A mutation in the gene encoding the vaccinia virus 37,000-M(r) protein confers resistance to an inhibitor of virus envelopment and release.

Plaque formation in vaccinia virus is inhibited by the compound N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine (IMCBH). We have isolated a mutant virus that forms wild-type plaques in the presence of the drug. Comparison of wild-type and mutant virus showed that both viruses produced similar amounts of infectious intracellular naked virus in the presence of the drug. In contrast to the mutant, no extracellular enveloped virus was obtained from IMCBH-treated cells infected with wild-type virus. Marker rescue experiments were used to map the mutation conferring IMCBH resistance to the mutant virus. The map position coincided with that of the gene encoding the viral envelope antigen of M(r) 37,000. Sequence analysis of both wild-type and mutant genes showed a single nucleotide change (G to T) in the mutant gene. In the deduced amino acid sequence, the mutation changes the codon for an acidic Asp residue in the wild-type gene to one for a polar noncharged Tyr residue in the mutant.

False negative apraclonidine test in two patients with Horner syndrome

BACKGROUND: Because of denervation supersensitivity, a miotic pupil in a sympathetically-denervated eye dilates in response to a dilute or weak alpha-1-agonist drug. A reversal of anisocoria after topical apraclonidine is considered as a positive test result that diagnoses a unilateral Horner syndrome. HISTORY AND SIGNS: Two women aged 34 and 46 years with a cocaine-confirmed oculosympathetic defect (Horner syndrome) were tested with 1 % topical apraclonidine on separate days. THERAPY AND OUTCOME: In one patient, her miotic Horner pupil dilated marginally but not enough to reverse the baseline anisocoria. Additionally, the upper lid on the same side retracted. There was no discernable effect of apraclonidine on the normal, contralateral eye. In the second patient, there was no pupillary response to apraclonidine but there was resolution of her ptosis. CONCLUSIONS: Neither patient demonstrated a reversal of anisocoria, the current criterion for diagnosing a Horner syndrome using apraclonidine. Thus, these two patients with an established oculosympathetic defect were said to have a "negative test" for Horner syndrome. Yet both women showed subtle pupil and/or lid changes in response to apraclonidine that were consistent with sympathetic denervation supersensitivity. Reversal of anisocoria following topical apraclonidine does not occur in all patients with a unilateral oculosympathetic defect and more specific parameters for defining a positive test result might optimize apraclonidine's utility as a diagnostic test for Horner syndrome

Polymorphic mutations associated with the emergence of the multinucleoside/tide resistance mutations 69 insertion and Q151M.

BACKGROUND: We hypothesized that polymorphic mutations exist that are associated with the emergence of the multinucleoside resistance mutations (MNR), 69 insertion and Q151M. METHODS: The Swiss HIV Cohort Study was screened, and the frequencies of polymorphic mutations in HIV-1 (subtype B) were compared between patients detected with the 69 insertion (n = 17), Q151M (n = 29), ≥2 thymidine analogue mutations (TAM) 1 (n = 400) or ≥2 TAM 2 (n = 249). Logistic regressions adjusted for the antiretroviral treatment history were performed to analyze the association of the polymorphic mutations with MNR. RESULTS: The 69 insertion and TAM 1 were strongly associated and occurred in 94.1% (16 of 17) together. The 69 insertion seemed to emerge as a consequence of the TAM 1 pathway (median years until detection: 6.8 compared with 4.4 for ≥2 TAM 1, P Wilcoxon = 0.009). Frequencies of 8 polymorphic mutations (K43E, V60I, S68G, S162C, T165I, I202V, R211K, F214L) were significantly different between groups. Logistic regression showed that F214L and V60I were associated with the emergence of Q151M/TAM 2 opposed to 69 insertion/TAM 1. S68G, T165I, and I202V were associated with Q151M instead of TAM 2. CONCLUSIONS: Besides antiretroviral therapy, polymorphic mutations may contribute to the emergence of specific MNR mutations.

Modulation of proteasomal activity required for the generation of a cytotoxic T lymphocyte-defined peptide derived from the tumor antigen MAGE-3.

