Amino acid sequence of guinea pig prostate kallikrein

The primary structure of the major arginine esteropeptidase from guinea pig prostate has been deduced from automated Edman degradation of peptides generated by clostripain, cyanogen bromide, endoproteinase Lys-C, and Staphylococcus aureus V8 protease digestion of the protein. The esteropeptidase is a single polypeptide chain comprised of 239 amino acids and contains 2 apparent sites of carbohydrate attachment, Asn-78 and Asn-169. Both occur in consensus sequences for N-linked glycosylation sites. The esteropeptidase exhibits approximately 35% homology with trypsin including conservation of the catalytic residues and the aspartic acid which confers specificity toward basic amino acids. The sequence identity, however, extends to greater than 60% with the kallikrein family of serine proteases. In addition to the overall homology, the guinea pig enzyme displays a number of features characteristic of kallikreins including 10 conserved half-cystine residues, a C-terminal proline, and the "kallikrein loop". On the basis of this structural relatedness, the enzyme has been designed as guinea pig prostate kallikrein. In contrast to many of the kallikreins of other species and tissues, this enzyme does not contain any sites within the kallikrein loop sensitive to proteases that result in internal breaks in the polypeptide chain.     

The removal of exogenous thiols from proteins by centrifugal column chromatography

Centrifugal column chromatography was shown to provide a rapid, efficient, and useful means of separation of various low molecular weight thiols from proteins. The single chromatographic step procedure employed standard 5 ml plastic syringes containing Sephadex G-25 as the bed matrix and required less than 5 min to produce average dilutions of 5000-, 980-, and 25-fold, respectively, from 5 to 200 mM initial concentrations of 2-mercaptoethanol, dithiothreitol, and reduced glutathione in the sample as measured by titration with 5,5'-dithiobis-(2-nitrobenzoic acid). Dihydrofolate reductase solutions of 0.07-0.08 mM were separated from 50 mM 2-mercaptoethanol, dithiothreitol, or reduced glutathione with a minimum 16,500-fold dilution of the thiol after centrifugal chromatography on two consecutive columns. Thymidylate synthase solutions of 0.06 mM were effectively separated from 50 mM 2-mercaptoethanol or dithiothreitol with a minimum average 5900-fold dilution of the thiol after consecutive column chromatography. There was no change in either the physical or chemical properties of the enzyme throughout the course of the experiments as determined by activity, active site sulfhydryl group titration, and binding assays. Recoveries of protein obtained in the load fraction were usually in excess of 70% of the protein loaded with virtually no dilution from the initial concentration. This method was developed in order to facilitate the study of the active site sulfhydryl groups in enzymes.     

Drought and the organization of tree-hole mosquito communities

Summary In southeastern North America (North Florida, USA), the duration, frequency, and timing of drought differentially affect the survivorship of pre-adult tree-hole mosquitoes. Drought affects survivorship both by the direct action of dehydration on developing larvae and pupae and by the indirect modulation of predation. The drought-susceptible species, Toxorhynchites rutilus, Orthopodomyia signifera, and Anopheles barberi co-occur in more permanent holes that are larger, with larger, more vertical openings, lower down in larger trees, and contain darker water with higher conductivity, pH, and tannin-lignin content than the holes occupied by Aedes triseriatus that has drought-resistant eggs and rapid larval development. Ovipositing mosquitoes cue on physical and chemical attributes of tree holes independently of host tree species. These same attributes differ among drought-prone and drought-resistant holes but mosquitoes track these attributes more faithfully than the attributes predict tree-hole stability.     

Tolerance ofHolcus lanatus andAgrostis stolonifera to sodium chloride in soil solution and saline spray

Summary Inland and sea cliff populations of bothAgrostis stolonifera L. andHolcus lanatus L. were subjected to soil NaCl treatments, of 100 and 200 mol m⁻³ NaCl, and tolerance examined using plant dry weight data. A parallel experiment subjected them to salt spray treatments of 2.5%, 5% and 10% NaCl in distilled water, and tolerance assessed from leaf damage. Both populations of each species were equally sensitive to soil NaCl. When subjected to salt spray the sea cliff populations however showed marked resistance to leaf damage. Soil salinity resistance and salt spray resistance thus appear to be independent characteristics in these two species.     

Salmonella infections in a marsupial, the quokka (Setonix brachyurus), in relation to seasonal changes in condition and environmental stress.

