Biomedical subjects

R G Ulrich

Publications and source records attributed to R G Ulrich.

At least 37 records · Page 2Linked to original sources

PubMed1996

Potentiation of hypoxic injury in cultured rabbit hepatocytes by the quinoxalinone anxiolytic, panadiplon.

The quinoxalinone anxiolytic, panadiplon, produces hepatic metabolic inhibition (mitochondrial impairment), microvesicular steatosis and centrilobular necrosis in rabbits. Metabolic inhibition occurs in cultured hepatocytes without cytotoxicity, suggesting that hepatic injury is influenced by additional factors. The present experiments were conducted to determine if metabolic inhibition by panadiplon predisposed hepatocytes to hypoxic injury. Injury (cell death) was evaluated by lactate dehydrogenase (LDH) release from cells; ATP and glycogen levels were also evaluated. Under hypoxic conditions, control cultures showed a 6.5-fold increase in LDH release compared to normoxic controls, with a coincident 80% decrease in ATP and 50% decrease in glycogen levels. Under normoxic conditions 10 microgram/ml panadiplon treatment for 48 h reduced ATP and glycogen levels by 40% but did not cause an increase in LDH leakage. Cells treated with panadiplon, then exposed to hypoxia conditions, showed a significant level of injury compared to normoxic control cultures, and a further reduction in ATP. No additional decrease in glycogen ws observed. In an attempt to prevent panadiplon-mediated injury, glycolytic substrates (dihydroxyacetone or pyruvate) were included during normoxic and hypoxic incubations. Both cotreatments reduced the level of LDH leakage produced by panadiplon during hypoxia. Cotreatment did not generally increase ATP or glycogen levels (compared to panadiplon treatment groups) during hypoxia, though individual experiments showed a slight increase in ATP levels. During normoxia both cotreatments with panadiplon resulted in significantly higher glycogen levels than in panadiplon cultures alone. These results suggest that cellular glycogen and subsequently ATP levels are reduced during panadiplon exposure, metabolically predisposing hepatocytes to hypoxic injury.

Adenosine Triphosphate↗

PubMed1996

Identification of dioxin-responsive genes in Hep G2 cells using differential mRNA display RT-PCR.

Differential mRNA display RT-PCR (DD RT-PCR) offers a tool to identify genes which are regulated or responsive to certain receptors or chemicals such as dioxin (TCDD). Treatment of Hep G2 cells with TCDD followed by DD analysis of a gel with series of different primers revealed a significantly different pattern from the control for a number of mRNAs. The differentially displayed mRNAs were isolated and reamplified. A GenBank search of four mRNAs revealed two known and two unknown sequences. Northern blot analysis revealed that two known sequences, fibrinogen gamma chain and plastin mRNAs were down regulated by TCDD in a time-dependent manner, whereas two unknown mRNAs were induced by TCDD treatment. The function of these genes in TCDD toxicity is not known; however, the application of DD RT-PCR in the studies of TCDD-induced responses could be very useful in the discovery of other unknown genes important for TCDD toxicity.

Base Sequence↗

PubMed1996

Superantigen vaccines: a comparative study of genetically attenuated receptor-binding mutants of staphylococcal enterotoxin A.

Superantigens exert their pathologic effects by direct binding to major histocompatibility complex (MHC) class II molecules and T cell antigen receptors (TCR), thus circumventing the normal, antigen-specific immune response. A direct link between disease and toxin suggests an excellent opportunity for vaccine intervention. Site-directed mutants of staphylococcal enterotoxin A (SEA) that have attenuated binding to either the TCR or the MHC class II molecule were developed. Both kinds of SEA mutants induced high levels of antibody against SEA when used as vaccines, and the immunized animals were fully protected when challenged with wild type toxin. However, a residual lethality was associated with the attenuated TCR-binding mutant. These results, combined with an understanding of the molecular nature of superantigen and receptor interactions, indicate that targeting MHC class II binding by site-directed mutagenesis will produce the most effective vaccine.

Animals↗

PubMed1996

Biotransformation of lifibrol (U-83860) to mixed glyceride metabolites by rat and human hepatocytes in primary culture.

