Biomedical subjects
Publications and source records attributed to D L Williams.
The gracilis muscle was excised from cold-acclimated rats, placed in vitro, and simultaneously perfused via its artery by high pO2 medium and superfused by low pO2 medium. With a doubling of the perfusion rate (from 50 to 100 microliters/min) phosphocreatine and ATP increased by 39% and 44%, respectively.
This report describes a method for the solubilization of micro-particulate (1-->3)-beta-D-glucan. Insoluble glucan is dissolved in methyl sulfoxide and urea (8 M) and partially sulfated at 100 degrees. The resulting water-soluble product is called glucan sulfate. The conversion rate is 98%, and the preparation is endotoxin free as determined by the Limulus lysate procedure. Glucan sulfate is composed of 34.06% C, 6.15% H, 50.30% O, 5.69% S and 3.23% N, and has a repeating unit empirical formula of (C6H10O5)8.3 SO3NH4+.4 H2O, suggesting that, on the average, a sulfate group is substituted on every third glucose subunit along the polymer. Molecular weight averages, polydispersity, and intrinsic viscosity were determined by aqueous high-performance size-exclusion chromatography (HPSEC). Two polymer peaks were resolved. Peak 1 (Mw = 1.25 x 10(6) g/mol) represents < 1% of the total polymer mass. Peak 2 (Mw = 1.45 x 10(4) g/mol) comprises > 99% of polymers. 13C NMR spectroscopy confirmed the beta-(1-->3) interchain linkage. In solution, glucan sulfate polymers self-associate in a triple helix. Glucan sulfate stimulates murine bone marrow proliferation following intravenous administration. The ability to prepare a immunologically active, water-soluble (1-->3)-beta-D-glucan preparation will greatly enhance the clinical utility of this class of compounds.
An unlinked anonymous HIV antibody study of neonates, using surplus dried blood spots on Guthrie cards, was introduced as a means of estimating the prevalence of HIV infection in childbearing women. From March 1990 to February 1992, blood spots from 14,520 infants were tested for anti-HIV, using an HIV IgG antibody capture particle-adherence test; only one sample gave a confirmed positive reaction. This type of study involves no interference in routine care and can be operated without difficulty in a district general hospital setting where neonatal metabolic screening is performed.
We have previously reported that steroidogenesis is dramatically reduced in mouse Y1 adrenocortical cells which express the human apolipoprotein E gene (Y1-E cells). This suppression results in part from inhibition of cAMP-mediated events. In this report we have examined the expression of protein kinase C (PKC) in the Y1-E cell lines. Total cellular PKC activity in vitro is increased 3-5-fold in the Y1-E cell lines. PKC activity in the particulate and cytosolic fractions is increased to the same relative extent. Increased PKC activity reflects increased levels of PKC mRNA, as determined by Northern blot analysis, and PKC protein, as determined by immunoblot analysis. Increased expression of PKC in the Y1-E cell lines is accompanied by a 2-3-fold increase in diacylglycerol, an in vivo activator of PKC. To determine the contribution of elevated PKC expression to the Y1-E cell phenotype, we utilized the PKC inhibitors, staurosporine and calphostin C. Upon treatment with staurosporine or calphostin C, expression of P450-cholesterol side chain cleavage mRNA is increased severalfold to a level equal to, or greater than, basal expression in the Y1-neo control cell line. Treatment with calphostin C also results in recovery of steroidogenesis in the Y1-E cells to a level comparable to the basal level observed in the Y1-neo control cell line. These results indicate that increased expression of PKC in the Y1-E cell lines decreases basal steroidogenesis by suppressing P450-cholesterol side chain cleavage mRNA expression. Inhibition of PKC, however, does not reverse the block in cAMP-stimulated steroidogenesis in Y1-E cells, suggesting that the pleiotropic effects of apoE expression are not mediated entirely through altered PKC expression.
The combination of a photometric reporter-gene assay, with transfection by electroporation, is potentially a rapid and sensitive tool for the study of genetic regulatory elements in many types of cells. We have found that the sensitivity, accuracy, and reproducibility of the technique is greatly improved by the inclusion of appropriately chosen carrier DNA as the primary DNA species present during electroporation. By using high levels of carrier, the activities of constructs of differing sizes can be quantitatively compared, active constructs can be assayed with sub-microgram amounts of plasmid, and the activities of the constructs are linear over a wide concentration of DNA. In addition, the activity of miniprep DNA can be screened without purification on CsCl gradients giving activities equal to CsCl-purified DNA. This is extremely useful when doing preliminary screening of large numbers of constructs for promoter or enhancer activities. We report the results of testing various types of DNA as carrier, and the parameters for optimizing its use.
