The crystal structure of the reduced, Zn2+-bound form of the B. subtilis Hsp33 chaperone and its implications for the activation mechanism

The bacterial heat shock protein Hsp33 is a redox-regulated chaperone activated by oxidative stress. In response to oxidation, four cysteines within a Zn2+ binding C-terminal domain form two disulfide bonds with concomitant release of the metal. This leads to the formation of the biologically active Hsp33 dimer. The crystal structure of the N-terminal domain of the E. coli protein has been reported, but neither the structure of the Zn2+ binding motif nor the nature of its regulatory interaction with the rest of the protein are known. Here we report the crystal structure of the full-length B. subtilis Hsp33 in the reduced form. The structure of the N-terminal, dimerization domain is similar to that of the E. coli protein, although there is no domain swapping. The Zn2+ binding domain is clearly resolved showing the details of the tetrahedral coordination of Zn2+ by four thiolates. We propose a structure-based activation pathway for Hsp33.     

Modulation of the human preadipocyte mitochondrial activity by beta-carotene

Increased ROS generation by the overload by metabolic substrates mitochondria paralleled by decrease of antioxidant activity are typical events found in metabolic syndrome and diabetes type 2. Metabolites of beta-carotene (BC) such as retinoic acid (RA), as well as low concentration of reactive oxygen species (ROS) modify the mitochondrial bioenergetic function. The aim of the study was to investigate the effect of beta-carotene on mitochondrial activity in human preadipocytes. BC used in concentrations, 10 or 30 μM, decreased mitochondrial membrane potential, inhibited mitochondrial respiration and decreased cellular ATP content. We conclude, that BC, the known antioxidant may decrease oxidative phosphorylation capacity of mitochondria.     

Expression of soluble recombinant TGF-beta type II receptor fused with the Fc portion of human IgG1 (sTbetaRII-Fc) in NS0 cells

We have constructed and expressed recombinant chimeric soluble TGF-beta type II receptor fused with the Fc portion of human IgG1 (sTbetaRII-Fc) in NS0 mouse myeloma cells and isolated cell lines constitutively secreting very high levels of biologically active protein. The GS-NS0 expression system takes advantage of the strong human cytomegalovirus immediate early promoter expression vector and glutamine synthetase as a selectable marker. The recombinant chimeric receptor could be produced in high amounts and efficiently purified by one step chromatography on a protein A column. Biochemical studies revealed that recombinant sTbetaRII-Fc binds native TGF-beta1 and TGF-beta3 isoforms and neutralizes their activity in vitro.     

Ca2+-independent binding and cellular expression profiles question a significant role of calmyrin in transduction of Ca2+-signals to Alzheimer's disease-related presenilin 2 in forebrain

The interaction between the EF-hand Ca(2+)-binding protein calmyrin and presenilin 2 (PS2) has been suggested to play a role in Alzheimer's disease (AD). We now report that calmyrin binds specifically endogenous PS2 and not PS1. However, binding appears to be Ca(2+)-independent and calmyrin does not exhibit a Ca(2+)-dependent translocation to intracellular membranes as demonstrated in a Ca(2+)-myristoyl switch assay. Moreover, calmyrin is only present at very low levels in brain areas associated with the onset of AD. In rat, forebrain calmyrin is localized only in a subset of principal neurons, similarly as in human forebrain. Finally, subcellular fractionation demonstrates only a limited overlap of calmyrin and PS2 at neuronal membranes. We therefore conclude that calmyrin will not contribute significantly as a Ca(2+)-sensor that transduces Ca(2+)-signaling events to PS2 in forebrain.     

Comparative Large-Scale Analysis of Interactions between Several Crop Species and the Effector Repertoires from Multiple Pathovars of Pseudomonas and Ralstonia

Bacterial plant pathogens manipulate their hosts by injection of numerous effector proteins into host cells via type III secretion systems. Recognition of these effectors by the host plant leads to the induction of a defense reaction that often culminates in a hypersensitive response manifested as cell death. Genes encoding effector proteins can be exchanged between different strains of bacteria via horizontal transfer, and often individual strains are capable of infecting multiple hosts. Host plant species express diverse repertoires of resistance proteins that mediate direct or indirect recognition of bacterial effectors. As a result, plants and their bacterial pathogens should be considered as two extensive coevolving groups rather than as individual host species coevolving with single pathovars. To dissect the complexity of this coevolution, we cloned 171 effector-encoding genes from several pathovars of Pseudomonas and Ralstonia. We used Agrobacterium tumefaciens-mediated transient assays to test the ability of each effector to induce a necrotic phenotype on 59 plant genotypes belonging to four plant families, including numerous diverse accessions of lettuce (Lactuca sativa) and tomato (Solanum lycopersicum). Known defense-inducing effectors (avirulence factors) and their homologs commonly induced extensive necrosis in many different plant species. Nonhost species reacted to multiple effector proteins from an individual pathovar more frequently and more intensely than host species. Both homologous and sequence-unrelated effectors could elicit necrosis in a similar spectrum of plants, suggesting common effector targets or targeting of the same pathways in the plant cell.     

