A developmental study of rat sperm and testis selenoproteins

Essentially all of the selenium in the rat spermatozoon is bound to a polypeptide of Mr 15,000-17,000 confined to the capsule that surrounds the sperm mitochondria. Isoelectric focussing of isolated 75Se-labelled, carboxymethylated mitochondrial capsule protein (MCP) reveals the presence of at least four radioactive components, with a predominant charge isomer at pI4.6. The sperm selenoprotein appears to be identical with MCP, as judged by the exact coincidence of radioactivity and protein stain during two-dimensional electrophoresis. The temporal pattern of 75Se-labelling of rat caput epididymal spermatozoa after intratesticular 75Se injection suggests that maximum incorporation of 75Se into MCP occurs in step 7-step 12 spermatids and that 75Se uptake ceases during step 15 of spermiogenesis. The developmental appearance of sperm selenoprotein in rat testis therefore appears to lag several days behind that reported for MCP in mouse testis, suggesting the presence of selenium-free MCP in immature germ cells. SDS gel electrophoretic analysis of testis subcellular fractions 24 h after 75Se injection into rat testis at 21, 28 and 90 days of age indicates that sperm selenoprotein first appears in very low concentration during late meiosis and that its concentration increases sharply during early spermiogenesis. Additional 75Se-labelled polypeptides were detected on the gels, most of them of higher molecular weight than MCP. At least two of these (Mr 47,000 and 54,000) displayed a marked decrease in labelling between 5 and 24 h after injection into adult testis, coincident with a comparable increase in 75Se-labelled MCP, indicating that they may be precursors of MCP.     

Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes

A shortened form of the self-splicing intervening sequence RNA of Tetrahymena acts as a sequence-specific endoribonuclease. Specificity of cleavage is determined by Watson-Crick base pairing between the active site of the RNA enzyme (ribozyme) and its RNA substrate [Zaug, A. J., Been, M. D., & Cech, T. R. (1986) Nature (London) 324, 429-433]. Surprisingly, single-base changes in the substrate RNA 3 nucleotides preceding the cleavage site, giving a mismatched substrate-ribozyme complex, enhance the rate of cleavage. Mismatched substrates show up to a 100-fold increase in kcat and, in some cases, in kcat/Km. A mismatch introduced by changing a nucleotide in the active site of the ribozyme has a similar effect. Addition of 2.5 M urea or 3.8 M formamide or decreasing the divalent metal ion concentration from 10 to 2 mM reverses the substrate specificity, allowing the ribozyme to discriminate against the mismatched substrate. The effect of urea is to decrease kcat and kcat/Km for cleavage of the mismatched substrate; Km is not significantly affected at 0-2.5 M urea. Thus, progressive destabilization of ribozyme-substrate pairing by mismatches or by addition of a denaturant such as urea first increases the rate of cleavage to an optimum value and then decreases the rate.     

DETERMINATION OF GUSTATORY SUCROSE THRESHOLD IN WATER BY USE OF A LINEAR SUCROSE GRADIENT

A novel experimental method was developed which allows the determination of the threshold concentration of sucrose by use of a linear sucrose gradient in water. With this method a continuous tasting of the test-liquid is possible. A panel of 15 persons experienced in taste-testing was used. Three gradients of different steepness were applied: 0 to 1.5% (w/w) sucrose in 2 min (I), 3 min (II) and 4 min (III). The results of the new method were compared with those of the standard method (DIN). With gradients I and II we found values which were significantly higher than those of the standard method (I: 0.49% (w/w); II: 0.46% (w/w); DIN: 0.31% (w/w)), whereas with gradient III the same threshold value was found as with the DIN-Method (III: 0.32% (w/w)).     

Biocalorimetry- supported analysis of fermentation processes

The close relation between metabolic activity and heat release means that calorimetry can be successfully applied for on-line monitoring of biological processes. Since the use of available calorimeters in biotechnology is difficult because of technical limitations, a new sensitive heat-flux calorimeter working as a laboratory fermenter was developed and tested for different aerobic and anaerobic fermentations with Saccharomyces cerevisiae and Zymommonas mobilis. The aim of the experiments was to demonstrate the abilities of the method for biotechnological purposes. Fermentations as well as the corresponding heat, substrate and product analyses were reproducible. During experiments the heat signal was used as a sensitive and fast indicator for the response of the organisms to changing conditions. One topic was the monitoring of diauxic growth phenomena during batch fermentations, which may affect process productivity. S. cerevisiae was used as the test organism and a protease-excreting Bacillus licheniformis strain as an industrial production system. Other experiments focused on heat measurements in continuous culture under substrate-limiting conditions in order to analyse bacterial nutrient requirements. Again, Z. mobilis was used as the test organism. Ammonium, phosphate, magnesium, biotin and panthothenate, as important substrate compounds, were varied. The results indicate that these nutrients are required in lower amounts for growth than formerly suggested. Thus, a combination of heat measurements and other methods may rapidly improve our knowledge of nutrient requirements even for a well-known microorganism like Z. mobilis. *** DIRECT SUPPORT *** AG903062 00004     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Delimitation of the maritime boundary in the gulf of Maine area

The compulsory dispute settlement regime included in the 1982 Law of the Sea Convention is recognized as one of the most comprehensive in a modern international convention. Yet, in the recent application of this regime, the question has arisen as to whether the procedural prerequisites associated with the LOS Convention's compulsory dispute settlement mechanism are so arduous as to avoid binding and compulsory jurisdiction in most instances. This article addresses that question by examining, in particular, the reasoning of the Southern Bluefin Tuna arbitration tribunal, which found Article 281 of Section 1 of the LOS Convention to bar jurisdiction to the compulsory dispute settlement mechanism prescribed by the Convention, and offers suggestions as to how states might distinguish or overcome the barriers imposed by the Southern Bluefin Tuna tribunal in future cases.     

