Development ofEimeria dispersa Tyzzer, 1929, from bobwhite quail (Colinis virginianus), in bovine kidney cell cultures

Summary The development ofEimeria dispersa Tyzzer, a parasite of bobwhite quail, in Madin-Darby bovine kidney cell cultures was investigated. Excysted sporozoites were inoculated into Leighton tubes containing cell monolayers on glass coverglasses and maintained in minimum essential medium supplemented with heat-inactivated fetal calf serum. Sporozoites became intracellular within 2 h. Sporozoite-shaped schizonts, schizonts with developing merozoites, and mature first-generation schizonts were seen 24 h postinoculation. Intracellular first-generation merozoites, second-generation trophozoites, and early second-generation schizonts containing two nuclei were first observed 72 h postinoculation. Second-generation schizonts containing developing merozoites as well as mature second-generation schizonts were first seen 96h postinoculation. Gametogony was not observed.DM developing merozoite - HN host nucleus - IM intracellular merozoite - M merozoite - N nucleus - R refractile body - RB residual body - V parasitophorous vacuole     

Effects of exercise training on responses to central injection of CRF and noise stress

The purpose of this study was to test the hypothesis that the cardiovascular and sympathoadrenal responses to acute environmental stress are attenuated by exercise training. Furthermore, we tested the hypothesis that the cardiovascular and sympathoadrenal responses to intracerebroventricular (ICV) administration of corticotropin-releasing factor (CRF) would be attenuated by training. Conscious, unrestrained, male Sprague-Dawley rats assigned to either a treadmill trained (16-26 m/min, 30-60 min/day, 5 days/week) or nontrained (16-26 m/min, 10 min/day, 1 day/week) group were studied. After 8-10 weeks of training, maximal oxygen uptake was significantly higher in the trained (108 +/- 3 ml/kg/min) vs. the nontrained (94 +/- 4 ml/min/kg) group. There were no significant differences in baseline mean arterial pressure, heart rate and plasma catecholamine levels associated with training. Trained rats exhibited significantly attenuated elevations in arterial pressure (20 +/- 3 vs. 36 +/- 2 mmHg for nontrained) and heart rate (-3 +/- 3 vs. 12 +/- 5 beats/min for nontrained) in response to acute noise stress. Twenty minutes after ICV administration of CRF, blood pressure (trained = 119 +/- 2 mmHg, nontrained = 127 +/- 2 mmHg), heart rate (trained = 408 +/- 8 beats/min, nontrained = 424 +/- 10 beats/min), plasma norepinephrine levels (trained = 757 +/- 54 pg/ml, nontrained = 775 +/- 100 pg/ml) and plasma epinephrine levels (trained = 266 +/- 29 pg/ml, nontrained = 225 +/- 42 pg/ml) were significantly elevated in both trained and nontrained groups. CRF-induced elevations of blood pressure, but not heart rate or plasma catecholamine levels, were significantly attenuated in the trained group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoblot reactivity of polyclonal and monoclonal antibodies with periplasmic flagellar proteins FlaA1 and FlaB of porcine Serpulina species.

The periplasmic-flagellum (PF) proteins of Triton X-100-soluble and Triton X-100-insoluble sodium dodecyl sulfate-treated fractions from reference and field strains of Serpulina hyodysenteriae, Serpulina innocens, and Serpulina pilosicoli were characterized by Western blotting with a rabbit polyclonal antibody (PAb) specific for the 44-kDa PF sheath protein of S. hyodysenteriae (Z. Li, F. Dumas, D. Dubreuil, and M. Jacques, J. Bacteriol. 175:8000-8007, 1993) and a murine monoclonal antibody (MAb), designated 7G2, specific for the PF core FlaB proteins of S. hyodysenteriae. The MAb 7G2 reacted with a conserved epitope present in the 37-, 34-, and 32-kDa PF core FlaB proteins of all Serpulina species. This suggested that the core FlaB proteins are conserved among porcine Serpulina species. An immunoreactive band of approximately 44 kDa was present with all S. hyodysenteriae, S. innocens, and S. pilosicoli strains that were reacted with the PAb. The specificities of the PAb and the MAb for the FlaA1 and FlaB proteins of Serpulina species were confirmed by N-terminal amino acid sequencing of 44- and 37-kDa proteins, respectively, of S. hyodysenteriae and S. pilosicoli. Results from this study provide further evidence that the 44-kDa protein FlaA1 and the 37-, 34-, and 32-kDa FlaB proteins are conserved among porcine Serpulina species.     

