"The VIHsibilite Project": HIV-positive people in the Quebec press and community responses.

The VIHsibilite Project is a community-based action-research initiative that examines newspaper coverage of HIV/AIDS issues in Quebec from 1988 to 2004. Using standard qualitative research methods, and in consultation with an advisory committee of people directly impacted by HIV/AIDS news coverage, the project discerns trends in reporting on HIV/AIDS and undertakes discursive content analysis of these, aiming to better understand in what normative ways seropositive people are represented in print media, and, ultimately, to reduce the stigma attendant upon HIV infection. Preliminary findings include indications that seropositive women tend to be represented markedly differently from men in the news.

Peptidomimetic inhibitors of herpes simplex virus ribonucleotide reductase with improved in vivo antiviral activity.

We have been investigating the potential of a new class of antiviral compounds. These peptidomimetic derivatives prevent association of the two subunits of herpes simplex virus (HSV) ribonucleotide reductase (RR), an enzyme necessary for efficient replication of viral DNA. The compounds disclosed in this paper build on our previously published work. Structure-activity studies reveal beneficial modifications that result in improved antiviral potency in cell culture in a murine ocular model of HSV-induced keratitis. These modifications include a stereochemically defined (2,6-dimethylcyclohexyl)amino N-terminus, two ketomethylene amide bond isosteres, and a (1-ethylneopentyl)amino C-terminus. These three modifications led to the preparation of BILD 1351, our most potent antiherpetic agent containing a ureido N-terminus. Incorporation of the C-terminal modification into our inhibitor series based on a (phenylpropionyl)valine N-terminus provided BILD 1357, a significantly more potent antiviral compound than our previously published best compound, BILD 1263.

Metabolism of beta-methyl-heptadecanoic acid in the perfused rat heart and liver.

The metabolism of beta-methyl-[1-14C]heptadecanoic acid, a potential myocardial imaging agent, was investigated in perfused hearts and livers from rats. Hepatic uptake is approximately 4.5 times greater than cardiac uptake. In the heart, 66% of beta-methyl-heptadecanoic acid metabolism occurs via omega-oxidation, 33% by esterification and less than 1% via alpha-oxidation. In contrast, 53% of hepatic metabolism of beta-methyl-heptadecanoic acid occurs via alpha-oxidation, 27% via omega-oxidation, and 20% via esterification. Perfusion of hearts and livers with concentrations of beta-methyl-heptadecanoic acid 100 to 1000 times greater than that used for myocardial imaging does not alter any of the physiological and biochemical parameters measured. In the perfused liver, 3-methyl-[1-14C]glutarate was identified as the principal hydrosoluble catabolite of beta-methyl-heptadecanoic acid.

Cutaneously applied acyclovir acts systemically in the treatment of herpetic infection in the hairless mouse.

Using the SKH-1 hairless mouse (HM) we have addressed the issue as to whether topically applied acyclovir (ACV) may mediate some of its antiviral actions by a systemic effect. When topically applied in a formulation consisting of polyvinyl alcohol (25% w/v):DMSO:cremophor EL:linoleic acid (63:16:16:5, v/v/v/v), ACV penetrated hairless mouse skin in a concentration-dependent manner and dose-dependently reduced cutaneous herpes simplex virus 1 (HSV-1) KOS infection. Topically applied ACV also effectively reduced the mortality associated with disseminated HSV-2 HG-52 infection. At 1 h following topical application of 1.7% w/v ACV the plasma and skin concentrations of ACV were 5.5 nmoles/ml and 120 nmoles/g. At 1 h following an oral dose of ACV with antiviral efficacy comparable to topically applied ACV (1.7% w/v) the plasma and skin concentrations of ACV were 21.3 nmoles/ml and 51 nmoles/g. These findings imply that when applied topically to the HM, ACV can mediate a portion of its antiviral activity through a systemic mode of action.

Acute murine cytomegalovirus infection: a model for determining antiviral activity against CMV induced hepatitis.

