Chronic upper respiratory tract disease of free-ranging desert tortoises (Xerobates agassizii)

Seventeen desert tortoises, Xerobates agassizii, with upper respiratory tract disease were examined; thirteen were euthanatized for necropsy. Four normal control desert tortoises from a clinically healthy population were similarly evaluated. Hemoglobin and phosphorus values were significantly (P less than or equal to 0.05) lower and serum sodium, urea, SGOT, and cholesterol values were significantly higher in ill tortoises compared to controls. No significant differences in concentrations of serum or liver vitamins A and E were found between the two groups. While no significant differences were found for concentrations of lead, copper, cadmium, and selenium, the livers of ill tortoises had higher concentrations of mercury and iron. Lesions were found consistently in the upper respiratory tract (URT) of ill tortoises. In all ill tortoises dense infiltrates of lymphocytes and histiocytes obscured the mucosal epithelium and underlying glands. The mucosal epithelium was variably dysplastic, hyperplastic, and occasionally ulcerated. Electron microscopic studies revealed small (350 to 900 nm), pleomorphic organisms resembling Mycoplasma sp., in close association with the surface epithelium of the URT of ill tortoises. Pasteurella testudinis was cultured from the nasal cavity of all ill tortoises and one of four control tortoises. A Mycoplasma sp. was cultured from the nasal passageways of four ill tortoises and was ultrastructurally similar to the pleomorphic organism present on the mucosa in tissue section.     

Impacts of section 404 permits requiring compensatory mitigation on wetlands in California (USA)

We analyzed data from Section 404 permits issued in California from January 1971 through November 1987 that involved impacts to wetlands and required compensatory mitigation (wetland creation, restoration, or preservation). The purpose of this study was to determine patterns and trends in permitting activity and to document cumulative effects of associated management decisions on the California wetland resource. The 324 permits examined documented that 387 compensatory wetlands (1255.9 ha) were required as mitigation for impacts to 368 wetlands (1176.3 ha). The utility of the data on wetland area was limited, however, since 38.0% of the impacted wetlands and 41.6% of the compensatory wetlands lacked acreage data. The wetland type most frequently impacted (37.8% of impacted wetlands) and used in compensation (38.2% of compensatory wetlands) was palustrine forested wetlands. Estuarine intertidal emergent wetlands had the most area impacted (52.3%) and compensated (62.5%). The majority of the wetlands were small (less than or equal to 4.0 ha in size). Wildlife habitat was the most frequently listed function of impacted wetlands (90.7% of the permits) and objective of compensatory wetlands (83.3%). Endangered species were listed as affected in 20.4% of impacted and 21.0% of compensatory projects. The number of permits requiring compensatory mitigation and the number of impacted and compensatory wetlands increased from 1971 to 1986.Documentation of the details of Section 404 permit decisions was inadequate for the permits we examined. Area information and specific locations of impacted and compensatory wetlands were lacking or of poor quality. Follow-up information was also inadequate. For example, project completion dates were specified in the permit for only 2.2% of compensatory wetlands. Furthermore, less than one-third (31.5%) of the permits required the compensatory wetland to be monitored by at least one site visit. We recommend improved documentation, regular reporting, and increased monitoring for better evaluation of the Section 404 permitting system.     

Identification and characterization of an {alpha}-mannosidase from Trypanosoma cruzi

In this report we describe the first purification and characterizationof the acid -mannosidase from the human parasite Trypanosomacruzi. The purified enzyme exhibited a native mol. wt of 240000 Da and is apparently composed of four identical subunitsof mol. wt 58 000 Da. Each of the four subunits contains oneN-linked high-mannose-type oligosaccharide. The -mannosidaseexhibited a pH optimum of 3.5 and a pI of 5.9. This low pH optimumand the ability of swainsonine to inhibit its activity suggestthat the -mannosidase is a lysosomal enzyme. Antibodies againstthe T.cruzi enzyme did not react with mammalian lysosomal -mannosidaseand, conversely, antibody against a rat lysosomal -mannosidasedid not react with the T.cruzi enzyme. Thus, the T.cruzi enzymeappears to be distinct from its mammalian counterpart. -mannosidase lysosomal enzyme Trypanosoma cruzi     

Probing the opioid receptor complex with (+)-trans-superfit. I. Evidence that [D-Pen2,D-Pen5]enkephalin interacts with high affinity at the delta cx binding site.