The stability of the mRNA for apolipoprotein (apo) II is regulated by estrogen [Gordon et al. (1988) J. Biol. Chem. 263, 2625-2631]. On the hypothesis tha estrogen regulation of apoII mRNA stability is mediated through mRNA-protein interaction, we have examined the messenger ribonucleoprotein particle (mRNP) for apoII mRNA following release from chicken liver polyribosomes. Polyribosomes containing undegraded apoII mRNA were obtained when tissue was homogenized without detergent, and polyribosomes were isolated following simultaneous addition of detergent and magnesium to a 20000g supernatant. ApoII mRNP released by EDTA sedimented at 12-18 S in sucrose gradients, and banded at rho = 1.4 g/mL in CsCl isopycnic centrifugation, indicative of a 3:1 ratio of protein to mRNA. A fraction in which apoII mRNP was enriched to 40-50% of total mRNP was prepared by successive size fractionation steps on sucrose gradients. Proteins associated with sucrose gradient enriched apoII mRNP were examined by iodination of UV-cross-linked proteins followed by SDS-polyacrylamide gel electrophoresis. Comparisons of proteins in highly enriched apoII mRNP to proteins in mRNP from non-estrogen-treated rooster liver did not reveal any differences. This result suggests that the major proteins associated with apoII mRNA are mRNP proteins also associated with the bulk of liver mRNAs.
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It has been postulated that Kupffer cells provide signals that regulate hepatocyte responses in sepsis and inflammation. Although in vitro data support such a hypothesis, to our knowledge, no in vivo evidence has been reported. We injected rats with lipopolysaccharide intraperitoneally to simulate sepsis or turpentine intramuscularly to mimic localized inflammation. Both treatments are known to induce the hepatic acute-phase response. Liver nonparenchymal cells and hepatocytes were isolated and placed in culture. Hepatocyte fibrinogen synthesis was measured as an indication of interleukin 6 exposure, while nonparenchymal interleukin 6 production was measured directly. Both lipopolysaccharide and turpentine stimulated a sharp increase in hepatocyte fibrinogen synthesis (turpentine greater than lipopolysaccharide). However, only lipopolysaccharide injection was associated with increased nonparenchymal cell interleukin 6 synthesis. Increased circulating levels of interleukin 6 could be found only after lipopolysaccharide injection. In addition, tumor necrosis factor synthesis was enhanced by lipopolysaccharide but not turpentine. Our data show that nonparenchymal cells are stimulated to provide the interleukin 6 signal to hepatocytes in endotoxemia but not in remote localized inflammation, even though both treatments stimulate the hepatic acute-phase response. Our findings support paracrine functions for liver sinusoidal cells in certain septic states.
A modified crossover surface coil with minimal B1 field penetration was used for collection of skin phosphorus NMR spectra. Projection imaging experiments show that the coil-sensitive volume is uniform at the phosphorus frequency, but strikingly nonuniform at the proton frequency. Experiments with an in vitro phosphorus phantom, designed to simulate skin and underlying tissue, demonstrated that 45.1% (+/- 1.2%) of total signal was derived from Sprague-Dawley rat skin and 19.3% (+/- 1.4%) of total signal was derived from Fischer-344 rat skin. 31P MR spectra of rat skin in vivo permitted resolution of four phosphorus compounds: nucleoside triphosphates, phosphocreatine (PCr), inorganic phosphate (Pi), and phosphomonoester. Spectra collected after skin flap surgery in Fischer-344 rats showed a 50.1% (+/- 7.6%) reduction in the ratio of PCr/Pi within 30 min of surgery, compared to presurgical PCr/Pi levels (P less than 0.01). Skin phosphorus spectra are potentially useful for assessment of skin flap and skin graft viability.