We have analyzed the presentation of human histocompatability leukocyte antigen-A*0201-associated tumor peptide antigen MAGE-3271-279 by melanoma cells. We show that specific cytotoxic T lymphocyte (CTL)-recognizing cells transfected with a minigene encoding the preprocessed fragment MAGE-3271-279 failed to recognize cells expressing the full length MAGE-3 protein. Digestion of synthetic peptides extended at the NH2 or COOH terminus of MAGE-3271-279 with purified human proteasome revealed that the generation of the COOH terminus of the antigenic peptide was impaired. Surprisingly, addition of lactacystin to purified proteasome, though partially inhibitory, resulted in the generation of the antigenic peptide. Furthermore, treatment of melanoma cells expressing the MAGE-3 protein with lactacystin resulted in efficient lysis by MAGE-3271-279-specific CTL. We therefore postulate that the generation of antigenic peptides by the proteasome in cells can be modulated by the selective inhibition of certain of its enzymaticactivities.

Insulin resistance in adult cardiomyocytes undergoing dedifferentiation: role of GLUT4 expression and translocation.

Myocardium undergoing remodeling in vivo exhibits insulin resistance that has been attributed to a shift from the insulin-sensitive glucose transporter GLUT4 to the fetal, less insulin-sensitive, isoform GLUT1. To elucidate the role of altered GLUT4 expression in myocardial insulin resistance, glucose uptake and the expression of the glucose transporter isoforms GLUT4 and GLUT1 were measured in adult rat cardiomyocytes (ARC). ARC in culture spontaneously undergo dedifferentiation, hypertrophy-like spreading, and return to a fetal-like gene expression pattern. Insulin stimulation of 2-deoxy-D-glucose uptake was completely abolished on day 2 and 3 of culture and recovered thereafter. Although GLUT4 protein level was reduced, the time-course of unresponsiveness to insulin did not correlate with altered expression of GLUT1 and GLUT4. However, translocation of GLUT4 to the sarcolemma in response to insulin was completely abolished during transient insulin resistance. Insulin-mediated phosphorylation of Akt was not reduced, indicating that activation of phosphatidylinositol 3-kinase (PI3K) was preserved. On the other hand, total and phosphorylated Cbl was reduced during insulin resistance, suggesting that activation of Cbl/CAP is essential for insulin-mediated GLUT4 translocation, in addition to activation of PI3K. Pharmacological inhibition of contraction in insulin-sensitive ARC reduced insulin sensitivity and lowered phosphorylated Cbl. The results suggest that transient insulin resistance in ARC is related to impairment of GLUT4 translocation. A defect in the PI3K-independent insulin signaling pathway involving Cbl seems to contribute to reduced insulin responsiveness and may be related to contractile arrest.

Cost effectiveness and cost utility of risedronate for osteoporosis treatment and fracture prevention in women: a Swiss perspective.

OBJECTIVES: To assess the incremental cost-effectiveness ratio (ICER) and incremental cost-utility ratio (ICUR) of risedronate compared to no intervention in postmenopausal osteoporotic women in a Swiss perspective. METHODS: A previously validated Markov model was populated with epidemiological and cost data specific to Switzerland and published utility values, and run on a population of 1,000 women of 70 years with established osteoporosis and previous vertebral fracture, treated over 5 years with risedronate 35 mg weekly or no intervention (base case), and five cohorts (according to age at therapy start) with eight risk factor distributions and three lengths of residual effects. RESULTS: In the base case population, the ICER of averting a hip fracture and the ICUR per quality-adjusted life year gained were both dominant. In the presence of a previous vertebral fracture, the ICUR was below euro45,000 (pound30,000) in all the scenarios. For all osteoporotic women>or=70 years of age with at least one risk factor, the ICUR was below euro45,000 or the intervention may even be cost saving. Age at the start of therapy and the fracture risk profile had a significant impact on results. CONCLUSION: Assuming a 2-year residual effect, that ICUR of risedronate in women with postmenopausal osteoporosis is below accepted thresholds from the age of 65 and even cost saving above the age of 70 with at least one risk factor.

Oral pregabalin as an add-on treatment for status epilepticus.

Oral antiepileptic drugs (AEDs) represent possible add-on options in refractory status epilepticus (SE). In this setting, pregabalin (PGB) has not been reported before. Over the last 42 months, we identified 11 SE episodes (10 patients) treated with PGB in our hospital. Its use was prompted by the favorable pharmacokinetic profile, devoid of drug-drug interactions. The patients mostly had refractory, partial SE. Only two patients were managed in the intensive care unit (ICU). We found a definite electroclinical response in 5 of 11, already evident 24 h after PGB introduction, and a possible response (concomitantly with other AEDs) in 3 of 11 of the episodes; 3/11 did not respond. The treatment was well tolerated. Partial SE appeared to better respond than generalized convulsive SE. PGB appears to be an interesting option as add-on treatment in refractory partial SE.

Murine alpha1-adrenoceptor subtypes. I. Radioligand binding studies.

