Temperature-sensitive strain of Cryptococcus neoformans producing hyphal elements in a feline nasal granuloma.

We report the isolation of a temperature-sensitive, serotype A, mating type alpha strain of Cryptococcus neoformans from a case of nasal cryptococcosis in a cat. The strain grew extremely slowly at 35 degrees C and failed to grow at 37 degrees C in vitro. Histopathological sections of the infected tissue revealed yeast cells producing hyphae up to several hundred micrometers in length, in addition to numerous encapsulated yeast cells typical of C. neoformans. The cultures grown on yeast extract-peptone-glucose agar at 35 degrees C also produced some yeast cells with germ tube-like hyphal elements up to 100 microm in length.

Localization of peroxidase mRNAs in soybean seeds by in situ hybridization.

The soybean Ep gene encodes an anionic peroxidase enzyme that accumulates in large amounts in seed coat tissues. We have isolated a second peroxidase gene, Prx2, that is also highly expressed in developing seed coat tissues. Sequence analysis of Prx2 cDNA indicates that this transcript encodes a cationic peroxidase isozyme that is far removed from Ep in peroxidase phylogeny. To determine the expression patterns for these two peroxidases in developing seeds, the abundance and localization of the Ep and Prx2 transcripts were compared by in situ hybridization. Results show the expression of Ep begins in a small number of cells flanking the vascular bundle in the seed coat, spreads to encircle the seed, and then migrates to the hourglass cells as they develop. Expression of Prx2 occurs throughout development in all cell layers of the seed coat, and is also evident in the pericarp and embryo. Nonetheless, the Ep-encoded enzyme accounts for virtually all of the peroxidase activity detected in mature seed coats. The Prx2 enzyme is either insoluble in a catalytically inactive form, or is subject to degradation during seed maturation.

Dynamic association of L-selectin with the lymphocyte cytoskeletal matrix.

L-selectin mediates lymphocyte extravasation into lymphoid tissues through binding to sialomucin-like receptors on the surface of high endothelial venules (HEV). This study examines the biochemical basis and regulation of interactions between L-selectin, an integral transmembrane protein, and the lymphocyte cytoskeleton. Using a detergent-based extraction procedure, constitutive associations between L-selectin and the insoluble cytoskeletal matrix could not be detected. However, engagement of the L-selectin lectin domain by Abs or by glycosylation-dependent cell adhesion molecule-1, an HEV-derived ligand for L-selectin, rapidly triggered redistribution of L-selectin to the detergent-insoluble cytoskeleton. L-selectin attachment to the cytoskeleton was not prevented by inhibitors of actin/microtubule polymerization (cytochalasin B, colchicine, or nocodozole) or serine/threonine and tyrosine kinase activity (staurosporine, calphostin C, or genistein), although L-selectin-mediated adhesion of human PBL was markedly suppressed by these agents. Exposure of human PBL or murine pre-B transfectants expressing full-length human L-selectin to fever-range hyperthermia also markedly increased L-selectin association with the cytoskeleton, directly correlating with enhanced L-selectin-mediated adhesion. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids failed to associate with the cytoskeletal matrix in response to Ab cross-linking or hyperthermia stimulation and did not support adhesion to HEV. These studies, when taken together with the previously demonstrated interaction between the L-selectin cytoplasmic domain and the cytoskeletal linker protein alpha-actinin, strongly implicate the actin-based cytoskeleton in dynamically controlling L-selectin adhesion.

Symmetric cutaneous necrosis of the hind feet and multicentric follicular lymphoma in a cat.

A 7-year-old castrated male domestic shorthair cat was admitted to the veterinary teaching hospital for evaluation of symmetric necrosis of the skin of its hind feet and high liver enzyme activities. Lymphoma was diagnosed on cytologic examination of a fine needle aspirate of the liver. The owner declined treatment for the lymphoma. On postmortem histologic examination, lymphoma was found in the liver, stomach, and multiple lymph nodes. Immunohistochemical staining revealed the neoplasm to have a mixed B- and T-cell follicular arrangement, and a diagnosis of multicentric follicular lymphoma was made. The distal portion of the feet were necrotic, but a neoplastic infiltrate was not seen on histologic examination. After thrombosis and vasculitis were excluded as causes, the ischemic necrosis of the feet of the cat in this report was considered a paraneoplastic syndrome, as can be seen in people with lymphoma or other internal malignancies.

Percutaneous absorption of vanilloids: in vivo and in vitro studies.

The percutaneous absorption of three highly lipophilic analogs of capsaicin--vanillylnonanamide (VN), olvanil, and NE-21610--was measured in vivo in the CD:VAF rat, and in vitro through excised CD: VAF and SkH:Fz rat skin and human cadaver skin. Absorption and skin metabolism were monitored by radiolabel techniques. The rank order of penetration in all species was VN > olvanil > NE-21610, in accordance with that expected from their physical properties. Rat skin was more permeable than human skin by factors ranging from 4 to 8 for VN, 10 to 20 for olvanil, and approximately 10 to 100 for NE-21610. All three compounds were extensively metabolized during passage through fresh SkH:Fz rat skin, with the primary route of degradation for at least two of the compounds involving hydrolysis of the amide bond (the metabolites of NE-21610 were not identified). For the in vitro studies a range of receptor solutions was employed to determine a set of conditions that best mimicked in vivo absorption. The results with phosphate-buffered saline containing a preservative and 1-6% polyoxyethylene-20 oleyl ether (Oleth-20) were in good agreement with in vivo results for all three compounds for periods up to 24 h post-dose; after this time, in vivo absorption rates declined but in vitro rates remained relatively constant. Buffered saline or saline containing 0.5% bovine serum albumin led to marked underestimates of in vivo penetration for olvanil and NE-21610, whereas a 1:1 ethanol: water solution led to gross overestimates of the in vivo absorption rates for all three compounds.

