Are ruminal bacteria protected against environmental stress by plant antioxidants?

AIMS: To investigate the activity response of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) of the rumen bacterium Streptococcus bovis following exposure to mercury(II) chloride (HgCl(2) in the presence of plant antioxidants. METHODS AND RESULTS: Streptococcus bovis was grown with 0 or 5 microg ml(-1) of HgCl(2) alone or together with antioxidant substances (AOS): seleno-l-methionine (Se), alpha-tocopherol (alpha toc), beta-carotene (beta car), melatonin (mel). The activities of SOD and GHPx were estimated in supernatants of disrupted bacterial cells. A significant decrease in the Strep. bovis SOD activity in the presence of HgCl(2) and tested AOS, except mel, was observed. The GSHPx activity of Strep. bovis was under the same cultivation conditions nonsignificantly changed and a significant decrease in the GSHPx activity was recorded only in the presence of beta car. CONCLUSIONS: The positive effect of Se, alpha toc and beta car on the elimination of environmental stress, evoked by mercury, in ruminal bacterium Strep. bovis in vitro was documented. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential role of plant antioxidants in elimination of the environmental stress of ruminal bacteria evoked by heavy metals is discussed.     

P-Rex1, a PtdIns(3,4,5)P3- and Gbetagamma-regulated guanine-nucleotide exchange factor for Rac

Rac, a member of the Rho family of monomeric GTPases, is an integrator of intracellular signaling in a wide range of cellular processes. We have purified a PtdIns(3,4,5)P3-sensitive activator of Rac from neutrophil cytosol. It is an abundant, 185 kDa guanine-nucleotide exchange factor (GEF), which we cloned and named P-Rex1. The recombinant enzyme has Rac-GEF activity that is directly, substantially, and synergistically activated by PtdIns(3,4,5)P3 and Gbetagammas both in vitro and in vivo. P-Rex1 antisense oligonucleotides reduced endogenous P-Rex1 expression and C5a-stimulated reactive oxygen species formation in a neutrophil-like cell line. P-Rex1 appears to be a coincidence detector in PtdIns(3,4,5)P3 and Gbetagamma signaling pathways that is particularly adapted to function downstream of heterotrimeric G proteins in neutrophils.     

Engineering the quaternary structure of an enzyme: construction and analysis of a monomeric form of malate dehydrogenase from Escherichia coli.

The citric acid cycle enzyme, malate dehydrogenase (MDH), is a dimer of identical subunits. In the crystal structures of 2 prokaryotic and 2 eukaryotic forms, the subunit interface is conformationally homologous. To determine whether or not the quaternary structure of MDH is linked to the catalytic activity, mutant forms of the enzyme from Escherichia coli have been constructed. Utilizing the high-resolution structure of E. coli MDH, the dimer interface was analyzed critically for side chains that were spatially constricted and needed for electrostatic interactions. Two such residues were found, D45 and S226. At their nearest point in the homodimer, they are in different subunits, hydrogen bond across the interface, and do not interact with any catalytic residues. Each residue was mutated to a tyrosine, which should disrupt the interface because of its large size. All mutants were cloned and purified to homogeneity from an mdh- E. coli strain (BHB111). Gel filtration of the mutants show that D45Y and D45Y/S226Y are both monomers, whereas the S226Y mutant remains a dimer. The monomeric D45Y and D45Y/S226Y mutants have 14,000- and 17,500-fold less specific activity, respectively, than the native enzyme. The dimeric S226Y has only 1.4-fold less specific activity. All forms crystallized, indicating they were not random coils. Data have been collected to 2.8 A resolution for the D45Y mutant. The mutant is not isomorphous with the native protein and work is underway to solve the structure by molecular replacement.     

Crystallographic studies of glutamate dehydrogenase. Preliminary crystal data.

Native and pyridoxal phosphate modified rat liver glutamate dehydrogenase crystals have been obtained and used for a preliminary x-ray crystallographic analysis. The space group is P6222 (P6422) having unit cell dimensions a = b = 101 A, c = 724 A and gamma = 120 A. The unit cell contains 36 subunits (six hexameric molecules) of molecular weight 56,000 and there is one half-molecule, i.e. three subunits, in the asymmetric unit. Packing considerations suggest that the glutamate dehydrogenase molecule has the point group symmetry 32 and that each subunit can be represented as a particle with approximate dimensions of 45 x 45 x 60 A.     

Rat MDC family of proteins: Sequence analysis,tissue distribution,and expression in prepubertal and adult rat testis

An increasing number of sequence-related, cysteine-rich membrane proteins containing metalloproteinase-like and disintegrin-like domains (the MDC protein family) have been identified in mammalian tissues. Here, we report the cloning and sequence analysis of cDNAs encoding several rat orthologues of this protein family, some of which are found to be expressed exclusively in the male reproductive tract, others exhibiting a broader tissue distribution. We also examine their expression in prepubertal and adult rat testis, which, in conjunction with the data on tissue distribution, form a necessary prelude to further studies aimed at establishing their individual functions. Mol. Reprod. Dev. 48:159–167, 1997. © 1997 Wiley-Liss, Inc.     

