Molecular cloning and tissue distribution of microsomal-1 and cytosolic prostaglandin E synthases in macaque

Prostaglandins derived from arachidonic acid are involved in a wide variety of physiological and pathological processes. The primary enzymes involved in the production of PGE2 from arachidonic acid are cyclooxygenases and prostaglandin E synthases. These enzymes have been identified in human, but only partially in the monkey where microsomal PGES-1 and cytosolic PGES have not been characterized. The present study was undertaken to clone these enzymes and to study their tissue distribution, along with mPGES-2. The coding sequence of Macaque mPGES-1 is 98% homologous to human mPGES-1 at the nucleic acid level and the deduced amino acid sequence has 98% homology with the human protein. The Macaque cPGES cDNA is more than 99% homologous to the human and the deduced amino acids sequence is identical to that of the human cPGES. By Northern blot analysis, we found that mPGES-2 and cPGES mRNA were expressed in the endometrium, myometrium, ovary and oviduct, albeit at different levels, while mPGES-1 mRNA was detected at a weak level, mainly in the oviduct. Western Blot analysis revealed that mPGES-2, mPGES-1 and cPGES proteins were present in all tissues tested. These results suggest that production of PGE2 in Macaque may involve more than one PGES and that further studies will be needed to fully understand the conditions under which each PGES contributes to PGE2 production.     

Antagonist Microorganisms with the Ability to Control <Emphasis Type="Italic">Pythium</Emphasis> Damping-off of Tomato Seeds in Rockwool

A total of 237 microorganisms were isolated from five different greenhouse tomato growing media. Of those, 40 microorganisms reduced the in vitro mycelial growth of both Pythium aphanidermatum and Pythium ultimum. The ability of these microorganisms to control damping-off was then tested in rockwool. As a result, Pseudomonas corrugata strains 1 and 3, Pseudomonas fluorescens subgroup F and G strains 1, 2, 3, 4 and 5, Pseudomonas marginalis, Pseudomonas putida subgroup B strain 1, Pseudomonas syringae strain 1 and Pseudomonas viridiflava significantly reduced damping-off caused by P. ultimum or P. aphanidermatum. Pseudomonas marginalis was the only microorganism that significantly reduced damping-off caused by both pathogens.     

Genetic screens for genes controlling motor nerve-muscle development and interactions

Motor growth cones navigate long and complex trajectories to connect with their muscle targets. Experimental studies have shown that this guidance process critically depends on extrinsic cues. In the zebrafish embryo, a subset of mesodermal cells, the adaxial cells, delineates the prospective path of pioneering motor growth cones. Genetic ablation of adaxial cells causes profound pathfinding defects, suggesting the existence of adaxial cell derived guidance factors. Intriguingly, adaxial cells are themselves migratory, and as growth cones approach they migrate away from the prospective axonal path to the lateral surface of the myotome, where they develop into slow-twitching muscle fibers. Genetic screens in embryos stained with an antibody cocktail identified mutants with specific defects in differentiation and migration of adaxial cells/slow muscle fibers, as well as mutants with specific defects in axonal pathfinding, including exit from the spinal cord and pathway selection. Together, the genes underlying these mutant phenotypes define pathways essential for nerve and muscle development and interactions between these two cell types.     

Drosophila myosin V is required for larval development and spermatid individualization

Class V myosins are multifunctional molecular motors implicated in vesicular traffic, RNA transport, and mechanochemical coupling of the actin and microtubule-based cytoskeletons. To assess the function of the single myosin V gene in Drosophila (MyoV), we have characterized both deletion and truncation alleles. Mutant animals exhibit no detectable defects during embryogenesis but are delayed in larval development; most die prior to 3rd instar. MyoV protein is widely distributed; however, there are no obvious cytological defects in mutant larval tissues where MyoV was normally highly expressed. Of the few adult MyoV mutant escapers, females were fertile but males were not. We examined the expression of MyoV during spermatogenesis. MyoV was associated with membranes, microtubule, and actin structures required for spermatid maturation; MyoV was strongly associated with the sperm nuclei during the maturation of the actin-rich investment cones that package spermatids in individual membranes. In MyoV mutant escaper males, the early stages of spermatogenesis were normal; however, in the later stages, the investment cones stained weakly for actin and their registration was disrupted; no mature sperm were produced. Our results suggest that MyoV contributes to the formation of the actin-based investment cones and acts to coordinate and/or anchor these structures and other components of the individualization complex.     

