Systematics within Gyps vultures: a clade at risk

Background  

Populations of the Oriental White-backed Vulture (Gyps bengalensis) have declined by over 95% within the past decade. This decline is largely due to incidental consumption of the non-steroidal anti-inflammatory veterinary pharmaceutical diclofenac, commonly used to treat domestic livestock. The conservation status of other Gyps vultures in southern Asia is also of immediate concern, given the lack of knowledge regarding status of their populations and the continuing existence of taxonomic uncertainties. In this study, we assess phylogenetic relationships for all recognized species and the majority of subspecies within the genus Gyps. The continuing veterinary use of diclofenac is an unknown but potential risk to related species with similar feeding habits to Gyps bengalensis. Therefore, an accurate assessment of the phylogenetic relationships among Gyps vultures should aid in their conservation by clarifying taxonomic uncertainties, and enabling inference of their respective relatedness to susceptible G. bengalensis.     

The extraction of a tissue collagenase associated with ovulation in the rat

A method has been developed to assay collagenase in ovarian extracts in the presence of tissue inhibitors. Rat ovarian tissue is first extracted with Triton X-100 and then heated to 60 degrees C in 50 mM Tris buffer containing 100 mM CaCl2. This extract contains collagenase activity and putative inhibitor(s). The inhibitory activity is removed by reduction with dithiothreitol and alkylation with iodoacetamide. Collagenase is then activated with aminophenylmercuric acetate and assayed using 3H-acetylated collagen from which the telopeptides have been removed. Identification of this activity as collagenase was performed by using the metalloprotease inhibitors EDTA and o-phenanthroline and by demonstration of the typical collagen cleavage fragments on sodium dodecyl sulfate-gel electrophoresis. To investigate the changes in collagenase activity associated with ovulation, immature rats received 20 IU of pregnant mare's serum gonadotropin and 52 h later 10 IU of human chorionic gonadotropin (hCG). After hCG administration, ovaries were removed at intervals from 0 to 20 h. Collagenase activity rose from 4.9 +/- 1.4% digestion of the 3H-collagen at 0 time to a maximum of 24.7 +/- 1.5% digestion at 8 h after hCG and remained high at 12 h (time of ovulation) and up to 20 h (18.7 +/- 1.9% and 16.1 +/- 1.6% digestion, respectively). These findings support a role of collagenase in the rupture of the follicle and they suggest a further role for this enzyme in the events following ovulation.     

Expression and Secretion of a Cellulomonas fimi Exoglucanase in Saccharomyces cerevisiae

We used the yeast MEL1 gene for secreted alpha-galactosidase to construct cartridges for the regulated expression of foreign proteins from Saccharomyces cerevisiae. The gene for a Cellulomonas fimi beta-1,4-exoglucanase was inserted into one cartridge to create a fusion of the alpha-galactosidase signal peptide to the exoglucanase. Yeast transformed with plasmids containing this construction produced active extracellular exoglucanase when grown under conditions appropriate to MEL1 promoter function. The cells also produced active intracellular enzyme. The secreted exoglucanase was N-glycosylated and was produced continuously during culture growth. It hydrolyzed xylan, carboxymethyl cellulose, 4-methylumbelliferyl-beta-d-cellobiose, and p-nitrophenyl-beta-d-cellobiose. A comparison of the recombinant S. cerevisiae enzyme with the native C. fimi enzyme showed the yeast version to have an identical K(m) and pH optimum but to be more thermostable.     

Regulation of prodynorphin gene expression in the ovary: distal DNA regulatory elements confer gonadotropin regulation of promoter activity.

Examination of the regulation of prodynorphin (pro-DYN) promoter activity is limited by the absence of a good cell model. The discovery of pro-DYN mRNA and derived peptides in the reproductive tract led us to examine the cellular localization and hormonal regulation of ovarian pro-DYN expression and to evaluate normal granulosa cells as a model for studying pro-DYN gene regulation. Ovarian pro-DYN mRNA levels were significantly elevated in PMSG-primed immature rats 12-24 h after receiving an ovulatory dose of hCG. Levels peaked 2 days after hCG, remained elevated throughout the ensuing pseudopregnancy, and rose again at the end of pseudopregnancy. In situ hybridization localized pro-DYN expression predominantly to granulosa and luteal cells. Transfection of primary cultures of granulosa cells revealed that the activity of the rat pro-DYN promoter [-1858 to 133 base pairs (bp)] was increased 18- to 19-fold by hCG and human FSH treatments and 7-fold by cAMP analog treatment. Deletion analysis identified a 358 bp fragment as the primary hormone-responsive sequence (-1858 to -1500 bp; containing three potential cAMP-responsive elements); its deletion resulted in severely reduced FSH responsiveness, and its ligation to hormone-unresponsive basal promoter sequences completely restored FSH responsiveness. This is an unusually distal position for cAMP-responsive elements compared to other cAMP-regulated genes. These data demonstrate specific expression of pro-DYN in granulosa and luteal cells, which is under sensitive gonadotropin regulation, and identify a distal hormone-responsive sequence within the promoter.     

Ca2+-dependent depolarization and burst firing of rat CA1 pyramidal neurones induced by N-methyl-D-aspartic acid and quinolinic acid: antagonism by 2-amino-5-phosphonovaleric and kynurenic acids

The excitatory effects of microiontophoretically applied quisqualic (QUIS), N-methyl-D-aspartic (NMDA), and quinolinic (QUIN) acids were investigated using intracellular recording from CAl pyramidal neurones in slices of rat hippocampus. QUIS evoked only simple action potentials superimposed upon a depolarization which attained a clear plateau. When this level had been reached, increased ejecting currents did not produce further depolarization. By contrast, with low currents NMDA and QUIN elicited small membrane depolarizations which triggered bursts of action potentials superimposed upon rhythmically occurring depolarizing shifts. Larger currents caused depolarization which if sufficiently large completely blocked spike activity. Tetrodotoxin (TTX) prevented the spikes evoked by QUIS and the bursts of action potentials seen with NMDA and QUIN, and the rhythmic depolarizing shifts then appeared as broad spikes of up to 50 mV in amplitude. These and the underlying membrane depolarization were blocked by Co2+, by the NMDA antagonist D(-)-2-amino-5-phosphonovaleric acid (DAPV), and by kynurenic acid (KYNU). It thus appears that the depolarization and burst firing of rat CAl pyramidal neurones elicited by NMDA and QUIN are Ca2+ dependent while the actions of QUIS are not.     

Escherichia coli expression and processing of human interleukin-1 beta fused to signal peptides

Escherichia coli expression, processing, and secretion of human interleukin-1 beta (IL-1 beta) fused to the signal peptide of E. coli OmpA or PhoA protein were studied. With fusion to either signal sequence, high-level expression was observed and the products accumulated to about 20% of total cell protein. In the fusion to OmpA leader sequence, more than 50% of the product has the OmpA signal peptide removed precisely. The majority of the processed material is not released by osmotic shock. On the other hand, very little of the material from the fusion to PhoA has the PhoA signal peptide removed. Use of the host with a mutation in prlA or prlF, variation of temperature for cell growth, and alteration of the amino acid residues around the cleavage site do not facilitate processing of the PhoA signal peptide. These results suggest that some component in the PhoA signal peptide, interacting with the Il-1 beta sequence, is interfering with the processing of the signal peptide.