Role of calcium channels in cadmium-induced disruption of cortisol synthesis in rainbow trout (Oncorhynchus mykiss)

The mechanisms of toxicity of cadmium (Cd(2+)) in adrenal steroidogenesis were investigated in vitro in adrenocortical cells of rainbow trout (Oncorhynchus mykiss). Toxicity of Cd(2+) was increased in absence of extracellular Ca(2+), but was prevented in Ca(2+)-supplemented medium. Pretreatment of cells with BAY K8644 (BAY), an agonist of voltage-dependent calcium channels, increased the Cd(2+)-mediated inhibition of ACTH-stimulated secretion but not pregnenolone (PREG)-stimulated secretion. Nicardipine, an antagonist of voltage-dependent calcium channels, also increased the inhibition of adrenocorticotropic hormone (ACTH)-stimulated secretion by Cd(2+). These results suggest that opening of voltage-dependent calcium channels with BAY may allow Cd(2+) entry at the same time as calcium, thus increasing toxicity of Cd(2+), however voltage-dependent calcium channels may not be the only way of entry into adrenocortical cells. The influx of Cd(2+), measured as intracellular Cd(2+) using Fluo-3 in PREG-stimulated adrenocortical cells, was significantly enhanced by the stimulation. These results suggest that the deleterious effect of Cd(2+) on cortisol steroidogenesis may be enhanced when the endocrine stress response is triggered.     

Complete genome of the mutualistic, N2-fixing grass endophyte Azoarcus sp. strain BH72

Azoarcus sp. strain BH72, a mutualistic endophyte of rice and other grasses, is of agrobiotechnological interest because it supplies biologically fixed nitrogen to its host and colonizes plants in remarkably high numbers without eliciting disease symptoms. The complete genome sequence is 4,376,040-bp long and contains 3,992 predicted protein-coding sequences. Genome comparison with the Azoarcus-related soil bacterium strain EbN1 revealed a surprisingly low degree of synteny. Coding sequences involved in the synthesis of surface components potentially important for plant-microbe interactions were more closely related to those of plant-associated bacteria. Strain BH72 appears to be 'disarmed' compared to plant pathogens, having only a few enzymes that degrade plant cell walls; it lacks type III and IV secretion systems, related toxins and an N-acyl homoserine lactones-based communication system. The genome contains remarkably few mobile elements, indicating a low rate of recent gene transfer that is presumably due to adaptation to a stable, low-stress microenvironment.     

Crotacetin, a novel snake venom C-type lectin homolog of convulxin, exhibits an unpredictable antimicrobial activity

Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins that are capable of interfering with blood stasis. A very well-studied svC-type lectin is the heterodimeric toxin, convulxin (CVX), from the venom of South American rattlesnake Crotalus durissus terrificus. CVX is able to activate platelets and induce their aggregation by acting via p62/GPVI collagen receptor. By using polymerase chain reaction homology screening, we have cloned several cDNA precursors of CVX subunit homologs. One of them, named crotacetin (CTC) β-subunit, predicts a polypeptide with a topology very similar to the tridimensional conformations of other subunits of CVX-like snake toxins, as determined by computational analysis. Using gel permeation and reverse-phase high-performance liquid chromatography, CTC was purified from C. durissus venoms. CTC can be isolated from the venom of several C. durissus subspecies, but its quantitative predominance is in the venom of C. durissus cascavella. Functional analysis indicates that CTC induces platelet aggregation, and, importantly, exhibits an antimicrobial activity against Gram-positive and-negative bacteria, comparable with CVX.     

7SL RNA, but not the 54-kd signal recognition particle protein, is an abundant component of both infectious HIV-1 and minimal virus-like particles

The virion incorporation of 7SL, the RNA component of the host signal recognition particle (SRP), has been shown for several simple retroviruses. Data here demonstrate that 7SL is also packaged by HIV-1, in sevenfold molar excess of genomic RNA. Viral determinants of HIV-1 genome and primer tRNA packaging were not required for 7SL incorporation, as virus-like particles with only minimal assembly components efficiently packaged 7SL. The majority of 7SL within cells resides in ribonucleoprotein complexes bound by SRP proteins, and most SRP protein exists in signal recognition particles. However, Western blot comparison of virion and cell samples revealed that there is at least 25-fold less SRP p54 protein per 7SL RNA in HIV-1 particles than in cells. Comparing 7SL:actin mRNA ratios in virions and cells revealed that 7SL RNA appears selectively enriched in virions.     