Alpha1-adrenoceptors were identified in murine tissues by [3H]prazosin saturation binding studies, with a rank order of cerebral cortex > cerebellum > liver > lung > kidney > heart > spleen, with the spleen not exhibiting detectable expression. Competition binding studies were performed with 5-methylurapidil, BMY 7378, methoxamine, (+)-niguldipine, noradrenaline, SB 216469 and tamsulosin. On the basis of monophasic low-affinity competition by BMY 7378, alpha1D-adrenoceptors were not detected at the protein level in any tissue. On the basis of competition studies with the alpha1A/alpha1B-discriminating drugs, alpha1B-adrenoceptors appeared to be the predominant or even the sole subtype in murine liver, lung and cerebellum, whereas murine cerebral cortex and kidney contained approximately 30% and 50% of alpha1A-adrenoceptors, respectively. The affinities of the various competitors in the murine tissues were quite similar to those reported from other species. The ratio of high- and low-affinity sites for tamsulosin did not in all cases match the percentages of alpha1A- and alpha1B-adrenoceptors detected by the other competitors; however, the low-affinity component of the tamsulosin competition curves was abolished in the cerebral cortex of alpha1B-adrenoceptor knockout mice. Treatment with chloroethylclonidine (10 microM, 30 min, 37 degrees C) inactivated the alpha1-adrenoceptors in all tissues by >75%. When the concentration-dependent inactivation of tissue alpha1B-adrenoceptors (liver) and tissue alpha1A-adrenoceptors (cerebral cortex from alpha1B-adrenoceptor knockout mice) was compared, alpha1A-adrenoceptors were only slightly less sensitive toward chloroethylclonidine than alpha1B-adrenoceptors. We conclude that murine tissues express alpha1A- and alpha1B-adrenoceptors, which are largely similar to those in other species. However, the tissue-specific distribution of subtypes may differ from that of other species.

Cyclooxygenase-2 controls energy homeostasis in mice by de novo recruitment of brown adipocytes.

Obesity results from chronic energy surplus and excess lipid storage in white adipose tissue (WAT). In contrast, brown adipose tissue (BAT) efficiently burns lipids through adaptive thermogenesis. Studying mouse models, we show that cyclooxygenase (COX)-2, a rate-limiting enzyme in prostaglandin (PG) synthesis, is a downstream effector of beta-adrenergic signaling in WAT and is required for the induction of BAT in WAT depots. PG shifted the differentiation of defined mesenchymal progenitors toward a brown adipocyte phenotype. Overexpression of COX-2 in WAT induced de novo BAT recruitment in WAT, increased systemic energy expenditure, and protected mice against high-fat diet-induced obesity. Thus, COX-2 appears integral to de novo BAT recruitment, which suggests that the PG pathway regulates systemic energy homeostasis.

Role of gluconic acid production in the regulation of biocontrol traits of Pseudomonas fluorescens CHA0.

The rhizobacterium Pseudomonas fluorescens CHA0 promotes the growth of various crop plants and protects them against root diseases caused by pathogenic fungi. The main mechanism of disease suppression by this strain is the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT). Direct plant growth promotion can be achieved through solubilization of inorganic phosphates by the production of organic acids, mainly gluconic acid, which is one of the principal acids produced by Pseudomonas spp. The aim of this study was to elucidate the role of gluconic acid production in CHA0. Therefore, mutants were created with deletions in the genes encoding glucose dehydrogenase (gcd) and gluconate dehydrogenase (gad), required for the conversion of glucose to gluconic acid and gluconic acid to 2-ketogluconate, respectively. These enzymes should be of predominant importance for rhizosphere-colonizing biocontrol bacteria, as major carbon sources provided by plant root exudates are made up of glucose. Our results show that the ability of strain CHA0 to acidify its environment and to solubilize mineral phosphate is strongly dependent on its ability to produce gluconic acid. Moreover, we provide evidence that the formation of gluconic acid by CHA0 completely inhibits the production of PLT and partially inhibits that of DAPG. In the Deltagcd mutant, which does not produce gluconic acid, the enhanced production of antifungal compounds was associated with improved biocontrol activity against take-all disease of wheat, caused by Gaeumannomyces graminis var. tritici. This study provides new evidence for a close association of gluconic acid metabolism with antifungal compound production and biocontrol activity in P. fluorescens CHA0.

Mutations in the epithelial Na+ channel ENaC outer pore disrupt amiloride block by increasing its dissociation rate.