Acute intraperitoneal infection of weanling BALB/c mice with murine cytomegalovirus (MCMV) resulted in an inoculum titer-dependent weight loss, mortality and elevation of plasma transaminases (ALT: alanine transaminase and AST: aspartate transaminase). Three days post infection (p.i.) with 10(4.85) plaque forming units (pfu) there was 90% mortality with a mean death day p.i. of 4.1 +/- 0.2. Plasma levels of ALT and AST were elevated 24- and 15-fold, respectively. Organ titers of virus (log10 pfu/g tissue) were 6.16 in the liver, 6.05 in the spleen, 4.0-4.7 in the lung, heart, kidney and intestine and undetectable in the muscle and brain. Organ concentrations (units/g wet-weight) of ALT were highest in the liver, whilst for AST the highest levels were found in the heart. The concentrations of ALT but not AST were reduced (35-55%) in the infected liver; the concentrations of ALT and AST were not changed in other infected organs. There were excellent correlations (r > 0.95) between viral titers in the liver, increases of plasma ALT and depletion of liver ALT. HPMPC and ganciclovir administered either p.o. or s.c. reduced mortality, increases in plasma transaminases and viral burdens in the liver and prevented depletion of liver ALT. HPMPC was approximately 10-fold more potent than ganciclovir. These results strongly suggest that intraperitoneal infection of the BALB/c mouse with MCMV represents an animal model of CMV hepatitis that can be monitored by measuring plasma ALT.

Gastric dilatation and volvulus in a brachycephalic dog with hiatal hernia.

A brachycephalic dog was presented with an acute onset of retching and abdominal discomfort. The dog had a chronic history of stertor and exercise intolerance suggestive of brachycephalic airway obstructive syndrome. Radiographs were consistent with a Type II hiatal hernia. The dog was referred and within hours of admission became acutely painful and developed tympanic abdominal distension. A right lateral abdominal radiograph confirmed gastric dilatation and volvulus with herniation of the pylorus through the hiatus. An emergency exploratory coeliotomy was performed, during which the stomach was derotated, and an incisional gastropexy, herniorrhaphy and splenectomy were performed. A staphylectomy was performed immediately following the exploratory coeliotomy. The dog recovered uneventfully. Gastric dilatation and volvulus is a potentially life-threatening complication that can occur in dogs with Type II hiatal hernia and should be considered a surgical emergency.

Metabolism of 2,3-butanediol stereoisomers in the perfused rat liver.

The identification of 2,3-butanediol in sera of alcoholics led to the hypothesis that it may be a specific marker of alcohol abuse. We have investigated the metabolism of the individual isomers of 2,3-butanediol (2R,3R-, 2S,3S-, meso-2,3-butanediol and racemic 2,3-butanediol) in perfused livers from fed rats. Rates of uptake of the isomers decrease in the order (i) 2R,3R-, (ii) meso-, (iii) 2S,3S-2,3-butanediol. We observed interconversion of isomers and oxidation to acetoin with 2R,3R- and meso- but not with 2S,3S-2,3-butanediol. In perfusions conducted in deuterium oxide, interconversion of isomers was accompanied by incorporation of deuterium. Thus, interconversion of isomers occurs via a reversible oxidation to acetoin with incorporation of hydrogen from water. In perfusions with either 2R,3R- or meso-[2-14C]2,3-butanediol, the substrates were converted to labeled acetate, R-3-hydroxybutyrate and CO2, suggesting that 2,3-butanediol is oxidized to acetyl-CoA via acetoin.

Nonhomogeneous labeling of liver mitochondrial acetyl-CoA.

