Effect of local cold application on glycogen recovery.

The purpose of this study was to investigate the effect of local cold application on muscle glycogen re-synthesis after exercise. Recreationally active male subjects (n=11) completed a 90-minute glycogen depleting ride, followed by 4 h of recovery. During recovery, ice was applied intermittently to one leg (IL) while the subjects other leg (CL) acted as a control. Intramuscular and rectal temperature was recorded continuously. A carbohydrate (1.8 g∙kg-1 bodyweight) beverage was supplied at 0 and 2 h post exercise. Muscle biopsies were taken immediately after exercise from the vastus lateralis and at 4 h post exercise for the analysis of muscle glycogen and muscle lactate. Leg circumference was measured 30, 60, 120, 180, and 240 minutes into recovery. The IL was colder than the CL from 15 minutes after initial ice application until the end recovery (P<0.05). Immediate post-exercise glycogen was similar between legs (55.3±7.4 vs. 56.1±7 mmol∙kg-1 wet weight for the iced vs. control, respectively). However, muscle glycogen was lower in the IL compared to the CL at 4 h post exercise (72±8.4 vs. 95±8.4 mmol∙kg-1 wet weight, respectively; P<0.05). Muscle lactate was lower in the IL after 4 h of recovery compared to the CL (1.6±.2 vs. 2.6±.2 mmol∙L-1, respectively; P<0.05). There was no difference in circumference between IL and CL. These data demonstrate a reduction in muscle glycogen re-synthesis with local cold application.

Human mRNA response to exercise and temperature.

The purpose of this research was to determine the mRNA response to exercise in different environmental temperatures. 9 recreationally active males (27±1 years, 77.4±2.7 kg, 13.5±1.5% fat, 4.49±0.15 L · min (-1) VO2 max) completed 3 trials consisting of 1 h cycling exercise at 60% Wmax followed by a 3 h recovery in the cold (7°C), room temperature (20°C), and hot (33°C) environments. Muscle biopsies were obtained pre, post, and 3 h post exercise for the analysis of glycogen and mRNA. Expired gases were collected to calculate substrate use. PGC-1α increased to a greater degree in the cold trial than in the room temperature trial (p=0.036) and the hot trial (p=0.006). PGC1-α mRNA was also higher after the room temperature trial than the hot trial (p=0.050). UCP3 and MFN2 mRNA increased with exercise (p<0.05), but were unaffected by temperature. COX was unaffected by exercise or temperature. Muscle glycogen decreased with exercise (p<0.05), but was no different among trials. Whole body VO2 was lower during exercise in the cold than exercise in the heat. However, VO2 was higher during recovery in the cold trial than in the room temperature and hot trials (p<0.05). This study presents evidence of PGC-1α temperature sensitivity in human skeletal muscle.

Review of the epidemiology, molecular characterization and tropism of the hepatitis G virus/GBV-C.

The hepatitis G virus and GBV-C are recently discovered variants of the same virus belonging to the family Flaviviridae (HGV/GBV-C). Although initially thought to be a hepatitis virus, it has been shown to have no association with liver disease. This paper reviews the data relating to the discovery, global prevalence, natural history, disease association, molecular features, replication and tissue tropism of HGV/GBV-C.

GBV-C/HGV genotypes: proposed nomenclature for genotypes 1-5.

The GB virus-C and hepatitis G virus (GBV-C/HGV) are variants of the same flavivirus. This proposal attempts to clarify the conflicting nomenclature for GBV-C/HGV genotypes. The first three genotypes described were genotype 1 (West Africa); genotype 2 (US/Europe) and genotype 3 (Asia). Subsequently, two groups published data from South Africa and Southeast Asia both stating the presence of a novel "4th genotype." These isolates are distinct phylogenetically. It is proposed that the nomenclature for genotypes 1-3 remains as per previous publications, and that the Southeast Asian isolates be known as genotype 4, and the South African isolates as genotype 5.

Evidence that the GBV-C/hepatitis G virus is primarily a lymphotropic virus.

