Antifungal Susceptibility,Morphological and Molecular Characterization of <Emphasis Type="Italic">Lasiodiplodia theobromae</Emphasis> Isolated from a Patient with Keratitis

Lasiodiplodia theobromae is a rare ocular pathogen. We report a patient with fungal keratitis caused by L. theobromae. The patient was a 75-year-old male, a farmer with diabetes type II, and no previous history of ocular trauma. Histopathology analysis revealed the presence fungi invading Descemet’s membrane of the cornea. The fungus was characterized by septate, highly bulged fungal filaments involving full corneal thickness in the corresponding histopathology specimens. A dematiaceous mold was isolated and initally identified as L. theobromae by microscopic and macroscopic morphology, and further confirmed by PCR-based determination of internal transcribed spacer (ITS) regions of ribosomal DNA. Antifungal susceptibility tests showed sensitivity to amphotericin B (AMB) and voriconazole ( VRC), and resistance to other azoles, including itraconazole (ITC) and fluconazole (FLC). Corneal transplant was performed. Despite in vitro itraconazole resistance, the patient was successfully treated with oral itraconazole, topical voriconazole and natamycin, combined with ocular injections of amphotericin B and voriconazole.     

Effect of the time of artificial insemination with frozen-thawed or fresh semen on embryo viability and early pregnancy rate in gilts

The aim of the present study was to evaluate the effect of artificial insemination time (before or after ovulation) using either fresh or frozen-thawed boar semen on embryo viability and early pregnancy rate. Seventy-seven prepubertal crossbred (Landrace x Large White x Duroc) gilts were inseminated in 4 treatments. Artificial inseminations were performed 6 h either after (A) or before (B) ovulation using frozenthawed (A-frozen, n = 19; B-frozen, n = 19) or fresh semen (A-fresh, n = 21; B-fresh, n = 18). The gilts were induced to puberty by administration of 400 IU of eCG and 200 IU hCG (sc) followed by 500 IU of hCG (sc) 72 h later. Ovulation was predicted to occur 42 h after the second injection. All animals were slaughtered 96 h after AI. Embryos were collected and classified as viable (5- to 8-cells, morulae, compacted morulae and early blastocysts) and nonviable (fragmented, degenerated and 1- to 4-cell embryos). The total embryo viability rate was: 64.3% (A-frozen), 54.2% (A-fresh), 76.0% (B-frozen), 91.9% (B-fresh); (A-fresh vs B-fresh, P = 0.018; A-frozen vs B-frozen, P = 0.094). It was observed that AI before ovulation resulted in a higher percentage of total viable embryos than AI after ovulation (P = 0.041). The early pregnancy rate, defined as presence of at least one viable embryo, was 78.9, 80.9, 84.2 and 94.4% for A-frozen, A-fresh, B-frozen, B-fresh, respectively. There was no significant difference in the early pregnancy rate among groups. In conclusion, there was a detrimental effect upon total embryo viability rate when AI was performed after ovulation with either frozen-thawed or fresh semen. The total embryo viability rate and the early pregancy rate were not affected by AI with either frozen-thawed or fresh semen regardless of the time of AI.     

Satellite DNA repeat sequence variation is low in three species of burying beetles in the genus Nicrophorus (Coleoptera: Silphidae)

Three satellite DNA families were identified in three species of burying beetles, Nicrophorus orbicollis, N. marginatus, and N. americanus. Southern hybridization and nucleotide sequence analysis of individual randomly cloned repeats shows that these satellite DNA families are highly abundant in the genome, are composed of unique repeats, and are species-specific. The repeats do not have identifiable core elements or substructures that are similar in all three families, and most interspecific sequence similarity is confined to homopolymeric runs of A and T. Satellite DNA from N. marginatus and N. americanus show single-base-pair indels among repeats, but single-nucleotide substitutions characterize most of the repeat variability. Although the repeat units are of similar lengths (342, 350, and 354 bp) and A + T composition (65%, 71%, and 71%, respectively), the average nucleotide divergence among sequenced repeats is very low (0.18%, 1.22%, and 0.71%, respectively). Transition/transversion ratios from the consensus sequence are 0.20, 0.69, and 0.70, respectively.      

MONOLAYER CULTURES DERIVED FROM NEONATAL HAMSTER PANCREAS : Light and Electron Microscopy

Cells derived by trypsinization of neonatal golden hamster pancreas were cultured in modified Eagle's medium for 120 h in the presence of glucose (0.8 mg/ml) and for an additional 48 h in medium containing glucose (0.8 or 3.1 mg/ml) or tolbutamide (1,000 µg/ml) plus glucose (0.8 mg/ml). At day 7, cultures were stained differentially for light microscopy or examined by electron microscopy. Immunoreactive insulin (IRI) and immunoreactive glucagon (IRG) in the culture medium were measured by standard immunoassay procedures. Staining properties and ultrastructural appearance of cultured cells were comparable to those of the intact neonatal hamster pancreas. Cultures consisted predominantly of cells possessing aldehyde fuchsin positive (AF⁺) cytoplasmic granules resembling ultrastructurally those of the intact neonatal pancreatic beta cells and additionally, those of fibroblastoid, acinar, acino-insular, and aldehyde fuchsin negative (AF^-) argyrophilic cells. IRI release rate by the cultured cells was increased in the presence of elevated glucose or tolbutamide which paralleled the loss of AF⁺ granulation, but IRG release rate was suppressed by elevated glucose concentration. These findings indicate that these monolayer cultures consist of most of the cell types occurring in the neonatal pancreas, including endocrinologically competent islet cells.     