The specific activity of carbons 1 and 2 of plasma acetoacetate has been used as a measure of the specific activity of liver mitochondrial acetyl-CoA in tracer studies. To test whether or not acetoacetate actually reflects acetyl-CoA, livers were perfused with a mixture of substrates that are converted to mitochondrial acetyl-CoA: 1 mM lactate, 0.2 mM pyruvate, 0.2 mM acetate, and, where indicated, 0.2 mM octanoate or 0.2 mM alpha-ketoisocaproate. In each experiment, one of these substrates was 13C-labeled. Labeling of mitochondrial acetyl-CoA was assessed by three methods: (i) molar percent enrichment of total tissue acetyl-CoA; (ii) molar percent enrichment of carbons 4 and 5 of tissue citrate, the precursor of which is acetyl-CoA; and (iii) molar percent enrichment of carbons 1 and 2 of perfusate ketone bodies. Nonhomogeneous labeling of liver mitochondrial acetyl-CoA occurred under most conditions, i.e. the enrichments of carbons 4 and 5 of citrate were different from enrichments of carbons 1 and 2 of ketone bodies. Thus, based upon our results obtained in perfused livers, we question the validity of measuring the labeling of carbons 1 and 2 of acetoacetate as a noninvasive probe of liver mitochondrial acetyl-CoA.

Characterization of intestinal smooth muscle responses and binding sites for endothelin.

The contractile activity of and binding sites for endothelin-1 (ET-1) were investigated in isolated guinea-pig ileal longitudinal smooth muscle (GPILM). ET-1 produced concentration-dependent contractions of GPILM that either slowly subsided in the continued presence of ET-1 or rapidly subsided following washing of the tissue. The ED50 value for ET-1 contractions was 4.2 +/- 1.3 x 10(-9) M. The removal of extracellular calcium or pretreatment with nifedipine produced a complete inhibition of the contractions to ET-1. The IC50 value of nifedipine for inhibition of ET-1 mediated contractions was 3.0 +/- 0.8 x 10(-8) M. ET-1 produced a marked prolonged homologous desensitization of its contractile response but did not affect the responses mediated by carbachol, histamine, serotonin, substance P, and PLA2. High-affinity binding sites for 125I-labelled ET-1 were identified on microsomal membranes prepared from GPILM with Kd and Bmax values obtained by Scatchard analysis of 3.5 +/- 0.6 x 10(-10) M and 2138 +/- 159 fmol/mg protein, respectively. The binding of 125I-labelled ET-1 to GPILM microsomes was characterized by a rapid association (kob value of 0.077 min-1 at a radioligand concentration of 0.45 nM and an extremely slow dissociation (k1 value of 0.011 min-1; t1/2 value of 793 min). The binding was unaffected by the calcium channel antagonists nifedipine, verapamil, and diltiazem (10(-6) M); the receptor antagonists phenoxybenzamine, atropine, and naloxone (10(-6) M) and propranolol; and the peripheral benzodiazepine receptor antagonists Ro 5-4864 and PK 11195 and psychotomimetic drug phencyclidine (10(-5) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Contribution of the shunt pathway of mevalonate metabolism to the regulation of cholesterol synthesis in rat liver.

The modulation of the shunt pathway of mevalonate metabolism (Edmond, J., and Popják, G. (1974) J. Biol. Chem. 249, 66-71) has been studied in livers from fed, starved, and diabetic rats perfused with a physiological concentration (300 nM) of [5-14C] + [5-3H]mevalonate. Shunt activity was measured by (i) production of 14CO2 (corrected for loss of label by exchange reactions) and (ii) production of 3H2O. Contribution of exogenous mevalonate to total mevalonate production (0.06-0.11%) was assessed in parallel experiments by the incorporation of 3H2O into sterols. Inhibition of non-saponifiable lipid synthesis by starvation and diabetes is not associated with an inhibition of mevalonate production but with a major increase in shunting (7-34%) of sterol-bound mevalonate. The shunt pathway of mevalonate metabolism appears to participate in the regulation of cholesterol synthesis.

Cardiac responses and binding sites for endothelin in normal and cardiomyopathic hamsters.