Lifibrol (U-83860), K12.148) is a lipid-lowering drug that has the potential to accumulate in the liver and induce hepatic peroxisome proliferation. To investigate the identity and potential human relevance of persistent lifibrol-related residues in rat liver, rat and human hepatocyte primary cultures were treated with 30 microM of [14C]lifibrol. After a steady uptake for 24 hr, cellular levels of radioactivity became stable for the next 24-48 hr. A nonradioactive lifibrol chase caused an efflux of intracellular radioactivity. Cellular autoradiography revealed the association of radioactivity with small lipid drops at 6 hr exposure and with large lipid drops at 24 hr exposure. HPLC analysis of media revealed that lifibrol acyl glucuronide and a t-butylcarboxylic acid metabolite (U-94613) were the major metabolites of rat and human hepatocytes, respectively. Using an HPLC method suitable for nonpolar metabolites, the analysis of rat and human cell extracts revealed a broad band of multiple, radioactive peaks that had a similar retention and UV spectrum to a synthetic standard of lifibrol cholesterol ester. Folch extracts of liver from rats treated with [14C]lifibrol or unlabeled lifibrol and [14C]acetate had a unique radioactive TLC band that had similar HPLC retention to hepatocyte residues. The group of nonpolar peaks from the hepatocytes was purified by HPLC. Conversion of the lifibrol sec-hydroxy group to a nicotinate ester afforded particle beam-electron impact mass spectra of the cholesterol ester standard and hepatocyte residues. The derivatized rat hepatocyte residue did not contain detectable lifibrol cholesterol ester (M+.816), but did contain molecular ion clusters corresponding to a mixed triglyceride of lifibrol and two fatty acids. Lifibrol-specific product ions of molecular ion clusters centered at M+.1021, 1047, and 1073 were observed at m/z 448, 430, and 310. The major lifibrol-containing triglycerides had a fatty acid composition of C16-C20 with 0-6 unsaturations.

Animals↗

PubMed1995

Method for measuring the activities of cholesteryl ester transfer protein (lipid transfer protein).

A continuous recording fluorescence assay was developed for cholesteryl ester transfer protein (CETP). The assay measures the increase in fluorescence accompanying the relocation of fluorescent lipids, cholesteryl esters and triglycerides, from a donor emulsion to an acceptor emulsion. In the absence of CETP, the quantum yields of the fluorescent lipids is low because their high concentrations in the donor emulsions result in self-quenching. CETP catalyzes the redistribution of the fluorescent lipids from the donor to the acceptor emulsions and fluorescence increases substantially. Efficient sonication and incorporation of apolipoproteins from human HDL into the emulsions significantly increased the transfer rates. Under optimal conditions, the redistribution of fluorescent compounds reaches equilibrium within < 30 min and the kinetics of this process are consistent with a simple, first-order reaction pathway. The redistribution kinetics support a mechanism of adsorption --> exchange --> desorption --> diffusion.

Boron Compounds↗

PubMed1995

Induction of a hepatic toxic syndrome in the Dutch-belted rabbit by a quinoxalinone anxiolytic.

The non-benzodiazepine anxiolytic, panadiplon, was discontinued from clinical development due to evidence of hepatic toxicity in human volunteers that was not predicted by rat or monkey preclinical development studies. The present study was conducted to examine potential toxicity in the rabbit. Three groups of female rabbits were administered vehicle, 10 mg/kg per day or 20 mg/kg per day of panadiplon by oral gavage for 14 days. Animals in the 20 mg/kg group lost weight, and 6/10 developed a profound lethargy. Hepatic toxicity was observed in treated animals, evidenced by dose- and time-related increases in serum transaminase activities, gross hepatic lesions and multifocal centrilobular necrosis. Hepatic microvesicular steatosis was evident in treated animals; lipid analysis revealed a 123% increase in hepatic triglyceride. A time-dependent increase in serum triglyceride levels was observed in the high-dose group beginning on day 4. Hepatic glycogen was reduced, and histochemical examination revealed the reduction to be heterogeneous across the lobule with some areas showing a complete absence of glycogen. One rabbit in each drug-treated group showed mild hypoglycemia at day 12, and 4/10 rabbits in the high-dose group showed hyperglycemia at days 12-14. We conclude that panadiplon produced a microvesicular steatosis and hepatic toxicity in the rabbit. The observed toxicity resembled a Reye's syndrome-like toxicity produced by a variety of mitochondrial fatty acid oxidation inhibitors.

Animals↗

PubMed1995

Upregulation of glucose-6-phosphate dehydrogenase in response to hepatocellular oxidative stress: studies with diquat.