Processing of radical prostatectomy specimens for correlation of data from histopathological, molecular biological, and radiological studies: a new whole organ technique

AIMS: To develop a method of processing non-formalin fixed prostate specimens removed at radical prostatectomy to obtain fresh tissue for research and for correlating diagnostic and molecular results with preoperative imaging. METHODS/RESULTS: The method involves a prostate slicing apparatus comprising a tissue slicer with a series of juxtaposed planar stainless steel blades linked to a support, and a cradle adapted to grip the tissue sample and receive the blades. The fresh prostate gland is held in the cradle and the blades are moved through the cradle slits to produce multiple 4 mm slices of the gland in a plane perpendicular to its posterior surface. One of the resulting slices is preserved in RNAlater. The areas comprising tumour and normal glands within this preserved slice can be identified by matching it to the haematoxylin and eosin stained sections of the adjacent slices that are formalin fixed and paraffin wax embedded. Intact RNA can be extracted from the identified tumour and normal glands within the RNAlater preserved slice. Preoperative imaging studies are acquired with the angulation of axial images chosen to be similar to the slicing axis, such that stained sections from the formalin fixed, paraffin wax embedded slices match their counterparts on imaging. CONCLUSIONS: A novel method of sampling fresh prostate removed at radical prostatectomy that allows tissue samples to be used both for diagnosis and molecular analysis is described. This method also allows the integration of preoperative imaging data with histopathological and molecular data obtained from the prostate tissue slices.     

Iron and fur regulation in Vibrio cholerae and the role of fur in virulence

Regulation of iron uptake and utilization is critical for bacterial growth and for prevention of iron toxicity. In many bacterial species, this regulation depends on the iron-responsive master regulator Fur. In this study we report the effects of iron and Fur on gene expression in Vibrio cholerae. We show that Fur has both positive and negative regulatory functions, and we demonstrate Fur-independent regulation of gene expression by iron. Nearly all of the known iron acquisition genes were repressed by Fur under iron-replete conditions. In addition, genes for two newly identified iron transport systems, Feo and Fbp, were found to be negatively regulated by iron and Fur. Other genes identified in this study as being induced in low iron and in the fur mutant include those encoding superoxide dismutase (sodA), fumarate dehydratase (fumC), bacterioferritin (bfr), bacterioferritin-associated ferredoxin (bfd), and multiple genes of unknown function. Several genes encoding iron-containing proteins were repressed in low iron and in the fur mutant, possibly reflecting the need to reserve available iron for the most critical functions. Also repressed in the fur mutant, but independently of iron, were genes located in the V. cholerae pathogenicity island, encoding the toxin-coregulated pilus (TCP), and genes within the V. cholerae mega-integron. The fur mutant exhibited very weak autoagglutination, indicating a possible defect in expression or assembly of the TCP, a major virulence factor of V. cholerae. Consistent with this observation, the fur mutant competed poorly with its wild-type parental strain for colonization of the infant mouse gut.     

Comparison of amphotericin B with fluconazole in the treatment of acute AIDS-associated cryptococcal meningitis. The NIAID Mycoses Study Group and the AIDS Clinical Trials Group.