The effects of endothelin-1 in the spontaneously beating atrium and the field-stimulated right ventricular strip preparation and the binding of [125I]endothelin-1 to membrane preparations from the atrium, ventricle, lung parenchyma, kidney and brain were compared using the 90-day-old dystrophic hamster strain CHF 147 (a strain displaying marked cardiomyopathologies at this age) and its age/genetically matched normal control, CHF 148. In the atrium, endothelin-1 produced dose-dependent positive inotropic and positive chronotropic effects in both CHF 148 and CHF 147 over the dose range of 10(-8) M to 3 x 10(-7) M. However, although no significant difference between CHF 148 and CHF 147 was observed for the positive chronotropic effects of endothelin-1, it produced a small, but significantly less positive inotropic effect in CHF 147 compared to CHF 148 at endothelin-1 concentrations of 2 x 10(-8) M and 3 x 10(-8) M. Field-stimulated right ventricular strips from CHF 148 contracted with a greater force compared to those from CHF 147. Endothelin-1 produced a dose-dependent decrease in the developed tension of the field-stimulated ventricular strip preparation, the extent of which did not differ between CHF 148 and CHF 147. [125I]Endothelin-1 bound with high affinity and in an apparently irreversible manner to membranes from both CHF 148 and CHF 147 atrium, ventricle, lung parenchyma, kidney and brain. No significant differences were noted between CHF 148 and CHF 147 for [125I]endothelin-1 binding to membranes prepared from the various tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of R- and S-1,3-butanediol in perfused livers from meal-fed and starved rats.

The metabolism of millimolar concentrations of R- or S-1,3-butanediol has been studied in perfused livers from fed and starved rats. Protocols were designed to measure in the same experiment (i) uptake of the diol, (ii) the contribution of the diol to ketogenesis, (iii) the contribution of the diol to total fatty acid plus sterol synthesis, and (iv) conversion of S-1,3-butanediol into S-3-hydroxybutyrate. Our data show that R- and S-1,3-butanediol are taken up by the liver at the same rate. Most of the metabolism of R-1,3-butanediol is accounted for by conversion to the physiological ketone bodies R-3-hydroxybutyrate and acetoacetate. Only 29-38% of S-1,3-butanediol uptake is accounted for by conversion into physiological ketone bodies. The balance of S-1,3-butanediol metabolism is conversion to S-3-hydroxybutyrate, lipids and CO2.

Determination of the concentration and specific activity of acetone in biological fluids.

The concentration of acetone dissolved in liver perfusion medium was determined by injection of the sample into a gas chromatograph equipped with a Carbopack/Carbowax-packed glass column. Interference from labile acetoacetate which readily decomposes to acetone was eliminated by treating the samples with NaBH4 prior to the analysis. Acetone was detected and quantified as 2-propanol. Separation of labeled 2-propanol in the sample by high-performance liquid chromatography allowed the determination of its specific activity. These methods make possible the convenient and rapid determination of acetone concentration and specific activity in biological samples.

Compartmentation of 14CO2 in the perfused rat liver.

The specific activity of the mitochondrial CO2 + bicarbonate system has been measured in perfused livers using the specific activities of urea and acetoacetate derived from 2-ketoisocaproate catabolism. Label was supplied either as NaH14CO3, 2-keto[1-14C]isocaproate, [1-14C]pyruvate, [1-14C]glutamine, or [14C]formate. With labeled bicarbonate, pyruvate, or 2-ketoisocaproate, the specific activities of effluent bicarbonate, urea, and acetoacetate were equal (acetoacetate was labeled only on C-1). In the presence of [14C]formate, the specific activity of acetoacetate was double that of urea. Acetazolamide (0.2 mM), an inhibitor of carbonic anhydrase, decreased the specific activities of urea and acetoacetate labeled from NaH14CO3 and increased the specific activities of urea and acetoacetate labeled from the other tracers. We conclude that: acetoacetate derived from 2-ketoisocaproate is, like urea, an index of the specific activity of mitochondrial CO2 in liver, carbonic anhydrase activity equalizes the specific activities of the CO2 + bicarbonate system on both sides of the mitochondrial membrane, and a fraction of [14C] formate-derived 14CO2 appears to be generated in a mitochondrial compartment, in the close vicinity of methylcrotonyl-CoA carboxylase.

Evaluation of a peptidomimetic ribonucleotide reductase inhibitor with a murine model of herpes simplex virus type 1 ocular disease.

