Temperature- and hydration-dependent protein dynamics in photosystem II of green plants studied by quasielastic neutron scattering

Protein dynamics in hydrated and vacuum-dried photosystem II (PS II) membrane fragments from spinach has been investigated by quasielastic neutron scattering (QENS) in the temperature range between 5 and 300 K. Three distinct temperature ranges can be clearly distinguished by active type(s) of protein dynamics: (A) At low temperatures (T < 120 K), the protein dynamics of both dry and hydrated PS II is characterized by harmonic vibrational motions. (B) In the intermediate temperature range (120 < T < 240 K), the total mean square displacement total slightly deviates from the predicted linear behavior. The QENS data indicate that this deviation, which is virtually independent of the extent of hydration, is due to a partial onset of diffusive protein motions. (C) At temperatures above 240 K, the protein flexibility drastically changes because of the onset of diffusive (large-amplitude) protein motions. This dynamical transition is clearly hydration-dependent since it is strongly suppressed in dry PS II. The thermally activated onset of protein flexibility as monitored by QENS is found to be strictly correlated with the temperature-dependent increase of the electron transport efficiency from Q(A)(-) to QB (Garbers et al. (1998) Biochemistry 37, 11399-11404). Analogously, the freezing of protein mobility by dehydration in dry PS II appears to be responsible for the blockage of Q(A)(-) reoxidation by Q(B) at hydration values lower than 45% r.h. (Kaminskaya et al. (2003) Biochemistry 42, 8119-8132). Similar effects were observed for reactions of the water-oxidizing complex as outlined in the Discussion section.     

Intracellular expression of the proliferative marker Ki-67 and viral proteins (NS3, NS5A and C) in chronic, long lasting hepatitis C virus (HCV) infection

Hepatitis C virus (HCV) continues to represent the main causative agent of the hepatitis, which leads to chronic transformation of the process in 60-80% individuals. It remains unclear how far cellular expression of HCV proteins in vivo may represent an index of progression of the disease and of proliferative activity in the liver in chronic hepatitis C. Aim of the studies included detection and subcellular localization of three HCV proteins (NS3, NS5A and C) in liver biopsies from adults (n=19) with chronic, long lasting hepatitis C as related to hepatocyte proliferative activity. The immunocytochemical ABC (avidin biotin-peroxidase complex) technique was applied, alone or associated with the ImmunoMax technique. Results of the immunocytochemical tests were compared to histological alterations in liver biopsies, proliferation index and with selected clinical data. A significantly higher expression of NS3 protein was noted, as compared to expressions of NS5A and C proteins. In all the patients, cytoplasmic localization of all proteins dominated over nuclear localization (p0.05). At the level of electron microscopy, protein localization in endoplasmic reticulum (ER) membranes, mitochondria, perinuclear region and/or in hepatocyte cell nucleus was observed. No direct relationships could be demonstrated between expressions of HCV proteins and of Ki-67 antigen. No correlations could also be demonstrated between cellular expression of any HCV protein on one hand and grading or staging, alanine transaminase (ALT), serum level of HCV RNA or alpha-fetoprotein (AFP) on the other. However, positive correlations were disclosed between proliferative activity of hepatocytes on one hand and patient's age, grading and staging on the other. Advanced hepatic fibrosis correlated also with serum levels of AFP. The studies were supplemented with data on subcellular localization of HCV proteins. Moreover, they indicated that in HCV infection grading and staging, proliferative activity of hepatocytes and serum AFP level represent more valuable indices of the disease progress than those provided by cellular expression of three potentially oncogenic HCV proteins in vivo.     

Effect of chronic Sildenafil treatment on the prostate of C57Bl/6 mice

Sildenafil is a potent and selective inhibitor of phosphodiesterase-5 (PDE5) and is considered first-line therapy for erectile dysfunction. Nowadays, Sildenafil is used extensively throughout the world on patients with pulmonary hypertension. However, few studies have evaluated the possible side effects of chronic Sildenafil treatment on the male reproductive system, specifically in the prostate. In the present study, it was demonstrated via morphological and ultrastructural analysis that chronic treatment with Sildenafil induced an enhancement of the glandular activity of the prostate. In addition, mice treated with Sildenafil showed a significant increase in testosterone serum levels. However, no statistically significant differences were observed in nitric oxide serum levels, or in sGC, eNOS, PSA and TGF-β prostatic expression. In conclusion, the present study suggests that chronic use of Sildenafil does not cause evident prostatic damage, and therefore, can be used pharmacologically to treat a variety of disorders.     