The expression of hepatic glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) is hypothesized to be modulated by free radicals during oxidative stress. The ability of diquat, a compound known to enhance oxidative stress through generation of reactive oxygen species, to modulate the expression of G6PDH in primary cultures of Fischer-rat hepatocytes was examined. Diquat-treated hepatocytes maintained in a chemically defined medium showed both a time- and concentration-dependent increase in G6PDH enzyme activity. This increase in enzyme activity was accounted for by an increase in both G6PDH mRNA and immunoreactive protein, suggesting control at a pretranslational level. The possibility that diquat increased transcription by transfecting cells with a chimeric gene containing 935 bp of the G6PDH promoter (-878 to +57) linked to the gene for chloramphenicol acetyl-transferase (CAT) was examined. Hepatocytes transiently transfected with this chimera, and subsequently treated with diquat, exhibited an increase in CAT activity. However, hepatocytes transfected with a chimera containing 287 bp of the G6PDH promoter (-230 to +57) exhibited only basal CAT activity in the presence of diquat. These results suggest that regions in the DNA sequences required for diquat-induced expression of G6PDH lie between base pairs -878 and -230 of the G6PDH gene. These findings are suggestive that oxidative stress in hepatocytes increased the expression of G6PDH activity and protein and that the increased expression is controlled at the transcriptional level.

Animals↗

PubMed1995

Divergence of human and nonhuman primate lymphocyte responses to bacterial superantigens.

We compared T cell responses of human, rhesus monkey (Macaca mulatta), and chimpanzee (Pan troglodytes) to four bacterial superantigens. When lymphocytes were cultured in media supplemented with species-specific sera, chimpanzee T cells were stimulated by lower doses of staphylococcal enterotoxin (SE) A and toxic shock syndrome toxin 1 (TSST1) than were human T cells, while chimpanzee responses to SEB and SEC1 were nearly equivalent to the human response. Interestingly, rhesus lymphocytes responded to 10,000 times lower amounts of SEA, SEB, and SEC1 and to 100 times lower concentrations of TSST1 than human cells. The greater sensitivity of rhesus T cells to these toxins was not a result of differences in class II binding affinities and was only partly attributable to the presence of anti-SE and TSST1 antibodies in human serum. These results suggest that rhesus T lymphocytes are more sensitive toward these bacterial superantigens than human T cells.

Animals↗

PubMed1995

Cultured hepatocytes as investigational models for hepatic toxicity: practical applications in drug discovery and development.

Drugs can fail at any phase during discovery, preclinical or clinical development due to unacceptable levels of toxicity, and liver is commonly the principle target organ. Investigational toxicology methods, using appropriate models and hypotheses, can often resolve problems, identify toxic chemical substituents and salvage therapeutic discovery programs. While in vivo models are used to investigate hepatic drug effects in the context of toxicokinetics and systemic influences, cell culture models provide in vitro systems for investigating specific mechanisms in a precisely controlled environment. Using primary hepatocytes isolated from laboratory animals, we have explored several drug-induced hepatic disorders that surfaced during different phases of drug discovery and development. Additionally, the use of human hepatocytes has allowed us to address concerns for human exposure, examine human relevance of animal data, and provide perspective on problems encountered in clinical trials.

Animals↗

PubMed1995

Bacterial superantigens in human disease: structure, function and diversity.

All bacterial superantigens use common structural strategies to bind to major histocompatibility complex class II receptors, while binding the T cell antigen receptor in different ways. Overstimulation of the immune response is responsible for the acute pathological effects, while reactivation of developmentally silenced T cells might result in autoimmune disease. Certain diseases might be controlled with superantigens or genetically attenuated vaccines.

Adjuvants, Immunologic↗

PubMed1995

Staphylococcal enterotoxins A and B share a common structural motif for binding class II major histocompatibility complex molecules.

A comparative site-directed mutagenesis study of staphylococcal enterotoxins A and B was undertaken to identify key amino-acid residues which govern interactions with major histocompatibility class II molecules. This involved generating a three-dimensional homology model for enterotoxin A in complex with the HLA-DR1 molecule, based on the reported X-ray crystal structures of enterotoxin B, both free and bound to HLA-DR1. A binding motif previously described for enterotoxin B was found to be conserved in enterotoxin A. An examination of the experimental data with the homology model clarifies how T-cell responses to enterotoxin A, and most bacterial superantigens, are likely to be mediated by variations of a structurally conserved HLA-DR alpha binding motif.

Amino Acid Sequence↗

PubMed1995

Staphylococcal enterotoxin A and toxic shock syndrome toxin compete with CD4 for human major histocompatibility complex class II binding.

