Disruption of redox homeostasis leads to oxidative DNA damage in spermatocytes of Wolbachia-infected Drosophila simulans.

Molecular interactions between symbiotic bacteria and their animal hosts are, as yet, poorly understood. The most widespread bacterial endosymbiont, Wolbachia pipientis, occurs in high density in testes of infected Drosophila simulans and causes cytoplasmic incompatibility (CI), a form of male-derived zygotic lethality. Wolbachia grow and divide within host vacuoles that generate reactive oxygen species (ROS), which in turn stimulate the up-regulation of antioxidant enzymes. These enzymes appear to protect the host from ROS-mediated damage, as there is no obvious fitness cost to Drosophila carrying Wolbachia infections. We have now determined that DNA from Wolbachia-infected mosquito Aedes albopictus (Aa23) cells shows a higher amount of the base 8-oxo-deoxyguanosine, a marker of oxidative DNA damage, than DNA from uninfected cells, and that Wolbachia infection in D. simulans is associated with an increase in DNA strand breaks in meiotic spermatocytes. Feeding exogenous antioxidants to male and female D. simulans dramatically increased Wolbachia numbers with no obvious effects on host fitness. These results suggest that ROS-induced DNA damage in sperm nuclei may contribute to the modification characteristic of CI expression in Wolbachia-infected males and that Wolbachia density is sensitive to redox balance in these flies.

Utilization of soybeans or corn milling by-products in beef heifer development diets.

Whole raw soybeans (SB), wet corn gluten feed (WCGF), and corn dried distillers grains (DDG) are sources of protein in heifer development rations. The objectives of this study were to compare puberty status before synchronization of estrus, response to synchronization, and AI and final pregnancy rates in heifers developed on diets containing SB, WCGF, or DDG that were formulated to be similar in energy and CP. These ingredients vary substantially in fat content, which may affect reproductive performance. Rate of gain during the feeding period and post-AI performance were also compared. In a preliminary experiment, 104 crossbred heifers were fed diets containing either 1.25 kg of SB/d or 2.0 kg of WCGF/d for 110 d (DM basis), beginning at 10 mo of age. In Exp. 1, 100 crossbred heifers received either 1.25 kg of SB/d or 2.5 kg of WCGF/d from approximately 7 to 10 mo of age (91 d; 4 pens/diet), and then were fed 1.25 kg of SB/d for an additional 114 d (4 pens/diet). In Exp. 2, 1.25 kg of SB/d or 1.25 kg of DDG/d was fed to 100 crossbred heifers for 226 d, beginning at 6 mo of age (4 pens/diet). At approximately 13 mo of age, heifers were fed melengestrol acetate (0.5 mg/d) for 14 d, followed by an i.m. injection of PGF(2 alpha) (25 mg) 19 d later to synchronize estrus. Heifers (14 mo of age) received AI for 5 d after PGF(2 alpha), at which time the dietary treatments were ended. Heifers were commingled while grazing on native pasture and were exposed to bulls for approximately 60 d beginning 10 d after the last day of AI. Pregnancy to AI was determined by ultrasound 45 d after the last day of AI. Heifers fed SB in the preliminary experiment had a lower (P < 0.05) synchronization rate (81 vs. 96%) and longer interval (P = 0.05) from PGF(2 alpha) to estrus (76.6 vs. 69.2 h) compared with heifers fed WCGF. In Exp. 1, the age at which the heifers were begun on SB diets did not alter (P > 0.10) the synchronization rate (79%) or timing of estrus after PGF(2 alpha) (77.8 h). In Exp. 2, the synchronization rate (86%) and timing of estrus after PGF(2 alpha) (69.3 h) did not differ (P > 0.10) because of diet. No differences (P > 0.10) were due to diet for AI conception rates (overall mean for each experiment: 76.5, 60, and 68.5%), percentage of all heifers becoming pregnant to AI (67, 46, and 59%), or final pregnancy rates (92, 90, and 90%) in the preliminary experiment, Exp. 1, or Exp. 2, respectively. In summary, SB, DDG, and WCGF can be used as sources of protein in heifer development diets at the inclusion rates used in these studies.

Monoclonal antibody to native P39 protein from Borrelia burgdorferi.