GB virus-C and the hepatitis G virus (GBV-C/HGV) are variants of the same positive sense RNA flavivirus, initially thought to be associated with hepatitis. The tissue tropism of GBV-C/HGV in normal subjects has not been evaluated to date using an extended tissue spectrum. Therefore, the sites of GBV-C/HGV replication were investigated in serum and twenty-three tissues collected during post-mortem examination of four apparently healthy individuals who died accidental deaths, who were infected with GBV-C/HGV. All were anti-HIV and anti-HCV negative and three out of four were HBsAg negative. Tissues were collected carefully to prevent cross contamination. A highly strand-specific RT-PCR assay was employed for the detection of either GBV-C/HGV positive strand RNA (virion) or negative strand RNA (replicative intermediary). Strand specificity of the RT-PCR assay was assessed with synthetic positive-and negative strand GBV-C/HGV RNA generated from a plasmid, using T7 and T3 RNA polymerases. The spleen and bone marrow biopsies were found to be uniformly positive for both negative-and positive strand GBV-C/HGV RNA. In addition, one cadaver was positive for both RNA strands in the kidney, and another positive for both in the liver. No negative strand RNA was detected in the following: brain, muscle, heart, thyroid, salivary gland, tonsil, lung, lymph nodes, gall bladder, pancreas, oesophagus, stomach, small bowel, large bowel, adrenal gland, gonad, aorta, skin and cartilage. This preliminary study concludes that GBV-C/HGV is a lymphotropic virus that replicates primarily in the spleen and bone marrow.

Intravascular lymphomatosis with bone marrow involvement.

We describe the case of a 56-year-old man who presented with numbness and tingling of the extremities, weakness, and fatigue. Laboratory findings included anemia and thrombocytopenia. A diagnosis of intravascular lymphomatosis was established when liver, omentum, and bone marrow samples were examined. A review of the literature reveals that most cases of intravascular lymphomatosis have cytopenias, mainly anemia and thrombocytopenia, but bone marrow involvement is rare. In our case, a subtle neoplastic infiltrate in the marrow sinusoids was highlighted with a B-cell marker. While immunohistochemical analysis was not performed in most reported cases in the literature, our studies suggest that a systematic search in bone marrow of cases of intravascular lymphomatosis may reveal unsuspected neoplastic cells. We conclude that bone marrow involvement in intravascular lymphomatosis appears to be rare, has subtle features, and is difficult to diagnose if unsuspected and not searched for.

Molecular characterization of the 5' non-coding region of South African GBV-C/HGV isolates: major deletion and evidence for a fourth genotype.

GB virus C/hepatitis G virus (GBV-C/HGV) has been characterised as a novel flavivirus, and to date three known genotypes have been cloned. Greater genetic variation of GBV-C/HGV has been demonstrated in West African isolates, but no major deletions have been shown in the 5' non-coding region (NCR). The 5'NCR regulates protein translation via an internal ribosomal entry site (IRES). We cloned, sequenced, and analysed a 344-bp polymerase chain reaction (PCR) product, representing >60% of the 5'NCR, from 32 GBV-C/HGV PCR-positive volunteers. Wild-type virus amplicons were detected in all samples. However, 5/32 (15.6%) also amplified another fragment of between 205 and 231 bp. Sequence analysis showed all cloned PCR fragments to be GBV-C/HGV-specific. A typical deletion of 113-131 bp with minor variation was detected in isolates generating the smaller bands. RNA secondary structure analysis showed the deletions to be over domains II and III. This finding suggests that nucleotides 303-444 may be non-essential for 5'NCR functioning. Phylogenetic analysis demonstrated a novel fourth South African genotype, distinct from genotypes 1-3 with DNA distances of >0.1000. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) values for the wild-type and mutant samples were normal. This study documents the first major deletion in the 5'NCR of GBV-C/HGV, and suggests that bases 303-444 may not be essential for viral replication and ribosomal entry. A fourth GBV-C/HGV genotype appears to predominate in South Africa.

Design and synthesis of a series of potent and orally bioavailable noncovalent thrombin inhibitors that utilize nonbasic groups in the P1 position.