Dystroglycan regulates structure, proliferation and differentiation of neuroepithelial cells in the developing vertebrate CNS

In the developing CNS alpha- and beta-dystroglycan are highly concentrated in the endfeet of radial neuroepithelial cells at the contact site to the basal lamina. We show that injection of anti-dystroglycan Fab fragments, knockdown of dystroglycan using RNAi, and overexpression of a dominant-negative dystroglycan protein by microelectroporation in neuroepithelial cells of the chick retina and optic tectum in vivo leads to the loss of their radial morphology, to hyperproliferation, to an increased number of postmitotic neurons, and to an altered distribution of several basally concentrated proteins. Moreover, these treatments also altered the oriented growth of axons from retinal ganglion cells and from tectal projection neurons. In contrast, expression of non-cleavable dystroglycan protein in neuroepithelial cells reduced their proliferation and their differentiation to postmitotic neurons. These results demonstrate that dystroglycan plays a key role in maintaining neuroepithelial cell morphology, and that interfering with dystroglycan function influences proliferation and differentiation of neuroepithelial cells. These data also suggest that an impaired dystroglycan function in neuroepithelial cells might be responsible for some of the severe brain abnormalities observed in certain forms of congenital muscular dystrophy.     

Pus1p-dependent tRNA pseudouridinylation becomes essential when tRNA biogenesis is compromised in yeast

Yeast Pus1p catalyzes the formation of pseudouridine (psi) at specific sites of several tRNAs, but its function is not essential for cell viability. We show here that Pus1p becomes essential when another tRNA:pseudouridine synthase, Pus4p, or the essential minor tRNA for glutamine are mutated. Strikingly, this mutant tRNA, which carries a mismatch in the T psi C arm, displays a nuclear export defect. Furthermore, nuclear export of at least one wild-type tRNA species becomes defective in the absence of Pus1p. Our data, thus, show that the modifications formed by Pus1p are essential when other aspects of tRNA biogenesis or function are compromised and suggest that impairment of nuclear tRNA export in the absence of Pus1p might contribute to this phenotype.     

Drosophila p24 homologues eclair and baiser are necessary for the activity of the maternally expressed Tkv receptor during early embryogenesis

p24 proteins are assumed to play an important role in the transport of secreted and transmembrane proteins into membranes. However, only few cargo proteins are known that partially, but in no case completely require p24 proteins for membrane transport. Here, we show that two p24 proteins are essential for dorsoventral patterning of Drosophila melanogaster embryo. Mutations in the genes, eclair (eca) and baiser (bai), encoding two p24 proteins reduce signalling by the TGF-beta homologue, Dpp, in early embryos. This effect is strictly maternal and specific to early embryogenesis, as Dpp signalling in other contexts is not notably affected. We provide genetic evidence that in the absence of eca or bai function in the oocyte, the maternally expressed type I TGF-beta receptor Tkv is not active. We propose that during early embryogenesis eca and bai are specifically required for the activity of the maternal Tkv, while the zygotic Tkv is not affected in the mutant embryos. Mutations in either eca or bai are sufficient for the depletion of Tkv activity and no enhancement of the phenotypes was observed in embryos derived from oocytes mutant for both genes. The dependence of maternal Tkv protein on the products of p24 genes may serve as an in vivo model for studying p24 proteins.     

Tyrosine dephosphorylation and ethanol inhibition of N-Methyl-D-aspartate receptor function

The inhibitory effect of ethanol on N-methyl-d-aspartate receptors (NMDARs) is well documented in several brain regions. However, the molecular mechanisms by which ethanol affects NMDARs are not well understood. In contrast to the inhibitory effect of ethanol, phosphorylation of the NMDAR potentiates channel currents (Lu, W. Y., Xiong, Z. G., Lei, S., Orser, B. A., Dudek, E., Browning, M. D., and MacDonald, J. F. (1999) Nat. Neurosci. 2, 331-338). We have previously shown that protein kinase C activators induce tyrosine phosphorylation and potentiation of the NMDAR (Grosshans, D. R., Clayton, D. R., Coultrap, S. J., and Browning, M. D. (2002) Nat. Neurosci. 5, 27-33). We therefore hypothesized that the ethanol inhibition of NMDARs might be due to changes in tyrosine phosphorylation of NMDAR subunits. In support of this hypothesis, we found that tyrosine phosphorylation of both NR2A and NR2B subunits was significantly reduced following in situ exposure of hippocampal slices to 100 mm ethanol. Specifically, phosphorylation of tyrosine 1472 on NR2B was reduced 23.5%. These data suggest a possible mechanism by which ethanol may inhibit the NMDAR via activation of a tyrosine phosphatase. Electrophysiological studies demonstrated that ethanol inhibited NMDAR field excitatory postsynaptic potential slope and amplitude to a similar degree as previously reported by our laboratory and others (Schummers, J., Bentz, S., and Browning, M. D. (1997) Alcohol Clin. Exp. Res. 21, 404-408). Inclusion of bpV(phen), a potent phosphotyrosine phosphatase inhibitor, in the recording chamber prior to and during ethanol exposure significantly reduced the inhibitory effect of ethanol on NMDAR field excitatory postsynaptic potentials. Taken together, these data suggest that phosphatase-mediated dephosphorylation of NMDAR subunits may play an important role in mediating the inhibitory effects of ethanol on the N-methyl-D-aspartate receptor.