A moderate threonine deficiency affects gene expression profile,paracellular permeability and glucose absorption capacity in the ileum of piglets

High dietary threonine extraction by the digestive tract suggests that threonine contributes to maintain gut physiology. In the present study, we evaluated the impact of a low (6.5 g of threonine/kg diet; LT group) or a control well-balanced threonine diet (9.3 g of threonine/kg diet; C group) given to piglets for 2 weeks on ileal permeability and Na+-dependant glucose absorption capacity in Ussing chambers. The paracellular permeability was significantly increased in the ileum of LT compared to C piglets (P=.017). The Na+-dependent glucose absorption capacity showed a nonsignificant increase in the LT piglets. In addition, we analysed ileal gene expression profiles in the LT and C groups using porcine multitissue cDNA microarrays. Compared to the C piglets, the expression of 324 genes was significantly modified in the ileum of the LT piglets: 214 genes were overexpressed (145 annotated) and 110 were down-expressed (79 annotated). Among them, some are involved in immune and defense responses, energy metabolism and protein synthesis. Furthermore, microarray analysis highlights changes in the expression of the gene encoding for the sodium/glucose cotransporter (SGLT1) and of genes involved in the regulation of paracellular permeability (ZO-1, cingulin and myosin light chain kinase). In conclusion, our results indicate that a moderate threonine deficiency affects intestinal functionality.     

Choline uptake across the ventricular membrane of neonate rat choroid plexus

The uptake of[³H]choline from thecerebrospinal fluid (CSF) side of the rat neonatal choroid plexus wascharacterized in primary cultures of the choroidal epithelium grown onsolid supports. Cell-to-medium concentration ratios were ~5 at 1 minand as high as 70 at 30 min. Apical choline uptake was facilitated; theK_m was ~50µM. Several organic cations (e.g., hemicholinium-3 and N¹-methylnicotinamide)inhibited uptake. The reduction or removal of externalNa⁺ or the addition of 5 mM LiClhad no effect on uptake. However, increasing externalK⁺ concentration from 3 to 30 mMdepolarized ventricular membrane potential (70 to 15 mV)and reduced uptake to 45% of that for the control. Treatment with 1 mMouabain or 2 mM BaCl₂ reduced uptake 45%, and intracellular acidification reduced uptake to ~90%of that for controls. These data indicate that the uptake of choline from CSF across the ventricular membrane of the neonatal choroidal epithelium is not directly coupled toNa⁺ influx but is sensitive toplasma membrane electrical potential.

     

Mapping the interaction between high molecular mass kininogen and the urokinase plasminogen activator receptor

The urokinase plasminogen activator receptor (uPAR) is a multifunctional, GPI-linked receptor that modulates cell adhesion/migration and fibrinolysis. We mapped the interaction sites between soluble uPAR (suPAR) and high molecular mass kininogen (HK). Binding of biotin-HK to suPAR was inhibited by HK, 56HKa, and 46HKa with an IC50 of 60, 110, and 8 nm, respectively. We identified two suPAR-binding sites, a higher affinity site in the light chain of HK and 46HKa (His477-Gly496) and a lower affinity site within the heavy chain (Cys333-Lys345). HK predominantly bound to suPAR fragments containing domains 2 and 3 (S-D2D3). Binding of HK to domain 1 (S-D1) was also detected, and the addition of S-D1 to S-D2D3 completely inhibited biotin-HK or -46HKa binding to suPAR. Using sequential and overlapping 20-amino acid peptides prepared from suPAR, two regions for HK binding were identified. One on the carboxyl-terminal end of D2 (Leu166-Thr195) blocked HK binding to suPAR and to human umbilical vein endothelial cells (HUVEC). This site overlapped with the urokinase-binding region, and urokinase inhibited the binding of HK to suPAR. A second region on the amino-terminal portion of D3 (Gln215-Asn255) also blocked HK binding to HUVEC. Peptides that blocked HK binding to uPAR also inhibited prekallikrein activation on HUVEC. Therefore, HK interacts with suPAR at several sites. HK binds to uPAR as part of its interaction with its multiprotein receptor complex on HUVEC, and the biological functions that depend upon this binding are modulated by urokinase.     

Paracoccus pantotrophus pseudoazurin is an electron donor to cytochrome c peroxidase

The gene for pseudoazurin was isolated from Paracoccus pantotrophus LMD 52.44 and expressed in a heterologous system with a yield of 54.3 mg of pure protein per liter of culture. The gene and protein were shown to be identical to those from P. pantotrophus LMD 82.5. The extinction coefficient of the protein was re-evaluated and was found to be 3.00 mM(-1) cm(-1) at 590 nm. It was confirmed that the oxidized protein is in a weak monomer/dimer equilibrium that is ionic-strength-dependent. The pseudoazurin was shown to be a highly active electron donor to cytochrome c peroxidase, and activity showed an ionic strength dependence consistent with an electrostatic interaction. The pseudoazurin has a very large dipole moment, the vector of which is positioned at the putative electron-transfer site, His81, and is conserved in this position across a wide range of blue copper proteins. Binding of the peroxidase to pseudoazurin causes perturbation of a set of NMR resonances associated with residues on the His81 face, including a ring of lysine residues. These lysines are associated with acidic residues just back from the rim, the resonances of which are also affected by binding to the peroxidase. We propose that these acidic residues moderate the electrostatic influence of the lysines and so ensure that specific charge interactions do not form across the interface with the peroxidase.