Two uncommon phospholipase D isoenzymes from poppy seedlings (Papaver somniferum L.)

Phospholipase D (PLD) has been detected in seedlings of Papaver somniferum L. cv. Lazúr (Papaveraceae). Purification of the enzyme revealed the existence of two forms of PLD (named as PLD-A and PLD-B). The two enzymes strongly differ in their catalytic properties. The pH optima were found at pH 8.0 for PLD-A and at pH 5.5 for PLD-B. While both enzymes show hydrolytic activity toward phosphatidylcholine (PC) and phosphatidyl-p-nitrophenol (PpNP), PLD-B only was able to catalyze the exchange of choline in PC by glycerol. Both enzymes were activated by Ca(2+) ions with an optimum concentration of 10 mM. In contrast to PLDs from other plants, PLD-B was still more activated by Zn(2+) ions with an optimum concentration of 5 mM. The apparent molecular masses of PLD-A and PLD-B, derived from sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), were estimated to be 116.4 and 114.1 kDa. N-terminal protein sequencing indicated N-terminal blockage in both cases. The isoelectric points were found to be 8.7 for PLD-A and 6.7 for PLD-B. Both enzymes were shown to be N-linked glycoproteins. This paper is the first report on PLD in poppy and indicates some important differences of the two enzyme forms to other PLDs known so far.     

Laminin alpha5 chain is required for intestinal smooth muscle development

Laminins (comprised of alpha, beta, and gamma chains) are heterotrimeric glycoproteins integral to all basement membranes. The function of the laminin alpha5 chain in the developing intestine was defined by analysing laminin alpha5(-/-) mutants and by grafting experiments. We show that laminin alpha5 plays a major role in smooth muscle organisation and differentiation, as excessive folding of intestinal loops and delay in the expression of specific markers are observed in laminin alpha5(-/-) mice. In the subepithelial basement membrane, loss of alpha5 expression was paralleled by ectopic or accelerated deposition of laminin alpha2 and alpha4 chains; this may explain why no obvious defects were observed in the villous form and enterocytic differentiation. This compensation process is attributable to mesenchyme-derived molecules as assessed by chick/mouse alpha5(-/-) grafted associations. Lack of the laminin alpha5 chain was accompanied by a decrease in epithelial alpha3beta1 integrin receptor expression adjacent to the epithelial basement membrane and of Lutheran blood group glycoprotein in the smooth muscle cells, indicating that these receptors are likely mediating interactions with laminin alpha5-containing molecules. Taken together, the data indicate that the laminin alpha5 chain is essential for normal development of the intestinal smooth muscle and point to possible mesenchyme-derived compensation to promote normal intestinal morphogenesis when laminin alpha5 is absent.     

Role for laminin-alpha5 chain LG4 module in epithelial branching morphogenesis

Laminin-alpha5 chain was localized in all epithelial basement membranes (BMs) of mouse submandibular gland (SMG) from the onset of branching morphogenesis and became restricted to BMs of epithelial ducts in the adult. To investigate whether the laminin-alpha5 chain plays a role in branching morphogenesis, a set of cell-adhesive peptides from the C-terminal globular domains (LG1-5) was tested for their effects in SMG organ cultures. One peptide, LVLFLNHGH (A5G77f), which represents a sequence located in the connecting loop between strands E and F of LG4, perturbed branching morphogenesis and resulted in irregularities in the contours of epithelial structures, with formation of deep clefts. The data suggest a role for the laminin-alpha5 LG4 module in the development of the duct system, rather than in the bifurcation of epithelial clusters. The epithelial BM of A5G77f-peptide-treated explants was continuous, which was in contrast to our previous finding of impaired epithelial BM assembly in explants treated with the laminin-alpha1 LG4 module peptide, or with a monoclonal antibody against this domain. A5G77f also perturbed in vitro development of lung and kidney. These results suggest a crucial role for the LG4 module of laminin-alpha5 in epithelial morphogenesis that is distinct from that of the laminin-alpha1 LG4.     