N-Glycosylation Regulates Fibroblast Growth Factor Receptor/EGL-15 Activity in Caenorhabditis elegans in Vivo

The regulation of cell function by fibroblast growth factors (FGFs) classically occurs through a dual receptor system of a tyrosine kinase receptor (FGFR) and a heparan sulfate proteoglycan co-receptor. Mutations in some consensus N-glycosylation sites in human FGFR result in skeletal disorders and craniosynostosis syndromes, and biophysical studies in vitro suggest that N-glycosylation of FGFR alters ligand and heparan sulfate binding properties. The evolutionarily conserved FGFR signaling system of Caenorhabditis elegans has been used to assess the role of N-glycosylation in the regulation of FGFR signaling in vivo. The C. elegans FGF receptor, EGL-15, is N-glycosylated in vivo, and genetic substitution of specific consensus N-glycosylation sites leads to defects in the maintenance of fluid homeostasis and differentiation of sex muscles, both of which are phenotypes previously associated with hyperactive EGL-15 signaling. These phenotypes are suppressed by hypoactive mutations in EGL-15 downstream signaling components or activating mutations in the phosphatidylinositol 3-kinase pathway, respectively. The results show that N-glycans negatively regulate FGFR activity in vivo supporting the notion that mutation of N-glycosylation sites in human FGFR may lead to inappropriate activation of the receptor.     

Mutation analysis of mitochondrial 12S rRNA gene in Polish patients with non-syndromic and aminoglycoside-induced hearing loss

Mutations in mitochondrial DNA have been reported as associated with non-syndromic and aminoglycoside-induced hearing loss. In the present study, we have performed mutational screening of entire 12S rRNA gene in 250 unrelated patients with non-syndromic and aminoglycoside-induced hearing loss. Twenty-one different homoplasmic sequence variants were identified, including eight common polymorphisms, one deafness-associated mutation m.1555 A>G and three putatively pathogenic variants: m.669 T>C, m.827 A>G, m.961 delT+C(n)ins. The incidence of m.1555 A>G was estimated for 3.6% (9/250); however, where aminoglycoside exposure was taken as a risk factor, the frequency was 5.5% (7/128). Substitution m.669 T>C was identified only in patients with hearing impairment and episode of aminoglycoside exposure, which may suggest that such additional risk factors must appear to induce clinical phenotype. Moreover, two 12S rRNA sequence variants: m.988 G>A and m.1453 A>G, localized at conserved sites and affected RNA secondary structure, may be new candidates for non-syndromic and aminoglycoside-induced hearing loss associated mutations.     

Time-related changes of motor unit properties in the rat medial gastrocnemius muscle after the spinal cord injury. II. Effects of a spinal cord hemisection

The contractile properties of motor units (MUs) were investigated in the medial gastrocnemius (MG) muscle in rats after the spinal cord hemisection at a low thoracic level. Hemisected animals were divided into 4 groups: 14, 30, 90 and 180 days after injury. Intact rats formed a control group. The mass of the MG muscle did not change significantly after spinal cord hemisection, hind limb locomotor pattern was almost unchanged starting from two weeks after injury, but contractile properties of MUs were however altered. Contraction time (CT) and half-relaxation time (HRT) of MUs were prolonged in all investigated groups of hemisected rats. The twitch-to-tetanus ratio (Tw/Tet) of fast MUs after the spinal cord hemisection increased. For slow MUs Tw/Tet values did not change in the early stage after the injury, but significantly decreased in rats 90 and 180 days after hemisection. As a result of hemisection the fatigue resistance especially of slow and fast resistant MU types was reduced, as well as fatigue index (Fat I) calculated for the whole examined population of MUs decreased progressively with the time. After spinal cord hemisection a reduced number of fast MUs presented the sag at frequencies 30 and 40 Hz, however more of them revealed sag in 20 Hz tetanus in comparison to control group. Due to considerable changes in twitch contraction time and disappearance of sag effect in unfused tetani of some MUs in hemisected animals, the classification of MUs in all groups of rats was based on the 20 Hz tetanus index (20 Hz Tet I) but not on the standard criteria usually applied for MUs classification. MU type differentiations demonstrated some clear changes in MG muscle composition in hemisected animals consisting of an increase in the proportion of slow MUs (likely due to an increased participation of the studied muscle in tonic antigravity activity) together with an increase in the percentage of fast fatigable MUs.     

Angiogenesis in Balb/c mice under beta-carotene supplementation in diet

Angiogenesis is a process of new blood vessel formation from pre-existing ones. The most important steps in angiogenesis include detachment, proliferation, migration, homing and differentiation of vascular wall cells, which are mainly endothelial cells and their progenitors. The study focused on the effect of beta-carotene (BC) supplementation (12,000 mg/kg) in the diet on angiogenesis in Balb/c mice. Female Balb/c mice were fed for 5 weeks with two different diets: with BC or without BC supplementation. After 4 weeks of feeding, Balb/c mice were injected subcutaneously with two matrigel plugs with or without basic fibroblast growth factor (bFGF). Six days later, the animals were killed, and the matrigel plugs were used for immunohistochemical staining with CD31 antibody and for gene expression analysis. Microarray and Real-Time PCR data showed down-regulation of genes involved in proliferation and up-regulation of genes encoding inhibitors of apoptosis, proteins regulating cell adhesion, matrix-degrading enzymes and proteins involved in the VEGF pathway. The results of this study demonstrated that BC proangiogenic activity (with or without bFGF) in vivo seemed to be more significantly associated with cells’ protection from apoptosis and their stimulation of chemotaxis/homing than cell proliferation.