Chitinase and protease activities in germinating zoospore cysts of a parasitic fungus,Aphanomyces astaci,Oomycetes

In germinating spores of the parasitic fungus, Aphanomyces astaci, chitinase was first demonstrated shortly before the germ-tube began to branch, in contrast to protease which was present in both ungerminated and germinated spores. The time at which chitinase would be required when this fungus penetrates the crayfish cuticle is correlated with that of the in vitro production of chitinase.     

A model for population regulation with density- and frequency-dependent selection

The life-cycle of a species with separate generations is divided into a reproduction phase and a growing-up phase. In the reproduction phase we assume random mating and selection due to genotype differences in fecundity of the parents and viability of the offspring. During the growing-up phase we assume a (deterministic) death process in continuous time with death rates for the genotypes which increase linearly with the genotype population sizes.In the absence of genotype differences the model gives logistic population regulation. With genotype differences the model generalizes the usual separate generations selection patterns. In addition to these we exhibit cases with three polymorphic equilibria or with a stable cycle.     

Splice Cassette II of Na+,HCO3? Cotransporter NBCn1 (slc4a7) Interacts with Calcineurin A: IMPLICATIONS FOR TRANSPORTER ACTIVITY AND INTRACELLULAR pH CONTROL DURING RAT ARTERY CONTRACTIONS*

Activation of Na⁺,HCO₃⁻ cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pH_i) control during artery contraction, but the signaling pathways involved have been unknown. We investigated whether physical and functional interactions between the Na⁺,HCO₃⁻ cotransporter NBCn1 (slc4a7) and the Ca²⁺/calmodulin-activated serine/threonine phosphatase calcineurin exist and play a role for pH_i control in VSMCs. Using a yeast two-hybrid screen, we found that splice cassette II from the N terminus of NBCn1 interacts with calcineurin Aβ. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTVVIH, the interaction was abolished. Native NBCn1 and calcineurin Aβ co-immunoprecipitated from A7r5 rat VSMCs. A peptide (acetyl-DDIPTVVIH-amide), which mimics the putative calcineurin binding motif, inhibited the co-immunoprecipitation whereas a mutated peptide (acetyl-DDIATAVAA-amide) did not. Na⁺,HCO₃⁻ cotransport activity was investigated in VSMCs of mesenteric arteries after an NH₄⁺ prepulse. During depolarization with 50 mm extracellular K⁺ to raise intracellular [Ca²⁺], Na⁺,HCO₃⁻ cotransport activity was inhibited 20–30% by calcineurin inhibitors (FK506 and cyclosporine A). FK506 did not affect Na⁺,HCO₃⁻ cotransport activity in VSMCs when cytosolic [Ca²⁺] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineurin Aβ. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin Aβ and the Na⁺/H⁺ exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin Aβ and cassette II of NBCn1. Intracellular Ca²⁺ activates Na⁺,HCO₃⁻ cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification.     

Effect of vegetation on density thresholds of adult desert locust gregarization from survey data in Mauritania

In the desert locust, Schistocerca gregaria (Forskål) (Orthoptera: Acrididae), the threshold density inducing the gregarization phenomenon has never been determined under natural conditions. The influence of environmental factors on this phenomenon has been studied mostly in controlled environments. Based on data collected during several years by the survey teams of the National Center for Locust Control in Mauritania, we analyzed the influence of locust density, vegetation cover, and vegetation status on the probability of observing gregarious locusts. We assumed that a probability to observe gregarious locusts of 0.5 corresponded to the density threshold of gregarization. The results showed in detail the change in the threshold of gregarization according to the cover and status of the vegetation. Low cover and dry vegetation led to a low density threshold of gregarization probably due to high probability of individuals to touch each other. Dense and green vegetation favored a high threshold of gregarization probably due to a dispersion of the individuals and a low probability of individual encounters. These findings should help the management of locusts and decision making during control operations.     

Site-specific chemical labeling of long RNA molecules

Site-specific labeling of RNA molecules is a valuable tool for studying their structure and function. Here, we describe a new site-specific RNA labeling method, which utilizes a DNA-templated chemical reaction to attach a label at a specific internal nucleotide in an RNA molecule. The method is nonenzymatic and based on the formation of a four-way junction, where a donor strand is chemically coupled to an acceptor strand at a specific position via an activated chemical group. A disulfide bond in the linker is subsequently cleaved under mild conditions leaving a thiol group attached to the acceptor-RNA strand. The site-specific thiol-modified target RNA can then be chemically labeled with an optional group, here demonstrated by coupling of a maleimide-functionalized fluorophore. The method is rapid and allows site specific labeling of both in vitro and in vivo synthesized RNA with a broad range of functional groups.     