Crystal structure of a statin bound to a class II hydroxymethylglutaryl-CoA reductase

Hydroxymethylglutaryl-CoA (HMG-CoA) reductase is the primary target in the current clinical treatment of hypercholesterolemias with specific inhibitors of the "statin" family. Statins are excellent inhibitors of the class I (human) enzyme but relatively poor inhibitors of the class II enzymes of important bacterial pathogens. To investigate the molecular basis for this difference we determined the x-ray structure of the class II Pseudomonas mevalonii HMG-CoA reductase in complex with the statin drug lovastatin. The structure shows lovastatin bound in the active site and its interactions with residues critically involved in catalysis and substrate binding. Binding of lovastatin also displaces the flap domain of the enzyme, which contains the catalytic residue His-381. Comparison with the structures of statins bound to the human enzyme revealed a similar mode of binding but marked differences in specific interactions that account for the observed differences in affinity. We suggest that these differences might be exploited to develop selective class II inhibitors for use as antibacterial agents against pathogenic microorganisms.     

Mechanism of sulfide-quinone reductase investigated using site-directed mutagenesis and sulfur analysis

Biological sulfide oxidation is a reaction occurring in all three domains of life. One enzyme responsible for this reaction in many bacteria has been identified as sulfide:quinone oxidoreductase (SQR). The enzyme from Rhodobacter capsulatus is a peripherally membrane-bound flavoprotein with a molecular mass of approximately 48 kDa, presumably acting as a homodimer. In this work, SQR from Rb. capsulatus has been modified with an N-terminal His tag and heterologously expressed in and purified from Escherichia coli. Three cysteine residues have been shown to be essential for the reductive half-reaction by site-directed mutagenesis. The catalytic activity has been nearly completely abolished after mutation of each of the cysteines to serine. A decrease in fluorescence on reduction by sulfide as observed for the wild-type enzyme has not been observed for any of the mutated enzymes. Mutation of a conserved valine residue to aspartate within the third flavin-binding domain led to a drastically reduced substrate affinity, for both sulfide and quinone. Two conserved histidine residues have been mutated individually to alanine. Both of the resulting enzymes exhibited a shift in the pH dependence of the SQR reaction. Polysulfide has been identified as a primary reaction product using spectroscopic and chromatographic methods. On the basis of these data, reaction mechanisms for sulfide-dependent reduction and quinone-dependent oxidation of the enzyme and for the formation of polysulfide are proposed.     

Pituitary adenylate cyclase-activating polypeptide stimulates nitric-oxide synthase type I expression and potentiates the cGMP response to gonadotropin-releasing hormone of rat pituitary gonadotrophs

Nitric-oxide synthase type I (NOS I) is expressed primarily in gonadotrophs and in folliculo-stellate cells of the anterior pituitary. In gonadotrophs, the expression and the activity of NOS I are stimulated by gonadotropin-releasing hormone (GnRH) under both experimental and physiological conditions. In the present study, we show that pituitary adenylate cyclase-activating polypeptide (PACAP) is twice as potent as GnRH at increasing NOS I levels in cultured rat anterior pituitary cells. The action of PACAP is detectable after 4-6 h and maximal at 24 h, this effect is mimicked by 8-bromo-cAMP and cholera toxin and suppressed by H89 suggesting a mediation through the cAMP pathway. Surprisingly, NADPH diaphorase staining revealed that these changes occurred in gonadotrophs exclusively although PACAP and cAMP, in contrast to GnRH, have the potential to target several types of pituitary cells including folliculo-stellate cells. There was no measurable alteration in NOS I mRNA levels after cAMP or PACAP induction. PACAP also stimulated cGMP synthesis, which was maximal within 15 min and independent of cAMP, however, only part resulted from NOS I/soluble guanylate cyclase activation implying that in contrast to GnRH, PACAP has a dual mechanism in cGMP production. Interestingly, induction of NOS I by PACAP markedly enhanced the capacity of gonadotrophs to produce cGMP in response to GnRH. The fact that PACAP may act on gonadotrophs to alter NOS I levels, generate cGMP, and potentiate the cGMP response to GnRH, suggests that cGMP could play important cellular functions.     

Accomplishments in HIV prevention science: implications for stemming the epidemic

The past two decades have witnessed substantial advances in the science of preventing HIV infection. Although important issues remain and there is a need for continuing research, arguably the biggest challenge in preventing HIV transmission is the full implementation of existing preventive interventions worldwide.     