A variety of data support the existence of an opioid receptor complex composed of distinct but interacting mu cx and delta cx binding sites, where "cx" indicates "in the complex." The ability of subantinociceptive doses of [Leu5]enkephalin and [Met5]enkephalin to potentiate and attenuate morphine-induced antinociception, respectively, is thought to be mediated via their binding to the delta cx binding site. [D-Pen2,D-Pen5]Enkephalin also modulates morphine-induced antinociception, but has very low affinity for the delta cx binding site in vitro. In the present study, membranes were depleted of their delta ncx binding sites by pretreatment with the site-directed acylating agent, (3S,4S)-(+)-trans-N-[1-[2-(4-isothiocyanato)phenyl)-ethyl]-3-methy l-4- piperidyl]-N-phenylpropaneamide hydrochloride, which permits selective labeling of the delta cx binding site with [3H][D-Ala2,D-Leu5]enkephalin. The major findings of this study are that with this preparation of rat brain membranes: a) there are striking differences between the delta cx and mu binding sites; and b) both [D-Pen2,D-Pen5]enkephalin and [D-Pen2,L-Pen5]enkephalin exhibit high affinity for the delta cx binding site.     

Three-dimensional structure of a mouse-adapted type 2/type 1 poliovirus chimera.

The crystal structure of V510, a chimeric type 2/type 1 poliovirus, has been determined at 2.6 A resolution. Unlike the parental Mahoney strain of type 1 poliovirus, V510 is able to replicate in the mouse central nervous system, due entirely to the replacement of six amino acids in the exposed BC loop of capsid protein VP1. Significant structural differences between the two strains cluster in a major antigenic site of the virus, located at the apex of the radial projection which surrounds the viral five-fold axis. Residues implicated in the mouse-virulence of poliovirus by genetic studies are located in this area, and include the residues which are responsible for stabilizing the conformation of the BC loop in V510. Despite evidence that this area is not involved in receptor binding in cultured primate cells, the genetic and structural observations suggest that this area plays a critical role in receptor interactions in the mouse central nervous system. These results provide a structural framework for further investigation of the molecular determinants of host and tissue tropism in viruses.     

Labeling methods for the study of poly- and mono(ADP-ribose) metabolism in cultured cells

Methods are described for the radiolabeling and determination of NAD+, poly(ADP-ribose), and protein-bound monomers of ADP-ribose in cultured mammalian cells. The adenine nucleotide pools of confluent monolayer cell cultures are radiolabeled using high-specific-activity [3H]adenine. Following any desired experimental manipulation, cultures are treated with trichloroacetic acid. Radiolabel in NAD+ can be rapidly determined from the acid-soluble fraction using dihydroxyboronyl Sepharose (DHB-Sepharose). The acid-insoluble material can be analyzed for radiolabeled polymers of ADP-ribose and protein-bound monomers of ADP-ribose. Polymers are separated from interfering material using dihydroxyboronyl-Bio-Rex 70 (DHB-Bio-Rex). Protein-bound monomers are separated from noncovalently bound ADP-ribose and different classes of (ADP-ribosyl) protein linkages are released by specific chemical treatments. The released ADP-ribose is then separated from interfering materials using DHB-Bio-Rex and DHB-Sepharose. Control experiments have demonstrated the sensitivity, selectivity, and precision of the methods. Major advantages of the methods are that they allow many simultaneous determinations and all components can be determined from material derived from a single dish of cultured cells. The methods should prove useful for detailed studies of the metabolism of both protein-bound monomers and polymers of ADP-ribose in cultured mammalian cells.     

Identification of a Putative Structural Gene for Cathepsin D in Caenorhabditis Elegans

Mutants of Caenorhabditis elegans having about 10% of wild-type activity of the aspartyl protease cathepsin D have been isolated by screening. Mutant homozygotes have normal growth rates and no obvious morphological or developmental abnormalities. The mutant gene (cad-1) has been mapped to the right extremity of linkage group II. Heterozygous animals (cad-1/+) show intermediate enzyme levels and animals heterozygous for chromosomal deficiencies of the right extremity of linkage group II have 50% of wild-type activity. Cathepsin D purified from a mutant strain has a lower activity per unit mass of pure enzyme. These data suggest that cad-1 is a structural gene for cathepsin D.     

Proteases of the nematode Caenorhabditis elegans

Crude homogenates of the soil nematode Caenorhabditis elegans exhibit strong proteolytic activity at acid pH. Several kinds of enzyme account for much of this activity: cathepsin D, a carboxyl protease which is inhibited by pepstatin and optimally active toward hemoglobin at pH 3; at least two isoelectrically distinct thiol proteases (cathepsins Ce1 and Ce2) which are inhibited by leupeptin and optimally active toward Z-Phe-Arg-7-amino-4-methylcoumarin amide at pH 5; and a thiol-independent leupeptin-insensitive protease (cathepsin Ce3) with optimal activity toward casein at pH 5.5. Cathepsin D is quantitatively most significant for digestion of macromolecular substrates in vitro, since proteolysis is inhibited greater than 95% by pepstatin. Cathepsin D and the leupeptin-sensitive proteases act synergistically, but the relative contribution of the leupeptin-sensitive proteases depends upon the protein substrate.