Sequence analysis of a variety of primate fertilin α genes: Evidence for non-functional genes in the gorilla and man

The sperm surface fertilin complex was first described in the guinea pig where it was found as a heterodimer of α and β subunits, both of which were proposed to play a role in sperm-oolemma recognition and plasma membrane fusion during fertilisation. Whilst the β subunit is apparently testis-specific, the finding of low levels of fertilin α in nonreproductive tissues has cast some doubt on a unique role in fertilisation. Moreover, the absence of a functional fertilin α gene in the human would imply that this gene product is not absolutely essential for fertilisation, although it could play a facilitatory role. We now describe the organisation and sequence of the fertilin α genes in a range of primates, including the great apes, and find that the gorilla gene, like that of the human, is non-functional. Mol. Reprod. Dev. 51:92–97, 1998. © 1998 Wiley-Liss, Inc.     

Are all urban green spaces a favourable habitat for pollinator communities? Bees,butterflies and hoverflies in different urban green areas

1. Urban ecosystems create suitable habitats for many plant and animal species, including pollinators. However, heterogenic habitats in city centres and suburban areas have various effects on pollinators due to variations in the composition of vegetation and in landscape management by humans. 2. This study compared the abundance and species richness of three main groups of pollinators – wild bees, butterflies, and hoverflies – in Poznań, western Poland, and in three different types of urban green areas – urban grasslands, urban parks, and green infrastructure in housing estates. 3. The total abundance of pollinators was higher in urban grasslands than in housing estates and urban parks. Species composition of pollinator communities differed between the three habitat types. 4. The study results showed that species richness and abundance of butterflies varied between habitat types, whereas no such differences were found in the case of wild bees and hoverflies. Cover of green area, vegetation structure, and plant height were important for the pollinator community; however, these variables had different effects depending on habitat type. 5. These findings revealed that not all urban green areas are equally valuable in terms of local biodiversity. High‐quality urban habitats such as urban grasslands are capable of supporting rich and abundant populations of pollinators. Therefore, it is important to protect high‐value urban green areas and simultaneously strive to improve intensively managed urban habitats through effective planning and new management practices.     

Lack of support for the association between GAD2 polymorphisms and severe human obesity

The demonstration of association between common genetic variants and chronic human diseases such as obesity could have profound implications for the prediction, prevention, and treatment of these conditions. Unequivocal proof of such an association, however, requires independent replication of initial positive findings. Recently, three (-243 A>G, +61450 C>A, and +83897 T>A) single nucleotide polymorphisms (SNPs) within glutamate decarboxylase 2 (GAD2) were found to be associated with class III obesity (body mass index > 40 kg/m2). The association was observed among 188 families (612 individuals) segregating the condition, and a case-control study of 575 cases and 646 lean controls. Functional data supporting a pathophysiological role for one of the SNPs (-243 A>G) were also presented. The gene GAD2 encodes the 65-kDa subunit of glutamic acid decarboxylase-GAD65. In the present study, we attempted to replicate this association in larger groups of individuals, and to extend the functional studies of the -243 A>G SNP. Among 2,359 individuals comprising 693 German nuclear families with severe, early-onset obesity, we found no evidence for a relationship between the three GAD2 SNPs and obesity, whether SNPs were studied individually or as haplotypes. In two independent case-control studies (a total of 680 class III obesity cases and 1,186 lean controls), there was no significant relationship between the -243 A>G SNP and obesity (OR = 0.99, 95% CI 0.83-1.18, p = 0.89) in the pooled sample. These negative findings were recapitulated in a meta-analysis, incorporating all published data for the association between the -243G allele and class III obesity, which yielded an OR of 1.11 (95% CI 0.90-1.36, p = 0.28) in a total sample of 1,252 class III obese cases and 1,800 lean controls. Moreover, analysis of common haplotypes encompassing the GAD2 locus revealed no association with severe obesity in families with the condition. We also obtained functional data for the -243 A>G SNP that does not support a pathophysiological role for this variant in obesity. Potential confounding variables in association studies involving common variants and complex diseases (low power to detect modest genetic effects, overinterpretation of marginal data, population stratification, and biological plausibility) are also discussed in the context of GAD2 and severe obesity.     

Membrane thinning due to antimicrobial peptide binding: an atomic force microscopy study of MSI-78 in lipid bilayers

The interaction of an antimicrobial peptide, MSI-78, with phospholipid bilayers has been investigated using atomic force microscopy, circular dichroism, and nuclear magnetic resonance (NMR). Binding of amphipathic peptide helices with their helical axis parallel to the membrane surface leads to membrane thinning. Atomic force microscopy of supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers in the presence of MSI-78 provides images of the membrane thinning process at a high spatial resolution. This data reveals that the membrane thickness is not reduced uniformly over the entire bilayer area. Instead, peptide binding leads to the formation of distinct domains where the bilayer thickness is reduced by 1.1 +/- 0.2 nm. The data is interpreted using a previously published geometric model for the structure of the peptide-lipid domains. In this model, the peptides reside at the hydrophilic-hydrophobic boundary in the lipid headgroup region, which leads to an increased distance between lipid headgroups. This picture is consistent with concentration-dependent 31P and 2H NMR spectra of MSI-78 in mechanically aligned DMPC bilayers. Furthermore, 2H NMR experiments on DMPC-d54 multilamellar vesicles indicate that the acyl chains of DMPC are highly disordered in the presence of the peptide as is to be expected for the proposed structure of the peptide-lipid assembly.