We have examined the role of the CD4 molecule in primary T-lymphocyte responses to the staphylococcal enterotoxins SEA, SEB, SEC1, and the toxic shock syndrome toxin TSST-1. Proliferating cells were predominantly CD4+; however, the responses to SEA and TSST-1 were most sensitive to inhibition by the anti-CD4 antibody Leu-3a. T-lymphocyte responses to the bacterial superantigens were inhibited by site-directed mutations of residues in the DR beta membrane-proximal domain (DR beta 2) that are also known to be important for interactions with CD4. SEA and TSST-1 binding to DR was reduced by the DR beta 2 mutations and by competition with soluble recombinant CD4. We propose that bacterial superantigens sequentially, or simultaneously with CD4, stabilize complexes of T-cell antigen receptors and major histocompatibility complex class II molecules. The superantigen qualities of these toxins may be due, in part, to a molecular mimicry of CD4 and other adhesion molecules.

Bacterial Toxins↗

PubMed1995

Inactivation of Serpulina hyodysenteriae flaA1 and flaB1 periplasmic flagellar genes by electroporation-mediated allelic exchange.

Serpulina hyodysenteriae, the etiologic agent of swine dysentery, contains complex periplasmic flagella which are composed of multiple class A and class B polypeptides. To examine the role these proteins play in flagellar synthesis, structure, and function and to develop strains which may provide insight into the importance of motility in the etiology of this pathogen, we constructed specific periplasmic flagellar mutations in S. hyodysenteriae B204. The cloned flaA1 and flaB1 genes were disrupted by replacement of internal fragments with chloramphenicol and/or kanamycin gene cassettes. Following delivery of these suicide plasmids into S. hyodysenteriae, homologous recombination and allelic exchange at the targeted chromosomal flaA1 and flaB1 genes was verified by PCR, sequence, and Southern analysis. The utility of a chloramphenicol resistance gene cassette for targeted gene disruption was demonstrated and found more amenable than kanamycin as a selective marker in S. hyodysenteriae. Immunoblots of cell lysates of the flagellar mutants with antiserum raised against purified FlaA or FlaB confirmed the absence of the corresponding sheath or core protein. Both mutations selectively abolished expression of the targeted gene without affecting synthesis of the other flagellar polypeptide. flaA1 and flaB1 mutant strains exhibited altered motility in vitro and were less efficient in movement through a liquid medium. Paradoxically, isogenic strains containing specifically disrupted flaA1 or flaB1 alleles were capable of assembling periplasmic flagella that were morphologically normal as evidenced by electron microscopy. This is the first report of specific inactivation of a motility-associated gene in spirochetes.

Bacterial Proteins↗

PubMed1995

Insulin secretory defect in Zucker fa/fa rats is improved by ameliorating insulin resistance.

The role of insulin resistance in the impaired glucose-stimulated insulin release of Zucker fatty rats was investigated using the insulin-sensitizing thiazolidinedione drug pioglitazone. Fatty rats had fasting hyperinsulinemia yet a blunted secretory response to intravenous glucose compared with lean age-matched controls. Islets from fatty rats secreted less insulin (based on islet DNA) in response to high glucose than islets from lean rats but secreted normal amounts of insulin when tolbutamide or alpha-ketoisocaproic acid (alpha-KIC) was the stimulus. Administering pioglitazone for 9 days diminished basal hyperinsulinemia and increased the insulin response to high glucose by fatty rats but not by lean controls. Pioglitazone pretreatment augmented the secretory response by isolated islets to high glucose, alpha-KIC, and tolbutamide. Augmentation of islet insulin release was not associated with reduced plasma glucose concentration, suggesting that altered glycemia was not involved. Pancreas and islet insulin content was greater in fatty rats than in lean controls and was decreased by pioglitazone; hence, insulin stores and glucose-stimulated insulin release did not correlate. Pioglitazone treatment did not affect the rate of islet glucose usage or ATP/ADP in the presence of 2.75 or 16 mmol/l glucose. These data indicate that ameliorating insulin resistance reverses defective glucose-stimulated insulin release by Zucker fa/fa rats. After pioglitazone administration, insulin secretion may be augmented by increased generation of a metabolic coupling factor from glucose or at a later step in the secretory process that is common to both glucose and nonglucose secretagogues.

Aging↗

PubMed1995

Strontium excitability of the inner mitochondrial membrane: regenerative strontium-induced strontium release.

