4TM-TRPM8 channels are new gatekeepers of the ER-mitochondria Ca2+ transfer

Calcium (Ca²⁺) release from the endoplasmic reticulum plays an important role in many cell-fate defining cellular processes. Traditionally, this Ca²⁺ release was associated with the ER Ca²⁺ release channels, inositol 1,4,5?triphosphate receptor (IP₃R) and ryanodine receptor (RyR). Lately, however, other calcium conductances have been found to be intracellularly localized and to participate in cell fate regulation. Nonetheless, molecular identity and functional properties of the ER Ca²⁺ release mechanisms associated with multiple diseases, e.g. prostate cancer, remain unknown. Here we identify a new family of transient receptor potential melastatine 8 (TRPM8) channel isoforms as functional ER Ca²⁺ release channels expressed in mitochondria-associated ER membranes (MAMs). These TRPM8 isoforms exhibit an unconventional structure with 4 transmembrane domains (TMs) instead of 6 TMs characteristic of the TRP channel archetype. We show that these 4TM-TRPM8 isoforms form functional channels in the ER and participate in regulation of the steady-state Ca²⁺ concentration ([Ca²⁺]) in mitochondria and the ER. Thus, our study identifies 4TM-TRPM8 isoforms as ER Ca²⁺ release mechanism distinct from classical Ca²⁺ release channels.     

Aberrant glycosylation of IgA from patients with IgA nephropathy

Despite the prominent role of IgA, particularly IgA₁, in the pathogenesis of IgA nephropathy (IgAN), the precise role of this molecule in the process remains unclear. Four biotin-conjugated lectins in sandwich-type enzyme-linked immunosorbent assays were devised to determine the glycosylation profiles of total IgA and its subclasses. We took advantage of differential binding properties of these lectins to sugar residues to dissect the oligosaccharide chainsO-linked to the hinge and thoseN-linked to the Fc region of total IgA and IgA subclasses in 47 patients with IgAN and an equal number of controls. The proportion of sialylated IgA₁ was higher in patients compared with controls (p<0.02), whereas IgA₂ in patients appeared less well sialylated. A reduction of galactose in pathological IgA as detected by RCA-I became significant after treatment of the molecule with neuraminidase (p<0.01). Defective galactosylation was also observed for patient IgA₁ when it was probed with ECL, a lectin that has a specificity for Gal 1,4N-acetylglucosamine groupings onN-linked oligosaccharides. The RCA and ECL results, therefore, suggest that increased sialylation on the IgA₁ is onO-linked oligosaccharides in the hinge region. This was partly confirmed by a small increase in the binding of PNA to IgA₁ from the patient group. This lectin binds preferentially to Gal 1,3N-acetylgalactosamine groups that are found onO-linked oligosaccharides.     

Food web modelling on the structure and functioning of a Mediterranean lentic system

A number of modelling results suggested thermocline shifts as a consequence of global climate change in stratifying lakes. Abundance and composition of the phytoplankton assemblage is strongly affected by the stratification patterns, and therefore, change in the thermocline position might have a substantial effect on this community or even on the whole lake ecosystem. In this study, thermocline depths in large mesocosms installed in Lake Stechlin (Germany) were deepened by 2 meters and phytoplankton changes were analysed by comparing changes to untreated mesocosms. Higher amounts of SRP were registered in the hypolimnion of treatment mesocosms than in the controls, and there were no differences in the epilimnion. Small but significant changes were observed on the phytoplankton community composition related to the effect of deepening the thermocline; however, it was weaker than the yearly successional changes. The most remarkable differences were caused by Planktothrix rubescens and by chlorophytes. P. rubescens became strongly dominant at the end of the experiment in the mesocosms, and in the open lake as well. The results of the experiment cannot clearly support the proliferation of cyanobacteria in general; however, the deepened thermocline can modify the behaviour of some species, as was observed in case of P. rubescens.     

Genomic organization of hsp90 gene family in Arabidopsis

We have isolated six members of the hsp90 gene family from Arabidopsis thaliana. Three genes designated hsp81.2, 81.3 and 81.4 are clustered within a 15 kb genomic region while two of these are 1.5 kb apart in a head-to-head orientation. The deduced amino acid sequence shows that the members can be divided into two types. The hsp81.1, 81.2, 81.3 and 81.4 genes comprise the cytosolic hsp90 type having few introns. However, the hsp88.1 and 89.1 genes comprising the organelle type are composed of 18 or 19 introns. Sequence comparison showed there is high homology among the cytosolic members while there is less homology among the organelle members. The expression of the hsp90 genes and mRNA accumulation in plants and calli is very low at control temperatures and is strongly induced by heat-shock. Arsenite stress strongly stimulates the expression of this gene family.     