The role of polymorphisms of genes CXCL12/CXCR4 and MIF in the risk development IBD the Polish population

Inflammatory bowel disease (IBD) are characterized recurrent inflammation of gastrointestinal tract. The etiology and pathogenesis this disease is currently unclear, but it has become evident that immune and genetic factors are involved in this process. The aim of this study was to determine whether gene polymorphisms: MIF-173 G/C; CXCL12-801 G/A and CXCR4 C/T exon 2 position of rs2228014 is associated with susceptibility to IBD. A total of 286 patients were examined with IBD, including 152 patients with ulcerative colitis and 134 with Crohn’s disease (CD) and 220 healthy subjects were recruited from the Polish population. Genotyping for polymorphisms in CXCL12/CXCR4 and MIF was performed by RFLP-PCR. Statistical significance was found for polymorphisms CXCR4, a receptor gene for CXCL12 genotypes and alleles in CD and for genotype C/T and T allele in ulcerative colitis with respect to control. This confirms the effect of CXCL12 gene. The interplay between CXCL12 and its receptor CXCR4 affects homeostasis and inflammation in the intestinal mucosa. Three-gene analysis in CD confirmed the association of genotype GGGGCT. Statistical analysis of clinical data of patients with ulcerative colitis showed significant differences in the distribution of genotype C/T and T allele for CXCR4 in the left-side colitis. Having CXCR4/CXCL12 chemokine axis polymorphisms may predispose to the development of IBD. Activation can also be their defensive reaction to the long-lasting inflammation.     

The RNase H-like superfamily: new members,comparative structural analysis and evolutionary classification

Ribonuclease H-like (RNHL) superfamily, also called the retroviral integrase superfamily, groups together numerous enzymes involved in nucleic acid metabolism and implicated in many biological processes, including replication, homologous recombination, DNA repair, transposition and RNA interference. The RNHL superfamily proteins show extensive divergence of sequences and structures. We conducted database searches to identify members of the RNHL superfamily (including those previously unknown), yielding >60 000 unique domain sequences. Our analysis led to the identification of new RNHL superfamily members, such as RRXRR (PF14239), DUF460 (PF04312, COG2433), DUF3010 (PF11215), DUF429 (PF04250 and COG2410, COG4328, COG4923), DUF1092 (PF06485), COG5558, OrfB_IS605 (PF01385, COG0675) and Peptidase_A17 (PF05380). Based on the clustering analysis we grouped all identified RNHL domain sequences into 152 families. Phylogenetic studies revealed relationships between these families, and suggested a possible history of the evolution of RNHL fold and its active site. Our results revealed clear division of the RNHL superfamily into exonucleases and endonucleases. Structural analyses of features characteristic for particular groups revealed a correlation between the orientation of the C-terminal helix with the exonuclease/endonuclease function and the architecture of the active site. Our analysis provides a comprehensive picture of sequence-structure-function relationships in the RNHL superfamily that may guide functional studies of the previously uncharacterized protein families.     

Is the Poly (L- Lactide- Co– Caprolactone) Nanofibrous Membrane Suitable for Urinary Bladder Regeneration?

The purpose of this study was to compare: a new five-layered poly (L–lactide–co–caprolactone) (PLC) membrane and small intestinal submucosa (SIS) as a control in rat urinary bladder wall regeneration. The five-layered poly (L–lactide–co–caprolactone) membrane was prepared by an electrospinning process. Adipose tissue was harvested from five 8-week old male Wistar rats. Adipose derived stem cells (ADSCs) were seeded in a density of 3×10⁶ cells/cm² onto PLC membrane and SIS scaffolds, and cultured for 5-7 days in the stem cell culture medium. Twenty male Wistar rats were randomly divided into five equal groups. Augmentation cystoplasty was performed in a previously created dome defect. Groups: (I) PLC+ 3×10⁶ADSCs; (II) SIS+ 3×10⁶ADSCs; (III) PLC; (IV) SIS; (V) control. Cystography was performed after three months. The reconstructed urinary bladders were evaluated in H&E and Masson''s trichrome staining. Regeneration of all components of the normal urinary bladder wall was observed in bladders augmented with cell-seeded SIS matrices. The urinary bladders augmented with SIS matrices without cells showed fibrosis and graft contraction. Bladder augmentation with the PLC membrane led to numerous undesirable events including: bladder wall perforation, fistula or diverticula formation, and incorporation of the reconstructed wall into the bladder lumen. The new five-layered poly (L–lactide–co–caprolactone) membrane possesses poorer potential for regenerating the urinary bladder wall compared with SIS scaffold.     