Oxomolybdenum(III, IV and V) complexes of thiofunctional ligands

Reaction of [MoO₂(acac)₂] with (S is a thioether, S′ a thiophenolate function) yielded the compound Li₇(thf)₁₇{MoO}₈ · 10thf · hexane, where {MoO}₈ represents one 1, three (2, linked, via the oxo group, to [Li(thf)₃]⁺) and two (3a, linked by two [Li(thf)₂]⁺).A mixed-valent variant of 3, (3b, with an additional[Li(thf)₃]⁺ attached to S′), was also identified. The compounds model features pertinent to oxo-transferases containing the molybdopterin cofactor.     

RNA interactions in the 5' region of the HIV-1 genome

The untranslated leader of the dimeric HIV-1 RNA genome is folded into a complex structure that plays multiple and essential roles in the viral replication cycle. Here, we have investigated secondary and tertiary structural elements within the 5' 744 nucleotides of the HIV-1 genome using a combination of bioinformatics, enzymatic probing, native gel electrophoresis, and UV-crosslinking experiments. We used a recently developed RNA folding algorithm (Pfold) to predict the common secondary structure of an alignment of 20 divergent HIV-1 sequences. Combining this analysis with biochemical data, we present a secondary structure model for the entire 744 nucleotide fragment, which incorporates previously recognized and novel structural elements. In particular, our data provided strong evidence for a long-distance interaction between the region encompassing the AUG Gag initiation codon and an upstream region and we demonstrate that this feature is highly conserved in distantly related human and animal retroviruses. To obtain information about tertiary interactions we applied an intramolecular UV-crosslinking strategy and identified a novel tertiary interaction within the PBS hairpin structure.     

The α2 isoform of the Na,K-pump is important for intercellular communication, agonist-induced contraction, and EDHF-like response in rat mesenteric arteries

The specific role of different isoforms of the Na,K-pump in the vascular wall is still under debate. We have previously suggested that the α(2) isoform of the Na,K-pump (α(2)), Na(+), Ca(2+)-exchange (NCX), and connexin43 form a regulatory microdomain in smooth muscle cells (SMCs), which controls intercellular communication and contractile properties of the vascular wall. We have tested this hypothesis by downregulating α(2) in cultured SMCs and in small arteries with siRNA in vivo. Intercellular communication was assessed by using membrane capacitance measurements. Arteries transfected in vivo were tested for isometric and isobaric force development in vitro; [Ca(2+)](i) was measured simultaneously. Cultured rat SMCs were well-coupled electrically, but 10 μM ouabain uncoupled them. Downregulation of α(2) reduced electrical coupling between SMCs and made them insensitive to ouabain. Downregulation of α(2) in small arteries was accompanied with significant reduction in NCX expression. Acetylcholine-induced relaxation was not different between the groups, but the endothelium-dependent hyperpolarizing factor-like component of the response was significantly diminished in α(2)-downregulated arteries. Micromolar ouabain reduced in a concentration-dependent manner the amplitude of norepinephrine (NE)-induced vasomotion. Sixty percent of the α(2)-downregulated arteries did not have vasomotion, and vasomotion in the remaining 40% was ouabain insensitive. Although ouabain increased the sensitivity to NE in the control arteries, it had no effect on α(2)-downregulated arteries. In the presence of a low NE concentration the α(2)-downregulated arteries had higher [Ca(2+)](i) and tone. However, the NE EC50 was reduced under isometric conditions, and maximal contraction was reduced under isometric and isobaric conditions. The latter was caused by a reduced Ca(2+)-sensitivity. The α(2)-downregulated arteries also had reduced contraction to vasopressin, whereas the contractile response to high K(+) was not affected. Our results demonstrate the importance of α(2) for intercellular coupling in the vascular wall and its involvement in the regulation of vascular tone.     

Human cornea proteome: identification and quantitation of the proteins of the three main layers including epithelium, stroma, and endothelium

Diseases of the cornea are common and refer to conditions like infections, injuries and genetic defects. Morphologically, many corneal diseases affect only certain layers of the cornea and separate analysis of the individual layers is therefore of interest to explore the basic molecular mechanisms involved in corneal health and disease. In this study, the three main layers including, the epithelium, stroma and endothelium of healthy human corneas were isolated. Prior to analysis by LC-MS/MS the proteins from the different layers were either (i) separated by SDS-PAGE followed by in-gel trypsinization, (ii) in-solution digested without prior protein separation or, (iii) in-solution digested followed by cation exchange chromatography. A total of 3250 unique Swiss-Prot annotated proteins were identified in human corneas, 2737 in the epithelium, 1679 in the stroma, and 880 in the endothelial layer. Of these, 1787 proteins have not previously been identified in the human cornea by mass spectrometry. In total, 771 proteins were quantified, 157 based on in-solution digestion and 770 based on SDS-PAGE separation followed by in-gel digestion of excised gel pieces. Protein analysis showed that many of the identified proteins are plasma proteins involved in defense responses.