Suppression of T cell cytotoxicity by nude mouse spleen cells: reversal by monosaccharides and interleukin 2

The effects of monosaccharides on the suppression of cytotoxic T cell generation by spleen cells from nu/nu mice were examined. Suppression of the B6 anti-BALB/c response and the B6 anti-C3H response was reversed by alpha-methyl-D-galactoside (alpha MG) but not other sugars, including beta MG. Suppression was associated with a decrease in the level of IL 2, which suggests competition; this decrease was also reversed by alpha MG.     

Characterization of IL-2-dependent cytotoxic T-cell clones. II. Cell-surface phenotypes, histochemical and ultrastructural properties

This report examines the histochemical staining patterns, ultrastructure, and cell-surface phenotypes of six antigen-specific T-cell clones. Histochemical analyses indicated that all cell lines expressed alpha-napthyl butyrate esterase characteristic of the monocytic isoenzyme, intense napthol AS-D chloroacetate reactivity characteristic of granulocytes, and were negative for leucoperoxidase, alkaline phosphatase, and Sudan black. Only one clone stained weakly for acid phosphatase. The esterase staining patterns became evident in newly established cell lines after growth for only 1 week in interleukin 2-conditioned medium. Ultrastructurally, the outstanding feature was numerous membrane-bound granules containing a complex-appearing globular material. The cell-surface phenotypes of the lines as determined by protein A-sheep red blood cell rosetting and indirect immunofluorescence was Ly-5+, T200+, Qa-5-, MAC-1-, and Lyt-1+,2,3- for the helper line and Lyt-1-2,3+ for the five cytotoxic lines. By quantitative absorption analyses, low levels of Lyt-1 antigens were detected on all examined cytotoxic lines. The results strengthen the view that long-term T-cell lines can retain normal T-cell characteristics while also expressing markers that are either absent or in undetectable levels on uncultured T lymphocytes. The presence of the esterases may be associated with an expanded functional role in the T-cell lines.     

Structural features and selective expression of three Ly-5+ cell-surface molecules

Conventional Ly-5 alloantisera precipitate cell-surface molecules of three sizes: 200K, 205K, and 220K. In SDS-PAGE, the rat monoclonal antibody 74/8 precipitates the same three molecules from both Ly-5.1 and Ly-5.2 cells. Cleveland mapping of the three molecules, precipitated by reaction of conventional Ly-5 alloantisera or 74/8 monoclonal antibody with lysates of ¹²⁵I-labeled cells, disclosed no differences among the three molecular forms, but markedly distinguished all three Ly-5.1 molecules from all three Ly-5.2 molecules. Each of the three molecular forms can be expressed independently of the other two by cloned culture lines of Ly-5⁺ cells of different hematopoietic lineage. All of the seven cloned lines tested expressed only one form. However, two of the seven uncloned culture lines tested, plasmacytoma MOPC-70A and the X.1 putative macrophage line which originated from an SJL tumor, yielded both 200K and 205K forms.American Cancer Society Research Professor of Cell Surface Immunogenetics     

Isolation and Purification of the Envelope Proteins of Newcastle Disease Virus

A procedure has been developed for the isolation of Newcastle disease virus (NDV) envelope proteins. The two surface glycoproteins and the non-glycosylated membrane protein were solubilized with 2% Triton X-100 and 1 m KCl. Removal of the KCl by dialysis yielded by precipitation a pure preparation of the non-glycosylated membrane protein, which is insoluble in solutions of low ionic strength. The soluble fraction consisting of the two glycoproteins possessed full neuraminidase and hemagglutinating activities. The two glycoproteins could be separated by rate zonal sedimentation in a sucrose gradient containing 1% Triton X-100 and 1 m KCl. Under these conditions, the sedimentation coefficient of the larger glycoprotein, virus protein 1, was 9.3s, and that of the smaller, virus protein 2, was 6.1s. Both hemagglutinating and neuraminidase activities were associated with virus protein 1; virus protein 2 had neither activity. The results suggest that both activities reside on a single NDV glycoprotein. Similar results were obtained previously with another paramyxovirus, simian virus 5. These findings suggest that the association of hemagglutinating and neuraminidase activities with one glycoprotein is a general property of the paramyxovirus group.     

Cardiorespiratory changes during HCl infusion unrelated to decreases in circulating blood pH

To test the hypothesis that infusion of HCl changes blood pressure and respiration independent of decreases in circulating blood pH, an extracorporeal arteriovenous shunt (20 ml/min) between the femoral artery and vein was installed in anesthetized cats. Into this loop, acid (0.25 M HCl) and, approximately 10 cm downstream, base (0.25 M NaOH) could be infused simultaneously. Likewise, either acid or base could be infused individually. Right ventricular (Prv) and arterial (Pa) blood pressure, tidal volume (VT), and respiratory frequency (fresp) were recorded as well as blood gases and pH in arterial, right ventricular, and shunt loop blood at the reentrance into the animal. When HCl and NaOH were infused simultaneously and at equimolar rates (0.2 mmol/min for 10 min), there was a large increase in Prv, with little change or decrease in Pa. Respiratory frequency was increased, but total ventilation was not elevated because of a concomitant fall in VT. The rise in Prv and increase in fresp were transient in that they could only be evoked during the first HCl-NaOH infusion in a given animal. Repetitive infusions of HCl-NaOH into the same animal failed to elicit the response. Similar transient acid effects were evoked when HCl was infused without NaOH but not when NaOH was infused without HCl. During the second and third infusion of HCl, ventilatory responses were elicited that were explainable by stimulation of known chemoreceptors. The transient rise in Prv and fresp evoked by acid infusion might be explained by release of an agent from blood elements at the tip of the HCl infusion catheter, which in turn would constrict pulmonary vessels and influence breathing.(ABSTRACT TRUNCATED AT 250 WORDS)