We have produced, by using a sonicate of Borrelia burgdorferi, a monoclonal antibody (MAb), NYSP39H, that is specific for the P39 protein band. This MAb reacted with 13 isolates of B. burgdorferi but not with eight different spirochetes (four borrelias, two leptospiras, and two treponemas). Surface labeling of B. burgdorferi with biotin and subsequent treatment with Nonidet P-40 showed that P39 was not biotinylated but was extracted with Nonidet P-40, indicating that it is present within the outer membrane, but not on the surface of the spirochete. Immunoelectron microscopy revealed the immunogold probe primarily at the cytoplasmic membrane region of the spirochete. The MAb detected B. burgdorferi in the indirect fluorescent-antibody test only when the spirochetes from a culture or in a tick homogenate were fixed with polylysine and not with acetone. NYSP39H appears to be an appropriate probe for use in the specific detection of B. burgdorferi.

The distribution of canine exposure to Borrelia burgdorferi in a Lyme-Disease endemic area.

OBJECTIVES: A serosurvey of canine exposure to Borrelia burgdorferi, the causative agent of human Lyme disease, was conducted in Westchester County, New York, to determine the distribution of exposure in an area endemic for Lyme disease. METHODS: A total of 1446 blood samples was collected from resident dogs and tested by modified enzyme-linked immunosorbent assay. Equivocal samples were further tested by immunoblot. A mean number of 57.8 samples was collected from each of 25 towns and cities. RESULTS: Seroprevalence rates for municipalities ranged from 6.5% to 85.2%. County seroprevalence was 49.2%. There was a significant difference among the rates for the northern (67.3%), central (45.2%), and southern (17.3%) regions. Multiple range analysis indicated homogeneity between the southern and central regions and the central and northern regions. CONCLUSIONS: Canine exposure to B burgdorferi increases in a south to north gradient within the county. Intensity of exposure, measured by enzyme-linked immunosorbent assay titers, indicates a similar pattern. The close association between dogs and humans suggests that human risk of acquiring Lyme disease within Westchester County is equally disparate and is inversely related to the degree of urbanization.

Adherence and entry of Borrelia burgdorferi in Vero cells.

Adherence to and entry of the parasite into the host is one of the essential elements of microbial pathogenicity. We investigated the adherence to and entry into primate kidney epithelial (Vero) cells of Borrelia burgdorferi by radiolabelling techniques, immunofluorescence and electronmicroscopy. The attachment to and subsequent entry of both untreated and heat (50 degrees C)-treated B. burgdorferi into Vero cells occurred at cell-surface sites associated with aggregated coated pits. In contrast, there was minimal attachment of spirochaetes heated at 60 degrees C. Radiometric studies showed that, with untreated cells, there was incorporation of both 14C-glucose-1-phosphate and 14C-thymidine, whereas with the 50 degrees C-treated spirochaetes only glucose-1-phosphate was incorporated, and with the 60 degrees C-treated spirochates neither radionuclide was incorporated. Spirochaetes heated at 50 degrees C or 60 degrees C did not grow at 35 degrees C in culture medium. These results suggest that the presence of certain metabolic activities of the spirochaete but not viability (ability to grow) are necessary for the attachment process. After entry of untreated B. burgdorferi, most of the spirochaetes were either free in the cytoplasm or tightly bound to the host membrane. In contrast, 50 degrees C-treated spirochaetes remained bound to host membrane in large phagosome-like vesicles.

Fluoroimmunoassay studies with solubilized antigens from Borrelia burgdorferi.

Sodium deoxycholate-solubilized Borrelia burgdorferi antigen was prepared for use in a solid-phase fluoroimmunoassay (FIA-L) to detect antibodies in Lyme disease. Serum specimens were tested by FIA-L and by a microimmunofluorescence test. The FIA-L results are comparable to those of the standard microimmunofluorescence test. The overall agreement was 0.98. Moreover, the FIA-L procedure is simple and rapid; fluorescence is objectively determined and is proportional to antibody titer.

Improved procedure for platelet freezing.

Platelets frozen in glycerol-glucose frequently aggregated on thawing or reconstitution. This has been eliminated by using a nonplasma diluent (pH 6.5) and a new freezing bag. The results were improved platelet 14C-serotonin uptake and readily demonstrable aggregation to ADP, epinephrine, collagen and thrombin. 14C-serotonin uptake was at maximum when platelets were frozen at thrombocrits of up to 15%. Hemostatic effectiveness was demonstrated in a severely thrombocytopenic patient with acute myelogenous leukemia in relapse, who was supported entirely with two autologous frozen platelet transfusions for the 13 days before bone marrow activity resumed.

Evaluation of a closed circuit television patient education program: structure, process and outcome.