Structural and Functional Characterization of DUF1471 Domains of Salmonella Proteins SrfN,YdgH/SssB,and YahO

Bacterial species in the Enterobacteriaceae typically contain multiple paralogues of a small domain of unknown function (DUF1471) from a family of conserved proteins also known as YhcN or BhsA/McbA. Proteins containing DUF1471 may have a single or three copies of this domain. Representatives of this family have been demonstrated to play roles in several cellular processes including stress response, biofilm formation, and pathogenesis. We have conducted NMR and X-ray crystallographic studies of four DUF1471 domains from Salmonella representing three different paralogous DUF1471 subfamilies: SrfN, YahO, and SssB/YdgH (two of its three DUF1471 domains: the N-terminal domain I (residues 21–91), and the C-terminal domain III (residues 244–314)). Notably, SrfN has been shown to have a role in intracellular infection by Salmonella Typhimurium. These domains share less than 35% pairwise sequence identity. Structures of all four domains show a mixed α+β fold that is most similar to that of bacterial lipoprotein RcsF. However, all four DUF1471 sequences lack the redox sensitive cysteine residues essential for RcsF activity in a phospho-relay pathway, suggesting that DUF1471 domains perform a different function(s). SrfN forms a dimer in contrast to YahO and SssB domains I and III, which are monomers in solution. A putative binding site for oxyanions such as phosphate and sulfate was identified in SrfN, and an interaction between the SrfN dimer and sulfated polysaccharides was demonstrated, suggesting a direct role for this DUF1471 domain at the host-pathogen interface.     

Conquerors or exiles? Impact of interference competition among invasive Ponto-Caspian gammarideans on their dispersal rates

Ponto-Caspian gammarids have invaded European waters, affecting local communities by predation and competition. Their ranges and dispersal rates vary across Europe, which may result from their interspecific interactions, accelerating or reducing migrations. We checked this hypothesis by testing interference competition among co-occurring invaders: Dikerogammarus villosus, D. haemobaphes and Pontogammarus robustoides. We used 140-cm long tanks (gravel substratum), divided into seven compartments. We introduced 25 “residents” into the outermost compartment, separated with a barrier. After 1 h, we introduced 25 “intruders”. After the next 1 h, we removed the barrier and the gammarids dispersed in the tank. After 4 or 20 h, we counted the gammarids in the compartments. We tested all pairwise species combinations and single-species controls. Dikerogammarus villosus displaced other species (P. robustoides only after 4 h) and reduced its own motility after 20 h in their presence. Pontogammarus robustoides stimulated the short-time migrations of D. villosus intruders and of D. haemobaphes. As P. robustoides migrated spontaneously much more than Dikerogammarus spp., its impact decreased after longer time. Dikerogammarus haemobaphes stimulated the short-time movement of P. robustoides intruders but reduced the long-time relocation of this species. In general, gammarid dispersal increased in the presence of stronger competitors (D. villosus and P. robustoides, especially residents) and decreased in response to weaker competitors (D. haemobaphes). Thus, competitive interactions may affect dispersal of invasive gammarids and contribute to the fastest spread of the weakest competitor, D. haemobaphes observed in the field, whereas the strongest species, D. villosus was the latest newcomer in many novel areas.     

Structure-activity relationships for the linker in a series of pyridinyl-alkynes that are antagonists of the metabotropic glutamate receptor 5 (mGluR5)

Studies of structure-activity relationships for the linker in a new series of metabotropic glutamate receptor 5 antagonists are presented together with in vitro and in vivo pharmacokinetic data.     

The phosphoramidate ProTide approach greatly enhances the activity of β-2′-C-methylguanosine against hepatitis C virus

β-2′-C-Methyl purines (1, 2) are known inhibitors of hepatitis C virus (HCV). We herein report the synthesis, biological and enzymatic evaluation of their 5′-phosphoramidate ProTides. Described herein are seven l-alanine phosphoramidate derivatives with variations to the amino acid ester. The 1-naphthyl phosphoramidate of β-2′-methylguanosine containing the benzyl ester (20) was the most active at 0.12 μM, an 84-fold of increase in activity compared to the parent nucleoside (2) with no increase of cytotoxicity. The carboxypeptidase mediated hydrolysis of several ProTides showed a predictive correlation with their activity versus HCV in replicon.