Recovery of Aujeszky's disease virus recombinants from experimentally co-infected swine.

Tissue homogenates were obtained from swine experimentally co-infected with two vaccine strains of Aujeszky's disease virus (ADV). Viral isolates were derived by serial plaque purification directly from tissue homogenates, without an intervening step of isolation and amplification on cell cultures. Use of limiting dilutions and recovery of virus isolates as individual plaques minimized the likelihood of in vitro recombination serving as a confounding source of recombinant ADV. The tyhmidine kinase and glycoprotein X gene sequences were classified as wildtype or deleted, using a battery of polymerase chain reaction assays. On the basis of pairwise combinations of the allelic forms of the thymidine kinase and glycoprotein X genes, the isolates were characterized as recombinant and parental genotypes. The results substantiate the observation that ADV vaccine strains can form genetic recombinants in vivo after experimentally induced co-infection. A full description of this study is available (Am. J. Vet. Res. 54 (4) 540-545, 1993).

Direct isolation and identification of recombinant pseudorabies virus strains from tissues of experimentally co-infected swine.

Tissue homogenates were obtained from swine co-infected with 2 vaccine strains of pseudorabies virus (PRV). Viral isolates derived by serial plaque purification directly from tissue homogenates, without an intervening step of isolation and amplification on cell cultures, were characterized as recombinant and parental PRV genotypes on the basis of thymidine kinase and glycoprotein X gene combinations. Use of limiting dilutions and recovery of virus isolates as individual plaques minimized the likelihood of in vitro recombination serving as a confounding source of recombinant PRV. The thymidine kinase and glycoprotein X gene sequences were classified as wild-type or deleted, using a battery of polymerase chain reaction assays. Results substantiate the observation that PRV vaccine strains can form genetic recombinants in vivo after experimentally induced co-infection.

Electrical analysis of fresh, excised human skin: a comparison with frozen skin.

Samples of human allograft skin prepared without freezing ("fresh skin") were found to have electrical and sodium ion transport properties which differed only slightly from those of skin which had been similarly treated but stored frozen ("frozen skin"). The fresh skin samples were less permeable to sodium ions during passive diffusion and less conductive than frozen skin at low current levels. They were more permselective for sodium versus chloride during constant-current iontophoresis and showed slightly more asymmetry in their current-voltage properties. Overall, the electrical behavior of the two tissues was similar enough to support the use of frozen tissue in iontophoresis studies. However, caution should be exercised when considering the use of frozen skin for applications, such as those based on electroosmosis, where the observed differences could have a major impact on the results.

DC electrical properties of frozen, excised human skin.

DC current-voltage relationships and sodium ion transport measurements for human allograft skin immersed in saline buffers have been determined using a four terminal potentiometric method and diffusion cells of our own design. About three-fourths of the skin samples were deemed suitable for study on the basis of their high resistivities and similar j-V characteristics. Most of these samples yielded sodium ion permeability coefficients less than or equal to those reported for human skin in vivo. The current-voltage relationship in these tissues was time dependent, highly nonlinear, and slightly asymmetric with respect to the sign of the applied potential. Skin resistance decreased as current or voltage increased. For current densities less than 15 microA/cm2 and exposure times of 10-20 min, this decrease was almost completely reversible; at higher current densities, both reversible and irreversible effects were observed. The overall dependence of current on voltage was nearly exponential and was satisfactorily described by an equation of the form j approximately sinh V. Diffusion potentials, sodium ion membrane transference numbers, and sodium ion flux enhancement factors during iontophoresis were measured for skin immersed both in normal saline solutions and in saline solutions of differing concentrations. The sign of the diffusion potentials and the value of the sodium ion transference number (0.51 in normal saline at pH 7.4) indicated a weak permselectivity of the skin for transport of sodium ion versus chloride. At a current density of 71 microA/cm2 and transmembrane potentials in the range of 1.1-1.6 V, the flux enhancement for sodium ion was three to five times greater than that predicted for an uncharged homogeneous membrane according to electrodiffusion theory. For transmembrane potentials less than 0.17 V, agreement of this theory with the data was better but still incomplete.

Application of liquid chromatography with on-line radiochemical detection to metabolism studies on a novel class of analgesics.

Vanilloids are a class of compounds structurally related to capsaicin, the pungent principle of hot peppers, which are under development as a novel class of analgesics. Vanilloids undergo extensive first-pass metabolism when dosed orally to rats and mice. These compounds, as well as capsaicin, would be anticipated to be susceptible to three major routes of metabolism: (omega, beta)-oxidation of the alkyl side chain, hydrolysis of the amide bond and conjugation of the phenolic group. Olvanil [N-(3-methoxy-4-hydroxybenzyl)oleamide], radiolabelled with either 14C at the benzylic carbon or 3H in the oleyl side chain, was studied in various in vitro, in situ and in vivo metabolism models to determine the major route(s) of intestinal and hepatic metabolism in rats for this new class of compounds. Models used in metabolism studies included isolated hydrolytic enzymes, cell-free intestinal and liver supernatants, hepatocytes, enterocytes, perfused intestine and whole animal studies. Reversed-phase liquid chromatography (LC) with on-line radiochemical detection was used to examine the metabolic profiles from the different models. The major metabolic route for olvanil in both the intestine and the liver was found to be hydrolysis of the amide bond. The benefits of selective 14C and 3H labels in conjunction with LC with on-line radiochemical detection are discussed.