The ribonucleotide reductase (RR) of herpes simplex virus type 1 (HSV-1) is an important virulence factor, being required for neurovirulence, ocular virulence, and reactivation from latency. The RR activity requires the association of two distinct homodimeric subunits, and the association of the subunits is inhibited in the presence of a peptide homologous to the carboxy terminus of the small subunit. A structural analog of the inhibitory peptide (BILD 1263) has been shown to inhibit the replication of HSV-1 at micromolar concentrations in vitro. We used a mouse model of HSV-1 ocular infection to determine the in vivo efficacy of topical BILD 1263. Treatment of HSV-1 KOS-infected mice resulted in significant reductions in the severity and incidence of stromal keratitis and corneal neovascularization. At higher concentrations (5%) BILD 1263 reduced the severity but not the incidence of blepharitis. Treatment with 5% BILD 1263 also reduced viral shedding from the cornea by 10- to 14-fold (P < 0.001). In uninfected mice treated with 5% BILD 1263, we found no evidence of corneal epithelial damage, conjunctivitis, or blepharitis, and histopathological studies revealed no changes in the corneas of these mice. These results show that the peptidomimetic RR inhibitor BILD 1263 is effective in preventing disease, has an antiviral effect in vivo, and has little or no toxicity.

Interference of 3-hydroxyisobutyrate with measurements of ketone body concentration and isotopic enrichment by gas chromatography-mass spectrometry.

Concentrations and 13C2 molar percentage enrichments of blood R-3-hydroxybutyrate and acetoacetate are measured by selected ion monitoring gas chromatography-mass spectrometry. Samples are treated with NaB2H4 to reduce unlabeled and labeled acetoacetate to corresponding deuterium-labeled RS-3-hydroxybutyrate species. Only the gas chromatographic peak for the tert-butyldimethylsilyl derivative of 3-hydroxybutyrate needs to be monitored. The various compounds are quantitated using an internal standard of RS-3-hydroxy-[2,2,3,4,4,4-2H6]-butyrate. Concentrations of ketone bodies are obtained by monitoring the m/z 159 to 163 fragments of tert-butyldimethylsilyl derivatives of labeled and unlabeled 3-hydroxybutyrate species. High correlations were obtained between ketone body concentrations assayed (i) enzymatically with R-3-hydroxybutyrate dehydrogenase and (ii) by gas chromatography-mass spectrometry. The limit of detection is about 10 nmol of substrate in blood samples. The current practice of monitoring the m/z 275 to 281 fragments overestimates the concentration of endogenous R-3-hydroxybutyrate, due to co-elution of 3-hydroxyisobutyrate, a valine metabolite. The method presented is used to measure ketone body turnover in vivo in 24-h-fasted dogs.

Pseudoketogenesis in the perfused rat heart.

Ketogenesis is usually measured in vivo by dilution of tracers of (3R)-hydroxybutyrate or acetoacetate. We show that, in perfused working rat hearts, the specific activities of (3R)-hydroxybutyrate and acetoacetate are diluted by isotopic exchanges in the absence of net ketogenesis. We call this process pseudoketogenesis. When hearts are perfused with buffer containing 2.3 mM of [4-3H]- plus [3-14C]acetoacetate, the specific activities of [4-3H] and [3-14C]acetoacetate decrease while C-1 of acetoacetate becomes progressively labeled with 14C. This is explained by the reversibility of reactions catalyzed by mitochondrial 3-oxoacid-CoA transferase and acetoacetyl-CoA thiolase. After activation of labeled acetoacetate, the specific activity of acetoacetyl-CoA is diluted by unlabeled acetoacetyl-CoA derived from endogenous fatty acids or glucose. Acetoacetyl-CoA thiolase partially exchanges 14C between C-1 and C-3 of acetoacetyl-CoA. Finally, 3-oxoacid-CoA transferase liberates weakly labeled acetoacetate which dilutes the specific activity of extracellular acetoacetate. An isotopic exchange in the reverse direction is observed when hearts are perfused with unlabeled acetoacetate plus [1-14C]-, [13-14C]-, or [15-14C]palmitate; here also, acetoacetate becomes labeled on C-1 and C-3. Computations of specific activities of (3R)-hydroxybutyrate, acetoacetate, and acetyl-CoA yield minimal rates of pseudoketogenesis ranging from 19 to 32% of the net uptake of (3R)-hydroxybutyrate plus acetoacetate by the heart.