As part of an ongoing effort to prepare therapeutically useful orally active thrombin inhibitors, we have synthesized a series of compounds that utilize nonbasic groups in the P1 position. The work is based on our previously reported lead structure, compound 1, which was discovered via a resin-based approach to varying P1. By minimizing the size and lipophilicity of the P3 group and by incorporating hydrogen-bonding groups on the N-terminus or on the 2-position of the P1 aromatic ring, we have prepared a number of derivatives in this series that exhibit subnanomolar enzyme potency combined with good in vivo antithrombotic and bioavailability profiles. The oxyacetic amide compound 14b exhibited the best overall profile of in vitro and in vivo activity, and crystallographic studies indicate a unique mode of binding in the thrombin active site.

Design of novel, potent, noncovalent inhibitors of thrombin with nonbasic P-1 substructures: rapid structure-activity studies by solid-phase synthesis.

Study of surface representations of the inhibitor-bound thrombin P-1 pocket revealed a lipophilic recess in this pocket which is not occupied by any known inhibitor. Solid-phase synthesis was used to generate benzylamides of D-diphenylAlaPro by aminolysis of Boc dipeptide Kaiser resin. The resulting amides inhibited thrombin in the range IC50 = 3-13,000 nM, and the structure-activity relationships and molecular modeling suggest a unique fit of the benzyl side chain into P-1 with the meta substituent occupying the recess.

Thyroid hormone concentrations in black bears (Ursus americanus): hibernation and pregnancy effects.

Previous studies on thyroid hormones in hibernating bears have used very few sampling periods, so that the time course of any change is poorly understood. In this study, plasma sampled from pregnant and nonpregnant black bears before and during hibernation (16 samples each at 10-day intervals) was assayed by radioimmunoassay for concentrations of thyroxine (T4) and triiodothyronine (T3). Only free T4 showed a difference (P = 0.019) between females that produced cubs and those that did not, but this appeared to be due to higher preimplantation values. Free T3, total T3, and free T4 varied (P = 0.001, 0.038, 0.002, respectively) among sampling periods: during December, bears had depressed concentrations. These lowered concentrations were maintained during hibernation for the free hormones. Our data confirm previous work showing that food restriction and/or physiological preparation for hibernation is coincident with depressed plasma concentrations of thyroid hormones. Hormonal changes associated with pregnancy were minor.

Identification and SAR for a selective, nonpeptidyl thrombin inhibitor.

A novel, nonpeptidyl thrombin inhibitor, L-636,619 (1), was identified via topological similarity searching over the Merck Corporate Sample Database. X-ray crystallographic studies determined the geometry for ligand binding to the enzyme. Chemical modification of the P1 and P3 segments of the ligand resulted in enhanced potency and improvement in the chemical stability of the lead. Analog 9 proved to be the most interesting lead from this structurally novel series.

High prevalence of GBV-C hepatitis G virus infection in a rural South African population.

A novel virus, GBV-C/hepatitis G virus (GBV-C/HGV), has been cloned and characterised recently. GBV-C/HGV global epidemiology and risk factors for acquisition are currently unclear. We aimed to establish the determinants of this infection in a rural South African (SA) population. The study population included two samples, namely a community-based sample, and consenting persons from a nonspecialist outpatient department in the same district. A questionnaire regarding demographic details and putative risk factors was administered; blood samples were taken on which a polymerase chain reaction (PCR) was performed for both 5'NCR and NS5a regions of GBV-C/HGV using commercially available primers and probes. Two hundred and forty-nine people were studied with a mean GBV-C/HGV prevalence of 10.4%. Outpatient department and community prevalences differed significantly (18.0% and 6.3%, respectively, P = 0.004). GBV-C/HGV infection was associated with excessive alcohol consumption (P = 0.02; OR, 4.18) and a lack of waterborne sewerage (P = 0.04). PCR amplification of the NS5a region of all but two South African GBV-C/HGV positive samples showed poor reactivity. The prevalence of GBV-C/HGV in rural SA appears to be higher than that reported from Europe and North America. Infection appeared to be associated with excess alcohol intake and a history of previous blood transfusion. The discrepant NS5a and 5'NCR PCR sensitivity in this study raises the possibility of genetic differences in southern African GBV-C/HGV.

Synthesis of a series of potent and orally bioavailable thrombin inhibitors that utilize 3,3-disubstituted propionic acid derivatives in the P3 position.