Few planners of a closed circuit television (CCTV) patient-education program have shared the evaluation of their program with other health agencies who are planning or considering such programs. This paper addresses the evaluation of the structure and process of developing and implementing such a program, and analyzes the outcome and cost effectiveness of one CCTV program. Two separate studies of patients at this hospital revealed that 6.5% and 1.7% of patients viewed the CCTV channel. The authors examined the possible factors contributing to the poor viewing habits of patients: (1) technical malfunctions, (2) low patient awareness, (3) minimal staff promotion, (4) nature of patient population. Planning processes and program structure are also examined to determine how they contributed to the outcomes. For example, incorrect planning assumptions and marketing strategies and failure to predict system barriers were possible factors. Finally, the costs involved in planning, implementing and monitoring the CCTV program are examined.

Characterization of insulin-degrading activity of intact and subcellular components of human fibroblasts.

We have studied insulin degrading activity (IDA) in cultured human fibroblasts and assessed the effect of various inhibitors of insulin processing on IDA. To evaluate the role of three enzymes of insulin degradation (neutral protease, microsomal glutathione insulin transhydrogenase, and lysosomal acid protease), we subfractionated homogenized fibroblasts into membrane (and nuclei) cytosol, mitochondria, microsomes, and lysosomes. Greater than 90% of IDA was found to be present in the cytosolar fraction containing neutral protease. IDA in intact fibroblasts was completely inhibited by 1 mM N-ethylmaleimide and partially by 0.5 mM dansylcadaverine (75%), 0.5 mM chloroquine (48%), 1 mg/ml bacitracin (32%) and Trasylol (30%). Lidocaine (5 mM) and glucagon (10(-6)M) exhibited about 15% inhibition with minimal inhibition (7%) by nonsuppressible insulin-like activity. Study of similar inhibitors on subfractionated components indicated inhibition of cytosolar enzyme by N-ethylmaleimide (100%), glucagon (30%), chloroquine (41%), nonsuppressible insulin-like activity (30%), Lidocaine (25%), dansylcadaverine (16%), and bacitracin (11%). Incubation of ammonium sulfate-fractionated cytosolar enzyme at 37 C with A14-125I-insulin resulted in generation of two intermediate peaks as early as 1 min. These peaks could be identified by HPLC but not by molecular sieve chromatography. These intermediates exhibited less immunoprecipitability with antiinsulin antibody and receptor binding with liver membrane preparations than intact insulin. Further incubation of A14-125I-insulin with the cytosolar enzyme(s) resulted in reduction of these peaks as well as insulin and formation of 125Iodotyrosine peak. We conclude that human fibroblast is capable of metabolizing cell-associated A14-125I-insulin in a time- and temperature-dependent manner. This process is inhibited by various inhibitors of insulin processing. The bulk of IDA consists of soluble neutral protease(s) with properties similar to other more purified neutral insulin protease preparations. This fraction, similar to the intact fibroblast degrades insulin to two intermediates with similar molecular weight to that of intact insulin but with more hydrophilicity and less binding affinity to antiinsulin antibody and liver membrane than intact insulin.

Early detection of degraded A14-125I-insulin in human fibroblasts by the use of high performance liquid chromatography.

We studied the metabolism of A14-125I-insulin in intact human fibroblasts using high performance liquid chromatography (HPLC) to detect and separate its early degradation products. The high resolving power of HPLC enabled us to separate what has been considered "intact insulin" by Sephadex G-50 chromatography or TCA precipitability into two additional peaks that had decreased biochemical properties with respect to immunoprecipitability and receptor binding but not decreased TCA precipitability. We conclude that human fibroblast is capable of metabolizing insulin within 2 min at 37 degrees C into intermediate molecules that can be detected by HPLC but not by TCA precipitability or molecular sieve chromatography.

The binding of cations to the surfaces of cells from early chick blastoderms.

Cells of the early chick blastoderms are either preparing for or undergoing regulated morphogenetic movements which culminate in the formation of a three-layered embryo. Information on the changes in the physical-chemical properties of cell surfaces may help in the understanding of this process. The binding of magnesium, manganese, strontium, barium and lanthanum to surfaces of early embryonic cells was estimated by the changes induced by these cations in the cells' electrophoretic mobilities (EPM). Cells show a positive EPM at concentrations of MgCl2 and MnCl2 at 3 X 10(-2) M while SrCl2 and BaCl2 were not able to reverse the cells' charge at concentrations up to 6 X 10(-2) M. CaCl3 reversed the cells' EPM at concentrations as low as 5 X 10(-3) M. Our results suggest that the surfaces of early embryonic cells have a high affinity for Mg and Mn. This is indicated by a reversal of polarity which cannot be detected in cells of differentiating or adult tissues at the cation concentrations used in these experiments.