RFLP-based analysis of three RbcS subfamilies in diploid and polyploid species of wheat

The RbcS multigene family of hexaploid (bread) wheat, Triticum aestivum (genome BBAADD), which encodes the small subunit of Rubisco, comprises at least 22 genes. Based on their 3′ non-coding sequences, these genes have been classified into four subfamilies (SFs), of which three (SF-2, SF-3 and SF-4) are located on chromosomes of homoeologous group 2 and one (SF-1) on homoeologous group 5. In the present study we hybridized three RbcS subfamily-specific probes (for SF-1, SF-2 and SF-3) to total DNA digested with four restriction enzymes and analyzed the RFLP patterns of these subfamilies in eight diploid species of Aegilops and Triticum, and in two tetraploid and one hexaploid species of wheat (the diploid species are the putative progenitors of the polyploid wheats). The three subfamilies varied in their level of polymorphism, with SF-2 being the most polymorphic in all species. In the diploids, the order of polymorphism was SF-2 > SF-3 > SF-1, and in the polyploids SF-2 > SF-1 > SF-3. The RbcS genes of the conserved SF-1 were previously reported to have the highest expression levels in all the wheat tissues studied, indicating a negative correlation between polymorphism and gene expression. Among the diploids, the species with the D and the S genomes were the most polymorphic and the A-genome species were the least polymorphic. The polyploids were less polymorphic than the diploids. Within the polyploids, the A genome was somewhat more polymorphic than the B genome, while the D genome was the most conserved. Among the diploid species with the A genome, the RFLP pattern of T. urartu was closer to that of the A genome of the common wheat cultivar Chinese Spring (CS) than to that of T. monococcum. The pattern in Ae. tauschii was similar to that of the D genome of CS. Only partial resemblance was found between the RFLP patterns of the species with the S genome and the B genome of CS. Received: 10 February 2000 / Accepted: 21 February 2000     

Bassoon,a Novel Zinc-finger CAG/Glutamine-repeat Protein Selectively Localized at the Active Zone of Presynaptic Nerve Terminals

The molecular architecture of the cytomatrix of presynaptic nerve terminals is poorly understood. Here we show that Bassoon, a novel protein of >400,000 M _r, is a new component of the presynaptic cytoskeleton. The murine bassoon gene maps to chromosome 9F. A comparison with the corresponding rat cDNA identified 10 exons within its protein-coding region. The Bassoon protein is predicted to contain two double-zinc fingers, several coiled-coil domains, and a stretch of polyglutamines (24 and 11 residues in rat and mouse, respectively). In some human proteins, e.g., Huntingtin, abnormal amplification of such poly-glutamine regions causes late-onset neurodegeneration. Bassoon is highly enriched in synaptic protein preparations. In cultured hippocampal neurons, Bassoon colocalizes with the synaptic vesicle protein synaptophysin and Piccolo, a presynaptic cytomatrix component. At the ultrastructural level, Bassoon is detected in axon terminals of hippocampal neurons where it is highly concentrated in the vicinity of the active zone. Immunogold labeling of synaptosomes revealed that Bassoon is associated with material interspersed between clear synaptic vesicles, and biochemical studies suggest a tight association with cytoskeletal structures. These data indicate that Bassoon is a strong candidate to be involved in cytomatrix organization at the site of neurotransmitter release.     

Activation of the Ca2+-sensing receptor stimulates the activity of the epithelial Ca2+ channel TRPV5

The extracellular Ca²⁺-sensing receptor (CaR) is a key-player in plasma Ca²⁺ homeostasis. It is essentially expressed in the parathyroid glands and along the kidney nephron. The distal convoluted tubules (DCT) and connecting tubules (CNT) in the kidney are involved in active Ca²⁺ reabsorption, but the function of the CaR has remained unclear in these segments. Here, the Ca²⁺-selective Transient Receptor Potential Vanilloid-subtype 5 channel (TRPV5) determines active Ca²⁺ reabsorption by forming the apical entry gate. In this study we show that the CaR and TRPV5 co-localize at the luminal membrane of DCT/CNT. Furthermore, by patch-clamp and Fura-2-ratiometric measurements we demonstrate that activation of the CaR leads to elevated TRPV5-mediated currents and increases intracellular Ca²⁺ concentrations in cells co-expressing TRPV5 and CaR. Activation of CaR initiated a signaling cascade that activated phorbol-12-myristate-13-acetate (PMA)-insensitive protein kinase C (PKC) isoforms. Importantly, mutation of two putative PKC phosphorylation sites, S299 and S654, in TRPV5 prevented the stimulatory effect of CaR activation on channel activity, as did a dominant negative CaR construct, CaR^R185Q. Interestingly, the activity of TRPV6, TRPV5′ closest homologue, was not affected by the activated CaR. We conclude that activation of the CaR stimulates TRPV5-mediated Ca²⁺ influx via a PMA-insensitive PKC isoform pathway.