The male seahorse incubates its young in a manner resembling that of a mammalian pregnancy. After the female deposits her eggs into the male's brood pouch they are fertilized and the embryos develop and grow for several weeks until they are able to withstand the external environmental conditions independently, at which point they are irreversibly released. Although the precise function of the brood pouch is not clear, it is probably related to providing a suitable protective and osmotic environment for the young. The aim of this project was to construct and characterize a cDNA library made from the tissue lining the pouch, in order to help understand the molecular mechanisms regulating its development and function. The library profile indicates expression of genes encoding proteins involved in metabolism and transport, as well as structural proteins, gene regulatory proteins, and other proteins whose function is unknown. However, a large portion of the library contained genes encoding C‐type lectins (CTLs), of which three full‐length proteins were identified and found to contain a signal peptide and a single C‐lectin domain, possessing all the conserved structural elements. We have produced recombinant protein for one of these and raised antisera; we have shown, using Western analysis and 2D electrophoresis, that this protein is secreted in significant quantities into the pouch fluid specifically during early pregnancy. Preliminary functional studies indicate that this CTL causes erythrocyte agglutination and may help to repress bacterial growth.
BackgroundPhosphatidic acid (PA) is a key regulated intermediate and precursor for de novo biosynthesis of all glycerophospholipids. PA can be synthesized through the acylation of lysophosphatidic acid (LPA) by 1-acyl-3-phosphate acyltransferase (also called lysophosphatidic acid acyltransferase, LPAAT). Recent findings have substantiated the essential roles of acyltransferases in various biological functions.Methodologies/Principal FindingsWe used a flow-injection-based lipidomic approach with ∼200 multiple reaction monitoring (MRM) transitions to pre-screen fatty acyl composition of phospholipids in the yeast Saccharomyces cerevisiae mutants. Dramatic changes were observed in fatty acyl composition in some yeast mutants including Slc1p, a well-characterized LPAAT, and Cst26p, a recently characterized phosphatidylinositol stearoyl incorporating 1 protein and putative LPAAT in S. cerevisiae. A comprehensive high-performance liquid chromatography–based multi-stage MRM approach (more than 500 MRM transitions) was developed and further applied to quantify individual phospholipids in both strains to confirm these changes. Our data suggest potential fatty acyl substrates as well as fatty acyls that compensate for defects in both Cst26p and Slc1p mutants. These results were consistent with those from a non-radioactive LPAAT enzymatic assay using C17-LPA and acyl-CoA donors as substrates.ConclusionsWe found that Slc1p utilized fatty acid (FA) 18:1 and FA 14:0 as substrates to synthesize corresponding PAs; moreover, it was probably the only acyltransferase responsible for acylation of saturated short-chain fatty acyls (12:0 and 10:0) in S. cerevisiae. We also identified FA 18:0, FA 16:0, FA 14:0 and exogenous FA 17:0 as preferred substrates for Cst26p because transformation with a GFP-tagged CST26 restored the phospholipid profile of a CST26 mutant. Our current findings expand the enzymes and existing scope of acyl-CoA donors for glycerophospholipid biosynthesis.
Glutamate-, aspartate-, N-methyl-D-aspartate receptor (NMDAR1 and 2 subunits)-, and nitric oxide synthase (NOS)-immunoreactive neurons were studied in the arcuate nucleus (AN) of mice treated neonatally with monosodium glutamate (MSG) which is known to cause extensive neuronal loss in this hypothalamic nucleus. It was found that intensely stained glutamate- and aspartate-immunoreactive neurons present in the AN of control mice were completely absent in the MSG-lesioned AN as well as the ventromedial nucleus lateral to the AN. Similarly, NMDAR1-immunoreactive neurons were mostly absent in the MSG-lesioned AN but remained intact in the ventromedial nucleus. There was also a substantial loss of NMDAR2 immunoreactivity within the AN. In contrast, NOS-immunoreactive neurons in the AN survived the neonatal glutamate treatment, although they appeared to be less intensely stained.
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