An unusual abundance of Salmonella infections was studied in an island population of a wild marsupial, the quokka (Setonix brachyurus), which experiences starvation in summer associated with significant mortality. The frequency of infections was found to vary seasonally over most parts of the island, with high infection rates (70 to 100%) in summer and low infection rates (0 to 30%) in winter. In some samples, there was an average of as many as two isolations per animal, and up to five isolations were made from a single animal. By the end of summer, virtually all animals excreted Salmonella spp., with a median rate of excretion of approximately 3,000 Salmonella organisms per g of feces. The seasonal changes occurred over intervals of only weeks. The infections are believed to be associated with disruption of the digestive physiology of the animals caused by the poor quality of feed available in summer. This conclusion was supported by a quantitative study of the infections and by a field manipulation experiment which delayed the initiation of the infections as long as a food supplement was available. The proliferation of Salmonella spp. is discussed in terms of the ecology of the quokka and of the use of Salmonella spp. as indicators of environmental stress acting on the animals.     

Isolation and characterization of the cytosolic and chloroplast forms of spinach leaf fructose diphosphate aldolase

Two different isoenzymes of fructose-P2 aldolase can be resolved by chromatography of crude spinach leaf extracts on DEAE-cellulose columns. The acidic isoenzyme comprises about 85% of the total leaf aldolase activity. The two forms differ in primary structure as judged by their distinctive amino acid compositions, tryptic peptide patterns, and immunological properties. Only the acidic isoenzyme was detected in extracts of isolated chloroplasts, suggesting that this molecule represents the chloroplast form of spinach leaf aldolase while the basic isoenzyme is of cytosolic origin. The cytosolic (basic) isoenzyme and chicken aldolase A4 are similar in the following respects. 1) They have similar specific catalytic activity (10-15 units/mg); 2) they are both highly sensitive to inactivation by very limited digestion with bovine pancreatic carboxypeptidase A; 3) they both have subunit molecular weights of 40,000; 4) they both have derivatized (blocked) NH2-terminal structures; 5) they are both resistant to thermal denaturation at 50 degrees C; and 6) they both regain catalytic activity following reversible denaturation at pH 2.3 or in 5.8 M urea. Also, the cytosolic aldolase cross-reacted immunologically with the single aldolases present in spinach seeds and in wheat germ. Further, this isoenzyme readily "hybridized" with chicken aldolase A4 in vitro. These observations demonstrate the close homology between the cytosolic aldolases derived from plant and animal origins. The chloroplast aldolase had a specific catalytic activity of about 8 units/mg and, like its cytosolic counterpart, was severely inactivated by limited digestion with carboxypeptidase A. However, this isoenzyme was distinct from the cytosolic aldolase in the following characteristics: 1) its "small" subunit size (Mr congruent to 38,000); 2) its underivatized NH2-terminal structure; 3) its high sensitivity to thermal denaturation at 50 degrees C; and 4) its inability to refold into an enzymatically active conformation following denaturation at pH 2.3 or in 5.8 M urea. The distinctive properties of the chloroplast aldolase may be expected for an enzyme which is synthesized as a higher molecular weight precursor on cytosolic polysomes and is then proteolytically processed to the "mature" form during its migration into the chloroplast organelle.     

Different acute effects of the tyrosine hydroxylase inhibitors alpha-methyl-p-tyrosine and 3-iodo-L-tyrosine on hypothalamic noradrenaline activity and adrenocorticotrophin release in the rat

Computerized gas chromatography-mass spectrometry techniques using selected ion monitoring and deuterated internal standards were used to assay simultaneously the medial basal hypothalamic concentrations of dopamine (DA) and noradrenaline (NA) and their major metabolites in individual rats 30 min after the administration of two different inhibitors of tyrosine hydroxylase, alpha-methyl-p-tyrosine (alpha-MT) and 3-iodo-L-tyrosine (MIT). Consistent with inhibition of DA synthesis, administration of both alpha-MT and MIT resulted in marked reductions (P less than 0.005) in the hypothalamic concentrations of DA and its metabolite homovanillic acid as well as in highly significant increases in prolactin secretion. alpha-MT administration, but not MIT, resulted in a highly significant decrease in NA concentration and a highly significant increase in the concentration of the NA metabolite 3,4-dihydroxyphenylethyleneglycol (DHPG). The hypothalamic ratio DHPG/NA was thus markedly increased (P less than 0.005) by alpha-MT indicating increased NA neuronal activity. alpha-MT administration also resulted in increased ACTH secretion (P less than 0.0005), an effect not observed following MIT. It is proposed that the effects on hypothalamic NA activity and ACTH secretion caused by alpha-MT are stress-mediated and unrelated to tyrosine hydroxylase inhibition. MIT is devoid of these effects but exhibits blockade activity, thus indicating it to be a preferable drug for the acute inhibition of tyrosine hydroxylase in neuroendocrine investigations.