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Adjuvants are commonly used in immunization protocols for the purpose of augmenting immune responses to antigens. The antigenic profile of Leishmania infantum (ex. Oklahoma) is described and the efficacies of three adjuvants delivered coincidentally with killed promastigotes are compared, by measuring relative serologic responses of Balb-C mice to specific antigenic determinants. Western blotting techniques were employed to visualize humoral responses to isolated antigens; serologic profiles were compared and contrasted. Four immunization protocols utilizing Freund's complete adjuvant, glucan adjuvant, lipovant adjuvant or phosphate-buffered saline, in conjunction with killed L. infantum (ex. Oklahoma) promastigotes were executed in parallel. All groups receiving adjuvant protocols developed enhanced serologic responsiveness. Similar profiles were observed in mice treated with glucan or lipovant. Animals receiving promastigotes in Freund's complete adjuvant also developed strong humoral responses, binding cross-reactive epitopes not recognized by other groups. Our findings indicate that glucan and lipovant present effective adjuvant alternatives, to Freund's complete adjuvant and may be of value in immunization against visceral leishmaniasis.
An improved protocol for PCR analysis of Mycobacterium leprae-infected tissues, based on enzymatic lysis, has been developed and used to demonstrate the feasibility of using PCR for detecting M. leprae in routine skin biopsies taken from leprosy patients throughout the clinical spectrum. Of 92 multibacillary patients tested, 99% were PCR-positive using gel electrophoresis or DNA hybridization to detect the amplified product. Similar analysis of paucibacillary patients, in which only one of 27 biopsies had demonstrable AFB microscopically, gave a positivity rate of 74%. No PCR signals were demonstrated from skin biopsies from seven patients with non-leprosy dermatoses and one AIDS patient with a disseminated atypical mycobacteriosis. Evaluation of leprosy patients with antileprosy drug therapy prior to biopsy demonstrated that PCR signals were either greatly diminished or absent after 2 months of continuous antibiotic therapy. PCR was also able to detect the presence of M. leprae in tissues of patients receiving antibacterial therapy when patients were suspected of harbouring drug-resistant M. leprae.
The effect of interleukin-2 (IL-2) on systolic blood pressure in Dahl salt-sensitive rats was investigated. The treatment group received human IL-2 injections (5000 units/kg). Control animals received subcutaneous saline injections. Both groups of animals were placed on a diet containing 1.5% sodium the day of the first injection and maintained on that diet for the duration of the study. Systolic blood pressure increased in both the IL-2 treated and the control groups (P = .0001) over 7 weeks. The increase in SBP was the same for both groups (P = .8823 for between group differences). At the end of 7 weeks, when SBP in both groups was elevated to a similar degree, the IL-2 group and the control group were each administered 5000 units/kg of IL-2. SBP in both groups remained elevated, showing no decrease over the next two weeks. These results indicate that perhaps unlike in SHR, IL-2 does not alter systolic blood pressure in Dahl salt-sensitive rats.
DNA hybridization studies using a 611-base pair (bp) probe, encoding the entire 18-kDa protein of Mycobacterium leprae, demonstrated that M. simiae, M. intracellulare, M. kansasii, M. terrae, ADM-2, M. avium, M. scrofulaceum, M. gordonae and M. chelonei appear to possess DNA sequences homologous to the 18-kDa protein gene of M. leprae. RFLP analysis revealed that the restriction sites in the M. leprae 18-kDa gene were not conserved in the putative gene homologs of M. simiae and M. intracellulare. The restriction patterns observed with the 611-bp probe were useful in differentiating M. intracellulare, M. simiae, and M. leprae from each other, as well as in distinguishing strains of M. simiae serovar 1. Finally, the presence of homologous sequences in various mycobacteria did not affect the specificity of a previously described PCR test for detection of M. leprae, based on the M. leprae 18-kDa protein gene.
The effects of standard fixatives (10% neutral buffered formalin, ethanol and mercury based) on the detection of Mycobacterium leprae DNA by the polymerase chain reaction (PCR) were studied. Mercury-based fixatives (Zenker's and Carnoy-Lebrun's fluids) strongly inhibited PCR amplification of M. leprae DNA. Ten percent neutral buffered formalin was inhibitory, but significant inhibition was observed only when fixation times exceeded 24 h. Ethanol-based fixatives provided the best medium for holding specimens for subsequent PCR with both free bacilli and skin biopsy specimens containing M. leprae. The M. leprae-specific, 360-bp region of the 18-kDa protein gene could be amplified from paraffin-embedded sections of formalin-fixed skin biopsy specimens from patients with either multibacillary or paucibacillary infections when proper fixation conditions were used. Results of the study demonstrate that tissues properly fixed with two standard fixatives (10% neutral buffered formalin and 50 or 70% ethanol) can be analyzed by PCR for the presence of M. leprae with no loss in specificity and only minimal diminution in sensitivity compared with the specificities and sensitivities obtained by use of freshly prepared, unfixed specimens.
