extends the use of mushrooms P. ostreatus and P. eryngii for human health.     

Complexes of Neutralizing and Non-Neutralizing Affinity Matured Fabs with a Mimetic of the Internal Trimeric Coiled-Coil of HIV-1 gp41

A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)₃ or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design.     

Restriction of de novo pyrimidine biosynthesis inhibits Th1 cell activation and promotes Th2 cell differentiation

Leflunomide, an inhibitor of de novo pyrimidine biosynthesis, has recently been introduced as a treatment for rheumatoid arthritis in an attempt to ameliorate inflammation by inhibiting lymphocyte activation. Although the immunosuppressive ability of leflunomide has been well described in several experimental animal models, the precise effects of a limited pyrimidine supply on T cell differentiation and effector functions have not been elucidated. We investigated the impact of restricted pyrimidine biosynthesis on the activation and differentiation of CD4 T cells in vivo and in vitro. Decreased activation of memory CD4 T cells in the presence of leflunomide resulted in impaired generation and outgrowth of Th1 effectors without an alteration of Th2 cell activation. Moreover, priming of naive T cells in the presence of leflunomide promoted Th2 differentiation from uncommitted precursors in vitro and enhanced Th2 effector functions in vivo, as indicated by an increase in Ag-specific Th2 cells and in the Th2-dependent Ag-specific Ig responses (IgG1) in immunized mice. The effects of leflunomide on T cell proliferation and differentiation could be antagonized by exogenous UTP, suggesting that they were related to a profound inhibition of de novo pyrimidine biosynthesis. These results indicate that leflunomide might exert its anti-inflammatory activities in the treatment of autoimmune diseases by preventing the generation of proinflammatory Th1 effectors and promoting Th2 cell differentiation. Moreover, the results further suggest that differentiation of CD4 T cells can be regulated at the level of nucleotide biosynthesis.     

The mechanisms of fatty-acid inhibition of electron transport in chloroplasts

Unsaturated fatty acids at concentrations of 1–2 μmol mg^-1 chlorophyll decrease the intensity of long-lived delayed fluorescence and inhibit the Hill reaction in Pisum sativum L. chloroplasts in a pH-dependent and reversible manner. A charged form of the fatty acids is two times more effective than an undissociated form. Fatty acids, anionic and cationic detergents and urea inhibit activity and decrease the temperature of heat inactivation of the water-spilitting system. Sucrose at a concentration of 2.5 M protects chloroplasts against the effects of these compounds. It is concluded that their action can be explained by the denaturation of the water-splitting protein.     

Leiomodin/tropomyosin interactions are isoform specific

Leiomodins are larger homologs of tropomodulin, a tropomyosin-binding, actin-capping protein. There are several leiomodin isoforms, one of them found in smooth muscles (Lmod1) and another one found in cardiac and skeletal muscles (Lmod2). In this work, the tropomyosin-binding abilities of these two isoforms were studied. The tropomyosin-binding sites were localized in the N-terminal regions of Lmod1 and Lmod2. The affinities of the leiomodin fragments containing the tropomyosin-binding sites for tropomyosin peptides containing N-termini of different tropomyosin isoforms, alpha, gamma and delta, were determined and compared using non-denaturing gel-electrophoresis and circular dichroism. It was shown that leiomodin/tropomyosin binding is isoform-specific and differs almost 100-fold for different tropomyosin isoforms.     

Complete genome sequence of Arcanobacterium haemolyticum type strain (11018)

Arcanobacterium haemolyticum (ex MacLean et al. 1946) Collins et al. 1983 is the type species of the genus Arcanobacterium, which belongs to the family Actinomycetaceae. The strain is of interest because it is an obligate parasite of the pharynx of humans and farm animal; occasionally, it causes pharyngeal or skin lesions. It is a Gram-positive, nonmotile and non-sporulating bacterium. The strain described in this study was isolated from infections amongst American soldiers of certain islands of the North and West Pacific. This is the first completed sequence of a member of the genus Arcanobacterium and the ninth type strain genome from the family Actinomycetaceae. The 1,986,154 bp long genome with its 1,821 protein-coding and 64 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of Ilyobacter polytropus type strain (CuHbu1)

Ilyobacter polytropus Stieb and Schink 1984 is the type species of the genus Ilyobacter, which belongs to the fusobacterial family Fusobacteriaceae. The species is of interest because its members are able to ferment quite a number of sugars and organic acids. I. polytropus has a broad versatility in using various fermentation pathways. Also, its members do not degrade poly-β-hydroxybutyrate but only the monomeric 3-hydroxybutyrate. This is the first completed genome sequence of a member of the genus Ilyobacter and the second sequence from the family Fusobacteriaceae. The 3,132,314 bp long genome with its 2,934 protein-coding and 108 RNA genes consists of two chromosomes (2 and 1 Mbp long) and one plasmid, and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.