BACKGROUND. Intravenous amphotericin B, with or without flucytosine, is usually standard therapy for cryptococcal meningitis in patients with the acquired immunodeficiency syndrome (AIDS). Fluconazole, an oral triazole agent, represents a promising new approach to the treatment of cryptococcal disease. METHODS. In a randomized multicenter trial, we compared intravenous amphotericin B with oral fluconazole as primary therapy for AIDS-associated acute cryptococcal meningitis. Eligible patients, in all of whom the diagnosis had been confirmed by culture, were randomly assigned in a 2:1 ratio to receive either fluconazole (200 mg per day) or amphotericin B. Treatment was considered successful if the patient had had two consecutive negative cerebrospinal fluid cultures by the end of the 10-week treatment period. RESULTS. Of the 194 eligible patients, 131 received fluconazole and 63 received amphotericin B (mean daily dose, 0.4 mg per kilogram of body weight in patients with successful treatment and 0.5 mg per kilogram in patients with treatment failure; P = 0.34). Treatment was successful in 25 of the 63 amphotericin B recipients (40 percent; 95 percent confidence interval, 26 percent to 53 percent) and in 44 of the 131 fluconazole recipients (34 percent; 95 percent confidence interval, 25 percent to 42 percent) (P = 0.40). There was no significant difference between the groups in overall mortality due to cryptococcosis (amphotericin vs. fluconazole, 9 of 63 [14 percent] vs. 24 of 131 [18 percent]; P = 0.48); however, mortality during the first two weeks of therapy was higher in the fluconazole group (15 percent vs. 8 percent; P = 0.25). The median length of time to the first negative cerebrospinal fluid culture was 42 days (95 percent confidence interval, 28 to 71) in the amphotericin B group and 64 days (95 percent confidence interval, 53 to 67) in the fluconazole group (P = 0.25). Multivariate analyses identified abnormal mental status (lethargy, somnolence, or obtundation) as the most important predictive factor of a high risk of death during therapy (P less than 0.0001). CONCLUSIONS. Fluconazole is an effective alternative to amphotericin B as primary treatment of cryptococcal meningitis in patients with AIDS. Single-drug therapy with either drug is most effective in patients who are at low risk for treatment failure. The optimal therapy for patients at high risk remains to be determined.     

Excessive glutamate as an inhibitor of excitatory transmission in rat hippocampal slice

Exposure of rat hippocampal slices to perfusate containing 1-2 mM glutamate (GLU) induces reversible and relatively selective blockade of excitatory transmission. Intracellular recordings from 20 region CA1 hippocampal cells demonstrated only transient and mild effects on resting membrane properties and action potentials. In contrast, in 2 mM GLU excitatory postsynaptic potentials declined to 28% of control (P less than 0.001); inhibitory postsynaptic potentials remained robust at 88% of control. This suggests that excess exposure to GLU may result in a selective 'down-regulation' of excitatory synaptic transmission, while preserving inhibitory pathways. These observations may have practical implications for development of new anticonvulsant drugs.     

Characteristics of bovine alveolar macrophage elastase

Lavage of isolated bovine lung lobes was used to retrieve pulmonary alveolar macrophages (PAM). Nominal yields of 72 million viable PAM were routinely obtained using 500 ml of calcium- and magnesium-free phosphate-buffered saline. Bovine PAM readily attached to glass coverslips and within 24 hours, provided cell monolayers consisting exclusively of macrophages. Bovine PAM synthesized and secreted a calcium-dependent elastase in serum-free media. Optimal proteolytic activity using a radiolabeled elastin substrate was observed at pH 7.6. The elastase was insensitive to synthetic peptide chloromethyl ketone elastase inhibitors and to the serine protease inhibitor, phenylmethylsulfonyl fluoride. Enzyme activity, however, was effectively inhibited by the metal chelator, ethylenediaminetetraacetic acid, or suppressed by inhibition of protein synthesis with cycloheximide.     

Studies of human myeloid antigens using monoclonal antibodies and variant lines from the promyeloid cell line HL60.

Monoclonal antibodies were produced using mice immunized with the human promyeloid cell line HL60. Two antibodies are described which identify antigens selectively expressed by myeloid cells. Studies using normal bone marrow and myeloid leukaemia cells demonstrated that one of these antibodies (AGF4.48) identifies an antigen expressed throughout the promyeloid to neutrophil stages of maturation. In contrast, the second antibody (AGF4.36) identifies an antigen expressed at the promyeloid to metamyeloid stages and is absent from most blood neutrophils. The HL60 line can be induced to differentiate into neutrophils by 1 . 25% dimethylsulphoxide (DMSO) (Collins et al., 1978). Variant lines from HL60, unresponsive to 1 . 25% DMSO, lack the 'transient' myeloid antigen (AGF4.36) and show a reduced expression of the myeloid antigen (AGF4.48). The variant lines can be induced to mature using higher DMSO concentrations (1 . 5-1 . 75%) and do not express the 'transient' antigen (AGF4.36) during their maturation. The use of these lines in studies of myelopoiesis is discussed.