Massive fungal contamination in animal care facilities traced to bedding supply.

During the course of immunologic studies involving the gastrointestinal colonization of mice with Candida albicans, it became apparent that the animals were being exposed to large numbers of Aspergillus fumigatus spores which interfered with the C. albicans colonization. To determine the source of the A. fumigatus exposure and the extent of fungal contamination of the medical school vivarium and four satellite facilities, fungal analyses of feed, bedding, and air were undertaken. Initial samples from the air were collected with 3-h settle plates; air sampling following cleanup was done with an Anderson air sampler. The source of contamination in the mouse rooms was determined to be Beta Chip bedding, which came from the manufacturer highly contaminated. Beta Chip bedding (1 g) obtained from the manufacturer just prior to testing contained 10(4) CFU of A. fumigatus, 20 CFU of a zygomycete, and 10 CFU of a Penicillium sp. Coarse-grade Beta Chip had approximately one-half those levels of contamination. Pure Cob bedding was highly contaminated also, but with a Fusarium sp. and a Cladosporium sp. Untreated and heat-treated Sani-Chip as well as all other heat-treated preparations obtained from the manufacturer contained no detectable spores. Rodent chow direct from the manufacturer had no A. fumigatus, although it did contain 150 CFU of fungus per g, including 80 CFU of a Rhodotorula sp., 60 CFU of Cryptococcus uniguttulatus, and 1 CFU of a Penicillium sp.(ABSTRACT TRUNCATED AT 250 WORDS)     

Engineering the substrate specificity of rhizopuspepsin: the role of Asp 77 of fungal aspartic proteinases in facilitating the cleavage of oligopeptide substrates with lysine in P1.

Rhizopuspepsin and other fungal aspartic proteinases are distinct from the mammalian enzymes in that they are able to cleave substrates with lysine in the P1 position. Sequence and structural comparisons suggest that two aspartic acid residues, Asp 30 and Asp 77 (pig pepsin numbering), may be responsible for generating this unique specificity. Asp 30 and Asp 77 were changed to the corresponding residues in porcine pepsin, Ile 30 and Thr 77, to create single and double mutants. The zymogen forms of the wild-type and mutant enzymes were overexpressed in Escherichia coli as inclusion bodies. Following solubilization, denaturation, refolding, activation, and purification to homogeneity, structural and kinetic comparisons were made. The mutant enzymes exhibited a high degree of structural similarity to the wild-type recombinant protein and a native isozyme. The catalytic activities of the recombinant proteins were analyzed with chromogenic substrates containing lysine in the P1, P2, or P3 positions. Mutation of Asp 77 resulted in a loss of 7 kcal mol-1 of transition-state stabilization energy in the hydrolysis of the substrate containing lysine in P1. An inhibitor containing the positively charged P1-lysine side chain inhibited only the enzymes containing Asp 77. Inhibition of the Asp 77 mutants of rhizopuspepsin and several mammalian enzymes was restored upon acetylation of the lysine side chain. These results suggest that an exploitation of the specific electrostatic interaction of Asp 77 in the active site of fungal enzymes may lead to the design of compounds that preferentially inhibit a variety of related Candida proteinases in immunocompromised patients.     

The synthesis, purification, and evaluation of a chromophoric substrate for pepsin and other aspartyl proteases: design of a substrate based on subsite preferences

A convenient chromophoric assay for porcine pepsin has been developed using a new synthetic substrate. The sequence of this substrate was chosen based on the known subsite preferences for this enzyme. The peptide contains a phenylalanyl-p-nitrophenylalanine sequence at the reactive site. Cleavage of this bond yields a change in absorbance at 310 nm of between 1700 and 2000 per mole. This allows kinetic data to be obtained readily and accurately. The products of cleavage have been identified by isolation of a peptide fragment by high-performance liquid chromatography. Values of kcat, Km, and kcat/Km of 94 +/- 6 s-1, 0.13 +/- .04 mM, and 815 +/- 210 s-1/mM-1 were obtained at pH 3.0 and 37 degrees C. The peptide is soluble over the pH range from 2 to 7, thus facilitating determination of the pH dependence of the kinetic parameters. The substrate is also valuable in studying the inhibition of pepsin.     

Association of gap junctions with endoplasmic reticulum in rat parotid glands

Summary Examination of the parotid gland of the rat has shown specific associations of cisterns of the endoplasmic reticulum with gap junctions. About 20% of the junctions are so intimately associated with cisterns of the endoplasmic reticulum that in freeze fractured material the cisternal membranes remain attached to the junctional membrane faces, obscuring most of the junctional array except for a thin ring of telltale particles. This association was seen only in the parotid gland of the rat, but not that of the other species examined.     

Production of erythropoietin in vitro: A review

Summary The in vitro production of the important regulatory of erythropoiesis, erythropoietin (Epo), is reviewed. It is concluded that it is possible to produce almost routinely small quantities of Epo in tissue culture. Although such procedures offer the potential to provide large quantities of the hormone for clinical use, the optimum culture conditions and mechanisms for triggering Epo production have yet to be resolved. This work was supported by Grants No. 74444 from The John A. Hartford Foundation, and HL 10567 from the National Institutes of Health.     