Regenerative Sr(++)-induced Sr++ release from isolated rat liver mitochondria was studied using ion-selective electrode techniques. Mitochondria, when exposed to a pulse of Sr++, demonstrated a reversible and transient increase in inner membrane permeability to K+ and H+ ions. The increase in permeability was an all-or-none process with a threshold dependent on the amplitude of the Sr++ pulse. The threshold concentration of Sr++ was lowered from 120-150 microM to 20-30 microM when mitochondria were preloaded with 100 nmoles Sr++/mg protein. Release of matrix-stored divalent cations provided a mechanism for amplification of the extramitochondrial Sr++ concentration (regenerative Sr(++)-induced Sr++ release). The mitochondrial inner membrane became refractory, since subsequent cycles of excitation could not be immediately induced. These experiments demonstrate that the inner mitochondrial membrane is an excitable membrane, with threshold-dependent, regenerative and subsequently refractile Sr(++)-induced Sr++ release characteristics.

Animals↗

PubMed1995

Nucleotides-induced cytosolic calcium transient and plasma membrane permeability in Chang human liver cells.

Extracellular nucleotides induce changes in cytosolic free Ca++ and also increase plasma membrane permeability to Ca++ ions in Chang human liver cells. Ca++ permeability induced by nucleotides is reversible and inactivated immediately upon removal of agonist. Stimulated cells transferred into the fresh medium and re-exposed to nucleotides demonstrate reopening of Ca++ channel without stimulation of Ca++ transient. Relative potencies of nucleotides to induce membrane permeability and Ca++ transient were: UTP > ATP > gamma-S-ATP > ADP (non-hydrolyzable ATP analogues and AMP had no effect). The permeability is not affected by specific inhibitors of voltage-operated calcium channels (verapamyl,cis-diltiazem and nifedipin). Nucleotides do not produce plasma membrane damages at concentrations up to 1 mM, as shown by exclusion of the propidium iodide. There are at least two types of nucleotides receptors in Chang cell membrane: P2y subtype receptors which is responsible for generation of the Ca++ transient, and P2x subtype receptors which lead to the opening of plasma membrane Ca++ channels upon activation.

Adenosine Diphosphate↗

PubMed1994

The development of fatty liver is accelerated in transgenic mice expressing cynomolgus monkey cholesteryl ester transfer protein.

Expression of cynomolgus monkey cholesteryl ester transfer protein (CETP) in C57BL/6 mice has been shown to have a profound effect on the lipoprotein profile in those animals. The objective of this study was to examine the effect of CETP expression on the hepatic lipids of the CETP transgenic mice. The triglyceride, cholesterol and phospholipid composition of livers from 6- and 12-month-old transgenic mice were evaluated and compared with those of age-matched C57BL/6 mice. Statistical analysis indicated that fatty liver was more severe in CETP transgenic mice than C57BL/6 controls (p < 0.01); progressed with age (p < 0.01); and developed more rapidly in males than females (p < 0.01). The lipid that accumulated was triglyceride. These data indicate that CETP expression accelerates the development of fatty liver in the C57BL/6 mouse and raise the possibility that CETP may also contribute to the process of hepatic steatosis in man.

Aging↗

PubMed1993

Toxicity of staphylococcal enterotoxins potentiated by lipopolysaccharide: major histocompatibility complex class II molecule dependency and cytokine release.

The biological effects of staphylococcal enterotoxins (SE), potentiated by bacterial lipopolysaccharide (LPS), were studied with mice. Control animals survived the maximum dose of either SE or LPS, while mice receiving both agents died. SEA was 43-fold more potent than SEB and 20-fold more potent than SEC1. The mechanism of toxicity was further examined with transgenic mice deficient in major histocompatibility complex class I or II expression. Class II-deficient mice were resistant to SEA or SEB. However, class I-deficient animals were less susceptible to SEA (30% lethality) than wild-type mice (93% lethality). In vitro stimulation of T cells from the three mouse phenotypes by SEA correlated well with toxicity. T cells from transgenic or wild-type mice were similarly responsive to SEA when presented by irradiated, wild-type mononuclear cells. These data confirmed that the toxicity of SE was mainly exerted through a mechanism dependent on the expression of major histocompatibility complex class II molecules. Toxicity was also linked to stimulated cytokine release. Levels in serum of tumor necrosis factor alpha, interleukin-6, and gamma interferon peaked 2 to 4 h after the potentiating dose of LPS but returned to normal within 10 h. Concentrations of interleukin-1 alpha were also maximal after 2 h but remained above the background for up to 22 h. Relative to the levels in mice given only SEA or LPS, the levels in serum of tumor necrosis factor alpha, interleukin-6, and gamma interferon increased 5-, 10-, and 15-fold, respectively, after injections of SEA plus LPS. There was only an additive effect of SEA and LPS on interleukin-1 alpha concentrations.

Animals↗