The crucial role of trehalose and structurally related oligosaccharides in the biosynthesis and transfer of mycolic acids in Corynebacterineae

Trehalose (alpha-D-glucopyranosyl-alpha'-D-glucopyranoside) is essential for the growth of the human pathogen Mycobacterium tuberculosis but not for the viability of the phylogenetically related corynebacteria. To determine the role of trehalose in the physiology of these bacteria, the so-called Corynebacterineae, mutant strains of Corynebacterium glutamicum unable to synthesize trehalose due to the knock-out of the genes of the three pathways of trehalose biosynthesis, were biochemically analyzed. We demonstrated that the synthesis of trehalose under standard conditions is a prerequisite for the production of mycolates, major and structurally important constituents of the cell envelope of Corynebacterineae. Consistently, the trehalose-less cells also lack the cell wall fracture plane that typifies mycolate-containing bacteria. Importantly, however, the mutants were able to synthesize mycolates when grown on glucose, maltose, and maltotriose but not on other carbon sources known to be used for the production of internal glucose phosphate such as fructose, acetate, and pyruvate. The mycoloyl residues synthesized by the mutants grown on alpha-D-glucopyranosyl-containing oligosaccharides were transferred both onto the cell wall and free sugar acceptors. A combination of chemical analytical approaches showed that the newly synthesized glycolipids consisted of 1 mol of mycolate located on carbon 6 of the non reducing glucopyranosyl unit. Additionally, experiments with radioactively labeled trehalose showed that the transfer of mycoloyl residues onto sugars occurs outside the plasma membrane. Finally, and in contradiction to published data, we demonstrated that trehalose 6-phosphate has no impact on mycolate synthesis in vivo.     

Heterologous expression of hexaheme fragments of a multidomain cytochrome from Geobacter sulfurreducens representing a novel class of cytochromes c

Multiheme cytochromes c are of great interest for researchers for a variety of reasons but difficult to obtain in quantities sufficient for the majority of studies. The genome of delta-proteobacterium Geobacter sulfurreducens contains more than a hundred genes coding for c-type cytochromes. Three of them represent a new class of multiheme cytochromes characterized by a mixed type of heme coordination and multidomain organization. We cloned and expressed in Escherichia coli three hexaheme fragments corresponding to two-domain fragments of one such protein containing 12 heme binding motifs and believed to consist of four triheme domains. Despite high sequence similarity among the fragments, expression levels varied significantly. Expression was optimized either by host strain variation or by reducing the rate of apoprotein synthesis. All three fragments were purified by cation exchange followed by gel filtration and were shown to contain six covalently attached hemes as confirmed by mass spectrometry. Their visible spectra are typical of c-type cytochromes. One of the fragments was crystallized and its preliminary X-ray structure shows two separate domains.     

Deep brain stimulation for treatment-resistant depression

Treatment-resistant depression is a severely disabling disorder with no proven treatment options once multiple medications, psychotherapy, and electroconvulsive therapy have failed. Based on our preliminary observation that the subgenual cingulate region (Brodmann area 25) is metabolically overactive in treatment-resistant depression, we studied whether the application of chronic deep brain stimulation to modulate BA25 could reduce this elevated activity and produce clinical benefit in six patients with refractory depression. Chronic stimulation of white matter tracts adjacent to the subgenual cingulate gyrus was associated with a striking and sustained remission of depression in four of six patients. Antidepressant effects were associated with a marked reduction in local cerebral blood flow as well as changes in downstream limbic and cortical sites, measured using positron emission tomography. These results suggest that disrupting focal pathological activity in limbic-cortical circuits using electrical stimulation of the subgenual cingulate white matter can effectively reverse symptoms in otherwise treatment-resistant depression.     

Carbonic anhydrase inhibitors. Interaction of isozymes I, II, IV, V, and IX with organic phosphates and phosphonates

The interaction of five human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, that is, hCA I, II, IV, V, and IX with a small library of phosphonic acids/organic phosphates, including methylphosphonic acid, MPA; phenylphosphonic acid, PPA; N-(phosphonoacetyl)-L-aspartic acid, PALA, methylene diphosphonic acid MDPA, the O-phosphates of serine (Ser-OP) and threonine (Thr-OP) as well as the antiviral phosphonate foscarnet has been studied. hCA I was activated by all these compounds, with the best activators being MPA and PPA (K(A)s of 0.10-1.20 microM). MPA and PPA were on the other hand nanomolar inhibitors of hCA II (K(I)s of 98-99 nM). PALA showed an affinity of 7.8 microM, whereas the other compounds were weak, millimolar inhibitors of this isozyme. The best hCA IV inhibitors were PALA (79 nM) and PPA (5.4 microM), whereas the other compounds showed K(I)s in the range of 0.31-5.34 mM. The mitochondrial isozyme was weakly inhibited by all these compounds (K(I)s in the range of 0.09-41.7 mM), similarly to the transmembrane, tumor-associated isozyme (K(I)s in the range of 0.86-2.25 mM). Thus, phosphonates may lead to CA inhibitors with selectivity against two physiologically relevant isozymes, the cytosolic hCA II or the membrane-bound hCA IV.