Canadian medical experiments on Shuttle flight 41-G.

During the 41-G mission, two payload specialist astronauts took part in six Canadian medical experiments designed to measure how the human nervous system adapts to weightlessness, and how this might contribute to space motion sickness. Similar tests conducted pre-flight provided base-line data, and post-flight experiments examined re-adaptation to the ground. No changes were detected in the vestibulo-ocular reflex during this 8-day mission. Pronounced proprioceptive illusions were experienced, especially immediately post-flight. Tactile acuity was normal in the fingers and toes, but the ability to judge limb position was degraded. Estimates of the locations of familiar targets were grossly distorted in the absence of vision. There were no differences in taste thresholds or olfaction. Despite pre-flight tests showing unusual susceptibility to motion sickness, the Canadian payload specialist turned out to be less susceptible than normal on-orbit. Re-adaptation to the normal gravity environment occurred within the first day after landing.

Rates of gluconeogenesis and citric acid cycle in perfused livers, assessed from the mass spectrometric assay of the 13C labeling pattern of glutamate.

Absolute rates of gluconeogenesis and of the citric acid cycle were assessed in livers isolated from 24-h starved rats, perfused with physiological concentrations of [3-13C]lactate and [3-13C]pyruvate +/- 0.2 mM octanoate. Calculations are based on (i) the 13C-labeling pattern of glutamate determined by gas chromatography-mass spectrometry combined with isotopomer analysis, (ii) substrate balance, and (iii) equations developed by Magnusson et al. (Magnusson, I., Schumann, W. C., Bartsch, G. E., Chandramouli, V., Kumaran, K., Wahren, J., and Landau, B. R. (1991) J. Biol. Chem. 266, 6975-6984) based on a citric acid cycle model proposed by Katz (Katz, J. (1985) Am. J. Physiol. 248, R391-R399). Glutamate, isolated from liver extracts, is enzymatically or chemically converted to gamma-aminobutyrate, alpha-hydroxyglutarate, isocitrate, and glutamine before mass spectrometric analysis. General equations have been developed ("Appendix I") to determine the isotopic enrichment of each carbon of glutamate from the isotopic enrichment of fragments obtained from the mass spectra of trimethylsilyl or t-butyldimethylsilyl derivatives of glutamate and of derived compounds ("Appendix II"). In the presence of octanoate, (i) the rate of the citric acid cycle decreases from 0.25 to 0.13 mumol/min x g wet weight which are one-third and one-sixth of the rate of pyruvate carboxylation, and (ii) the rate of gluconeogenesis increases from 0.65 to 0.83 mumol/min x g wet weight. The rate of pyruvate carboxylation is 13 and 34-fold faster than that of pyruvate dehydrogenation in the absence or presence of octanoate, respectively. The rate of oxaloacetate to fumarate interconversion is at least six times greater than that of the citric acid cycle. Our data closely agree with those obtained by Magnusson et al. who used a non-invasive "chemical biopsy" of the human liver and support the use of labeled lactate and/or pyruvate for tracing hepatic metabolism in vivo.

Production of acetone and conversion of acetone to acetate in the perfused rat liver.

The utilization of millimolar concentrations of [2-14C]acetone and the production of acetone from acetoacetate were studied in perfused livers from 48-h starved rats. We devised a procedure for determining, in a perfused liver system, the first-order rate constant for the decarboxylation of acetoacetate (0.29 +/- 0.09 h-1, S.E., n = 8). After perfusion of livers with [2-14C]acetone, labeled acetate was isolated from the perfusion medium and characterized as [1-14C]acetate. No radioactivity was found in lactate or 3-hydroxybutyrate. After 90 min of perfusion with [2-14C]acetone, the specific activity of acetate was 30 +/- 4% (n = 13) of the initial specific activity of acetone. We conclude that, in perfused livers from 2-day starved rats, acetone metabolism occurs for the most part via free acetate.