The epithelial Na+ channel ENaC mediates transepithelial Na+ transport in the distal kidney, the colon, and the lung and is a key element for the maintenance of Na+ balance and the regulation of blood pressure. Mutagenesis studies have identified residues alphaS583 and the homologous betaG525 and gammaG537 in the outer pore entrance that are critical for ENaC block by the K+-sparing diuretic amiloride. The aim of the present study was to determine first, whether these residues are part of the amiloride binding site, and second, whether they are general determinants of ENaC block by amiloride and its derivatives. Kinetic analysis of the association and dissociation rates of amiloride and benzamil to ENaC showed that mutation of residue alphaS583C and the homologous betaG525C increased the dissociation rate of the drugs from the binding site, with little changes in their association rate. Thus, these mutations destabilize the binding interaction between the blockers and the receptor on the channel, favoring the unbinding of the ligand. This strongly suggests that they are part of the binding site. Because mutations of alphaS583, betaG525, and gammaG537 have similar effects on amiloride, benzamil, and triamterene block, we conclude that these three ENaC blockers share a common receptor within the ion channel pore.

Transport of the organic cation N1-methylnicotinamide by the rabbit proximal tubule. I. Accumulation in the isolated nonperfused tubule.

Isolated nonperfused rabbit renal proximal tubules were used to investigate the basolateral step of transport of the organic cation N1-methylnicotinamide (NMN). NMN accumulation was highest and saturable in S2 and S3 segments, but lowest and nonsaturable in S1 segments. In S1 segments, accumulation of [3H]-NMN (0.5-8 microM in the bath) resulted in an average tubular water/medium concentration ratio (T/M) of 8.2, whereas in S2 and S3 segments T/M averaged 19.5 and 18.6, respectively. At these concentrations, about 30% of the label was attached in all segments to a metabolite comigrating with nicotinamide. KCN (10(-2) M) or ouabain (10(-4) M) reduced T/M to about 8 for all segments. NMN accumulation was inhibited (to a T/M of about 3 with mepiperphenidol) by other organic cations (10(-5)-10(-3) M) with the potency sequence mepiperphenidol greater than tetraethylammonium = quinine greater than morphine, these organic cations having no effect on p-aminohippurate accumulation, except for the highest concentration of quinine (10(-3) M). After correction for metabolism, NMN accumulation could be accounted for by simple electrochemical equilibrium across the basolateral membrane. The basolateral step of NMN transport appears therefore to be a carrier-mediated diffusion, in opposition to the active basolateral accumulation described for tetraethylammonium.

Use of duplex ultrasonography in the treatment of thromboangiitis obliterans with iloprost.

We present a 34-year-old patient with digital necrosis due to thromboangiitis obliterans. He was successfully treated with iloprost, a prostaglandin analogue. Duplex ultrasonography was performed during the perfusion of iloprost to optimize the doses and the treatment duration. A complete revascularization was observed after 10 days. Iloprost perfusions were stopped, and a slow regression of the necroses was observed in the subsequent days. With the use of duplex ultrasonography, unnecessary high doses of iloprost and long periods of treatment can be avoided reducing side effects and treatment costs.

Changes in protein turnover and resting energy expenditure after treatment of malaria in Gambian children.

To explore the changes in resting energy expenditure (REE) and whole body protein turnover induced by malaria, 23 children aged 6 to 14 y (23.9 +/- 1.0 kg, 1.3 +/- 0.02 m) were studied on three separate days after treatment (d 1, d 2, and 15 d later). REE was assessed by indirect calorimetry (hood), whereas whole body protein turnover was estimated using a single dose of [15N]glycine administered p.o. by measuring the isotopic enrichment of [15N]ammonia in urine over 12 h. Within the first 3.5 h after treatment, the body temperature dropped from 39.8 +/- 0.1 to 37.8 +/- 0.1 degrees C (p < 0.0001), and REE followed the same pattern, decreasing rapidly from 223 +/- 6 to 187 +/- 4 kJ/kg/d (p < 0.0001). Whole body protein synthesis and breakdown were significantly higher during the 1st day (5.65 +/- 0.38 and 6.21 +/- 0.43 g/kg/d, respectively) than at d 15 (2.95 +/- 0.17 and 2.77 +/- 0.2 g/kg/d). It is concluded that Gambian children suffering from an acute episode of malaria have an increased REE averaging 37% of the control value (d 15) and that this was associated with a substantial increase (by a factor of 2) in whole body protein turnover. A rapid normalization of the hypermetabolism and protein hypercatabolism states after treatment was observed.

Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro.

Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-3 cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P)H oxidase isoform 1, NAD(P)H oxidase isoform 2, p22(phox), p40(phox), p47(phox), p67(phox), xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable SOD but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.