Hepatocytes are known to synthesize nitric oxide (NO) from L-arginine via an inducible NO synthase. Studies were performed to determine the relationship between hepatocyte NO production and the stimulation of hepatocyte soluble guanylate cyclase. A combination of lipopolysaccharide (LPS), interferon-gamma, tumor necrosis factor, and interleukin-1 stimulates the biosynthesis of large quantities of nitrite and nitrate (NO2- + NO3-). Hepatocyte NO2- + NO3- production was associated with only small increases in intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels but much greater increases in extracellular cGMP release over an 18-h time period. This cGMP synthesis was dependent on the L-arginine concentration and was inhibited in a reversible manner by NG-monomethyl-L-arginine. The cytokines or LPS added alone induced small increases in nitrogen oxide production and concomitant minor elevations in cGMP release. Atrial natriuretic peptide also stimulated the release of cGMP by hepatocytes which appeared to be independent of the cytokine+LPS-induced cGMP release. The addition of probenecid reduced the cGMP release by 66%, while cell damage was excluded as a cause for the extracellular release. Addition of 3-isobutyl-1-methylxanthine, but not M&B 22948, increased hepatocyte intra- and extracellular cGMP levels after cytokine+LPS stimulation. Induction of nitrogen oxide synthesis by hepatocytes in vivo by injecting rats with killed Corynebacterium parvum resulted in increased cGMP levels in freshly isolated hepatocytes and increased cGMP release by the hepatocytes when placed in culture.(ABSTRACT TRUNCATED AT 250 WORDS)
The purpose of the study was to employ psychoeducational, neuropsychological, and sociobehavioral (Conners Rating Scale) variables in determining if definable subtypes exist within a diverse population of subjects with learning disabilities (LD) and documented brain damage. The sample of 95 subjects (27% female and 73% male) had been referred for neuropsychological assessment at a large, Midwestern medical center. Mean age was 10.6 years. Brain damage (BD) was documented for 45% of the sample. The first cluster analysis employed neuropsychological, psychoeducational, and sociobehavioral data and revealed four interpretable clusters. A second cluster analysis excluded sociobehavioral data and yielded two interpretable clusters. In neither analysis did a cluster consist exclusively of BD or LD subjects. Results were interpreted as supporting the importance of the sociobehavioral component in LD subtyping, as well as supporting the contention that parallels may exist in cerebral function and/or structure between the LD and BD classifications.
Previous studies showed that apolipoprotein-E (apoE) mRNA is regulated in rat adrenal gland by treatments that alter adrenal gland cholesterol content and steroidogenesis. In the present study cell types expressing apoE mRNA were determined by in situ hybridizations using an [alpha-35S]UTP-labeled RNA probe. Autoradiographic grains were counted to compare apoE expression in adrenal glands from control and experimentally treated animals. In control adrenal gland, zona (z.) fasciculata and z. reticularis exhibited the highest level of apoE mRNA expression, with lower levels in z. glomerulosa and medulla. Dexamethasone (DEX) treatment selectively increased apoE mRNA 3-fold in outer z. fasciculata, but not in other adrenal zones. ApoE mRNA expression appeared to be lower in adrenal glands from 4-aminopyrazolopyrimidine-treated rats, in that differences among adrenal gland zones were abolished. DEX treatment increased adrenal gland cholesteryl ester and oil red O staining in z. fasciculata cells in which the apoE mRNA concentration was increased as well as in other cortical cells in which apoE mRNA was unchanged. Aminoglutethimide administration led to a large increase in oil red O staining throughout the cortex, including z. fasciculata, without affecting apoE mRNA expression. These data suggest that adrenal gland apoE mRNA expression is not closely coupled to cellular cholesterol concentrations. Increased apoE mRNA expression in z. fasciculata of DEX-treated animals suggests an inverse relationship between apoE mRNA concentration and the level of steroidogenesis. This result is consistent with the proposal that apoE may play a role in regulating the utilization of cholesterol for steroid production.