Vanilloid receptor agonists and antagonists are mitochondrial inhibitors: how vanilloids cause non-vanilloid receptor mediated cell death

Time-lapse photomicroscopy of human H460 lung cancer cells demonstrated of the transient receptor potential V1 (TRPV1) channel agonists, (E)-capsaicin and resiniferatoxin, and the TRPV1 antagonists, capsazepine, and SB366791, were able to bring about morphological changes characteristic of apoptosis and/or necrosis. Immunoblot analysis identified immunoreactivity for the transient receptor potential V1 (TRPV1) channel in rat brain samples, but not in rat heart mitochondria or in H460 cells. In isolated rat heart mitochondria, all four ligands caused concentration-dependent decreases in oxygen consumption and mitochondrial membrane potential. (E)-Capsaicin and capsazepine evoked concentration-dependent increases and decreases, respectively, in mitochondrial hydrogen peroxide production, whilst resiniferatoxin and SB366791 were without significant effect. These data support the hypothesis that (E)-capsaicin, resiniferatoxin, capsazepine, and SB366791 are all mitochondrial inhibitors, able to activate apoptosis and/or necrosis via non-receptor mediated mechanisms, and also support the use of TRPV1 ligands as anti-cancer agents.     

Factors Influencing Non-<Emphasis Type="Italic">albicans</Emphasis> Candidemia: A Case–Case–Control Study

The study identified factors predisposing to non-albicans candidemia with special interest to prior antimicrobial treatment. A retrospective, case–case–control study was performed at the University Hospital of Heraklion, Greece, from November 2007 through September 2011 including adult patients. The study had three groups. The first included 58 patients with non-albicans candidemia, the second 48 with C. albicans candidemia, while the third (control) 104 without candidemia. Each of the two candidemia groups was compared with the control using multivariate logistic regression model. The mean (SD) age of the non-albicans, the albicans and the control patients was 67 (12), 67 (18) and 59 (19) years, respectively. The most common non-albicans Candida spp. isolated were C. parapsilosis in 19 patients (33%), C. glabrata in 17 (29%) and C. tropicalis in 15 (26%). Independent risk factors for non-albicans candidemia were prior treatment with quinolones (p < 0.001), b-lactam-b-lactamase inhibitors (p = 0.011) and presence of central venous catheter (p = 0.05), while for C. albicans candidemia were prior treatment with quinolones (p < 0.001), carbapenems (p = 0.003) along with cardiac disease (p < 0.001). Neither duration of hospitalization nor in-hospital mortality [41% for the non-albicans vs 29% for C. albicans group (p = 0.192)] was significantly different between the two candidemia groups. The study reveals the role of antimicrobial exposure as a risk factor for candidemia caused by different species. Prior treatment with b-lactam-b-lactamase inhibitors was associated with non-albicans, while with carbapenems with C. albicans candidemia. Prior use of quinolones was associated with candidemia in general.     

Development of a Canine Care and Welfare Knowledge Scale: Implications for Welfare and Preventing Dogfighting

The objective of this study was to develop a Canine Care and Welfare Knowledge (CCWK) Scale for use in educational intervention development and evaluation and validate the instrument. The study population was 504 children, aged 11 to 19 years old, from Detroit, MI. In this cross-sectional study, exploratory factor analysis (EFA) and confirmatory factor analysis (CFA) were used for scale development. The EFA and CFA of the CCWK Scale revealed a 2nd-order model with 6 factors to be a good fit of the data (chi-square [df = 269] = 433, p < .05, Comparative Fit Index = .94, Tucker-Lewis Index = .93, root mean square error of approximation = .05) with a Cronbach’s alpha of .78. The scale is valid and reliable to assess the study population’s CCWK.     

Structural and Functional Studies on the Overproduced L11 Protein from Thermus thermophilus

The L11 ribosomal protein from Thermus thermophilus (TthL11) has been overproduced and purified to homogeneity using a two-step purification protocol. The overproduced protein carries a similar methylation pattern at Lys-3 as does its homolog from Escherichia coli. Chymotrypsin digested only a small part of the TthL11 protein and did not cleave TthL11 into two peptides, as in the case of EcoL11, but produced only a single N-terminal peptide. Tryptic digestion of TthL11 also produced an N-terminal peptide, in contrast to the C-terminal peptide obtained with L11 from Bacillus stearothermophilus. The recombinant protein forms a specific complex with a 55-nt 23S rRNA fragment known to interact with members of the L11 family from several organisms. Cooperative binding of TthL11 and thiostrepton to 23S rRNA leads to an increased protection of TthL11 from tryptic digestion. The similar structural and biochemical properties as well as the significant homology between L11 from E. coli and B. stearothermophilus with the corresponding protein from Thermus thermophilus indicate an evolutionarily conserved protein important for ribosome function.