Possible growth hormone regulation of rat liver glutamine synthetase activity.

Hypophysectomy results in a marked decrease in glutamine synthetase activity of rat liver homogenates. The enzyme is affected to a lesser extent in the kidneys and is not influenced in the brain. Bovine growth hormone treatment of hypophysectomized rats elevates the diminished glutamine synthetase activity in liver and kidneys but has no effect on the brain enzyme. Adrenalectomy also results in decreased liver glutamine synthetase activity although less than the decline seen with hypophysectomy. Cortisol treatment has no effect on glutamine synthetase activity in hypophysectomized animals. Our results suggest that growth hormone is involved in the regulation of liver glutamine synthetase activity. This regulation may be important in the utilization of α-amino nitrogen from glucogenic amino acids associated with growth hormone enhanced glucose production.     

Restriction endonuclease mapping of the colicin Ib plasmid

Summary A cleavage site map of the colicin Ib plasmid (ColIb) has been determined for the enzymes Sall, XhoI, and HindIII by analysis of partial digests, double digests, DNA-DNA hybridization, and Tn5-induced insertion mutants. The site of the colicin gene has been determined by probing with cloned DNA coding for colicin production, as well as by analysis of a colicin negative ColIb:Tn5.     

Changes in the circulating hemocyte population of Manduca sexta larvae following injection of bacteria

A technique for the collection of stable hemolymph from larvae of Manduca sexta has been developed. The method avoids the cell clumping and melanization reactions commonly encountered with insect hemolymph by minimizing contact between hemocytes and surfaces which provoke defensive or repair responses. The circulating hemocyte population of second-day, fifth-instar larvae (2dL5) of M. sexta consisted of 4.5 ± 2.5 × 10⁶ cells/ml (n = 15, range 2–7 × 10⁶ cells/ml) and contained five cell types: prohemocytes, plasmatocytes, granulocytes, spherulocytes, and oenocytoids. Two strains of Pseudomonas aeruginosa which differ in pathogenicity (P11-1 and 9027) and Escherichia coli D31 grew well at 26°C in cell-free hemolymph prepared from naive (nonimmunized) 2dL5 M. sexta. When viable cells of any of the three bacteria were injected into M. sexta larvae, changes in both the total hemocyte count (THC) and differential hemocyte count were observed. Viable bacteria were not required to produce these changes since formalin-killed cells of P. aeruginosa 9027 produced a qualitatively and quantitatively similar response. Following injection of bacteria, the THC increased, reaching a maximal level at 1 hr postinjection, and remained elevated for at least 4 hr after injection. While prohemocytes, plasmatocytes, granulocytes, and spherulocytes all increased in number, 80% of the increased cell population at 1 hr postinjection of bacteria were the latter two cell types. Granulocytes and spherulocytes are cells with recognized defensive capabilities. The increased numbers of these cells in circulation soon after injection of bacteria may confer an advantage on M. sexta larvae in dealing with bacterial infections. This could explain in part the unusual resistance of M. sexta to certain bacterial pathogens.     

Seasonal patterns of daily net photosynthesis,transpiration and net primary productivity of Juncus roemerianus and Spartina alterniflora in a Georgia salt marsh

Summary Studies of the seasonal CO₂ and water vapor exchange patterns of Juncus roemerianus and Spartina alterniflora were conducted in an undisturbed marsh community on Sapelo Island, Georgia. Daily patterns of net photosynthesis, transpiration, leaf diffusive conductance and water-use efficiency in response to ambient conditions were monitored on intact, in situ plants. Net primary productivity was calculated from the daytime CO₂ fixation totals, nighttime CO₂ loss, leaf standing stock and aboveground to belowground biomass ratios for each plant type.The tall form of S. alterniflora had higher rates of photosynthesis and higher water-use efficiency values which, in conjunction with low respiratory losses and large leaf standing crop, results in high values of net primary productivity. The environmental factors in the marsh which permit these physiological responses occur in less than 10% of the marsh. Overall, the physiological capabilities of the short form of S. alterniflora were reduced in comparison to the tall form, but the combination of environmental factors which determine the physiological responses of this form occur in a much greater portion of the marsh, and the short form of S. alterniflora dominates the Sapelo Island marshes.The response patterns of the C₃ species, J. roemerianus, differed somewhat from the height forms of S. alterniflora. A large, seasonally constant leaf standing crop coupled with moderate rates of photosynthesis resulted in a net primary productivity value which was between the tall and short height forms of S. alterniflora. However, as with the tall S. alterniflora, the environmental conditions under which this high productivity and high water loss rate can be sustained are restricted to specific regions of the environmental gradient in the marsh.Contribution No. 435 from the University of Georgia Marine Institute