Central peptidergic mechanisms controlling reproductive hormone secretion: novel methodology reveals a role for the natriuretic peptides.

A variety of neural factors can influence reproductive hormone secretion by neuromodulatory actions within the hypothalamus or neuroendocrine actions within the anterior pituitary gland. Passive immunoneutralization and antagonist administration protocols have suggested physiological roles for a number of these factors; however, both experimental approaches have severe technical limitations. We have developed novel methodology utilizing cytotoxin cell targeting with neuropeptides linked to the toxic A chain of the plant cytotoxin ricin. With this methodology we can target and destroy in vivo or in vitro cells bearing receptors for that peptide. Ricin A chain conjugated to atrial natriuretic peptide (ANP), a neuropeptide known to pharmacologically inhibit luteinizing hormone-releasing hormone (LHRH) release, was injected into the cerebroventricular system of intact, cycling rats and ovariectomized rats. Cytotoxin conjugate treatment significantly lengthened the estrous cycle. In ovariectomized rats the luteinizing hormone surge induced by steroid priming was completely inhibited. LHRH content of the median eminences of these rats was not significantly altered. These data suggest that ANP binding to clearance receptors in the hypothalamus displaces the C-type natriuretic peptide (CNP) from the shared clearance receptor, making more CNP available to inhibit LHRH release. In the absence of cells bearing the clearance receptor all available CNP binds to the ANPR-B receptor and exerts its effect via an inhibitory interneuron, since LHRH fibers are spared by this treatment.     

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.     

Identification and Properties of Methyltransferases That Synthesize Phosphatidylcholine in Rat Brain Synaptosomes

Abstract: Rat brain was found to enzymatically methylate phospholipids to form phosphatidylcholine with S -adenosyl- l -methionine serving as the methyl donor. Methyltransferase activity was localized in the microsomes and synaptosomes. In synaptosomes, at least two enzymes were found to be involved in the formation of phosphatidylcholine. The first methyltransferase which catalyzes the methylation of phosphatidylethanolamine to form phosphatidyl- N -monomethylethanolamine was found to have a pH optimum of 7.5, a low K_m for 5-adenosyl- l -methionine and a partial requirement for Mg². Methyltransferase I is tightly bound to membranes. The second methyltransferase (II) catalyzes the successive methylations of phosphatidyl- N -monomethylethanolamine to phosphatidyl- N , N -dimethylethanolamine and then to phosphatidylcholine. In contrast to methyltransferase I, methyltransferase II has a pH optimum of 10.5, a high apparent K_m for S -adenosyl- l -methionine and no requirement for Mg². Methyltransferase II is easily solubilized by sonication. The highest specific activity for both enzymes was found in the synaptosomal plasma membrane.     

Characterization of a plasma membrane-associated plasminogen activator on thymocytes

Plasma membranes isolated from normal thymocytes of hamster and rats were found to exhibit neutral protease activity toward 125I-labeled casein. The plasma membrane-associated proteases were completely inhibited by the serine protease inhibitors, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride and p-nitrophenyl-p-guanidinobenzoate, partially inhibited by soybean trypsin inhibitor and antipain, but were only weakly inhibited by L-1-tosylamino-2-phenylethyl chloromethyl ketone. The plasma membrane-associated proteases were also completely inhibited by ZnCl2 (75--100 mu M), but they were not affected by several other divalent cations. The plasma membrane fraction contained a plasminogen activator activity which was specifically localized in this fraction. The plasma membrane-associated plasminogen activator activity was inhibited by all of the inhibitors which inhibited plasma membrane-associated proteases except L-1-tosylamido-2-phenylethyl chloromethyl ketone. Labeling of plasma membrane-associated serine esterases with [3H] diisopropyl fluorophosphate followed by separation of the proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that this fraction contained a single major 3H-labeled protein of Mr 105 000. Both the plasminogen activator and the Mr 105 000 esterase were shown to be glycoproteins by affinity chromatography on lentil lectin-Sepharose. These results indicate that the plasminogen activator of thymocytes is a glycosylated serine protease with an active site-containing subunit of Mr 105 000 which is specifically localized in the plasma membrane.     

Origin of hydrogen in methane produced by Methanobacterium thermoautotrophicum.

The production of deuterated methane by Methanobacterium thermoautotrophicum in H2O-D2O mixtures was examined by high-resolution mass spectrometry. The hydrogen in the methane arose solely from water and not from hydrogen gas. Hydrogen gas served only as an electron source in methanogenesis. A whole-cell product isotope discrimination of 1.5 favoring hydrogen over deuterium was observed in methane production in 81 atom% deuterated water. The distribution of deuterated methane species is described by a simple model of the overall reaction.     

Managing and mining protein crystallization data

The crystallization of macromolecules remains a major bottleneck in structural biology. The routine screening of more than one thousand crystallization conditions and subsequent optimization by fine screening presents a challenge to conventional laboratory notebook keeping. In addition, the development of high-throughput robotic crystallization and imaging systems presents a pressing need for low-cost laboratory information management system (LIMS). Here we describe CLIMS2, a crystallization LIMS that features a simple, user-friendly graphical interface, allowing the storage, management, retrieval and mining of crystallization data. The CLIMS2 executable and documentation is freely available at http://clims.med.monash.edu.au.     

Species traits and climate velocity explain geographic range shifts in an ocean‐warming hotspot

Species' ranges are shifting globally in response to climate warming, with substantial variability among taxa, even within regions. Relationships between range dynamics and intrinsic species traits may be particularly apparent in the ocean, where temperature more directly shapes species' distributions. Here, we test for a role of species traits and climate velocity in driving range extensions in the ocean‐warming hotspot of southeast Australia. Climate velocity explained some variation in range shifts, however, including species traits more than doubled the variation explained. Swimming ability, omnivory and latitudinal range size all had positive relationships with range extension rate, supporting hypotheses that increased dispersal capacity and ecological generalism promote extensions. We find independent support for the hypothesis that species with narrow latitudinal ranges are limited by factors other than climate. Our findings suggest that small‐ranging species are in double jeopardy, with limited ability to escape warming and greater intrinsic vulnerability to stochastic disturbances.     

Two alpha-L-arabinofuranosidase genes in Arabidopsis thaliana are differentially expressed during vegetative growth and flower development

Glycosyl hydrolases are important mediators of plant cell wall modification during plant development. These enzymes catalyse the hydrolytic release of specific sugars, such as L-arabinose, from the polysaccharide-rich cell wall matrix. The cloning and expression analysis of two genes, AtASD1 and AtASD2, encoding putative alpha-L-arabinofuranosidases in Arabidopsis thaliana are reported here. AtASD1 and AtASD2 identities were assigned on the basis of homology to plant and microbial family 51 glycoside hydrolases. Using RT-PCR, RNA gel blot analysis and reporter gene expression analysis, AtASD1 and AtASD2 were shown to have different developmental expression profiles. High levels of AtASD1 promoter activity are present in multiple tissues during vegetative and reproductive growth. AtASD1 expression is particularly intense in zones of cell proliferation, the vascular system, developing and regressing floral tissues, and floral abscission zones. By comparison, AtASD2 expression is limited to the vasculature of older root tissue and to some floral organs and floral abscission zones.