As part of an effort to prepare efficacious and orally bioavailable analogs of the previously reported thrombin inhibitors 1a, b, we have synthesized a series of compounds that utilize 3,3-disubstituted propionic acid derivatives as P3 ligands. By removing the N-terminal amino group, the general oral bioavailability of this class of compounds was enhanced without excessively increasing the lipophilicity of the compounds. The overall properties of the molecules could be drastically altered depending on the nature of the groups substituted onto the 3-position of the P3 propionic acid moiety. A number of the compounds exhibited good oral bioavailability in rats and dogs, and numerous compounds were efficacious in a rat FeCl3-induced model of arterial thrombosis. Compound 7, the 3,3-diphenylpropionic acid derivative, showed the best overall profile of in vivo and in vitro activity. Molecular modeling studies suggest that these compounds bind in the thrombin active site in a manner essentially identical to that previously reported for compound 1a.

Potent noncovalent thrombin inhibitors that utilize the unique amino acid D-dicyclohexylalanine in the P3 position. Implications on oral bioavailability and antithrombotic efficacy.

In an effort to prepare orally bioavailable analogs of our previously reported thrombin inhibitor 1, we have synthesized a series of compounds that utilize the unique amino acid D-dicyclohexylalanine as a P3 ligand. The resulting compounds are extremely potent and selective thrombin inhibitors, and the N-terminal Boc derivative 8 exhibited excellent oral bioavailability and pharmacokinetics in both rats and dogs. The des-Boc analog 6 was not orally bioavailable in rats. The high level of oral bioavailability observed with 8 appears to be a direct function of its increased lipophilicity versus other close analogs. Although increased lipophilicity may serve to increase the oral absorption of tripeptide thrombin inhibitors, it also appears to have detrimental effects on the antithrombotic properties observed with the compounds. Compound 6 performed extremely well in our in vivo antithrombotic assay, while the much more lipophilic but essentially equipotent analog 8 performed poorly. We have found that in general with this series of thrombin inhibitors as well as with other unreported series, increased lipophilicity and the associated increases in plasma protein binding have detrimental effects on 2X APTT values and subsequent performance in in vivo antithrombotic models.

Hepatitis C virus infection rate in volunteer blood donors from the Western Cape--comparison of screening tests and PCR.

INTRODUCTION: Hepatitis C virus (HCV) antibody seroprevalence studies overestimate the true infection rate. No data exist on the incidence of HCV or its clinical features in blood donors of sub-Saharan Africa. AIMS: To establish the true incidence of HCV infection in volunteer blood donors in the Western Cape, and compare risk factors and clinical and biochemical features of viraemic and non-viraemic subjects. METHODS: All donors attending the Western Province Blood Transfusion Service between December 1992 and August 1994 were screened prospectively for anti-HCV using the Abbott second-generation assay. Positive donors were evaluated clinically and biochemically. Their sera were examined for HCV-RNA by the polymerase chain reaction (PCR). RESULTS: Of 66314 donors screened, 275 (0.41%) were anti-HCV-positive. Of these 13.6% were PCR-positive (0.056% of all donors). PCR-positive patients had more risk factors for HCV acquisition (P < 0.01), symptoms of hepatitis (P = 0.02) and clinical signs of liver disease (P = 0.05) and higher alanine (P < 0.0001) and aspartate aminotransferase levels (P < 0.0001) than PCR-negative donors. However, clinical and biochemical features did not discriminate adequately between PCR-positive and negative donors. Liver biopsies performed in 9 of 13 PCR-positive cases showed mild inflammation, but no cirrhosis.

Hepatitis E in South Africa: evidence for sporadic spread and increased seroprevalence in rural areas.

Hepatitis E virus (HEV) is a major cause of non-A, non-B hepatitis in developing countries. Factors influencing sporadic spread of hepatitis E are unclear. We examined anti-HEV seroprevalence and demographic data from 407 urban and 360 rural black South African adults living in formal housing, squatter camps, or mud huts. Anti-HEV sero-prevalence ranged from 5.8% to 19.1% (mean 10.7%) in the different regions. Mean urban and rural rates were 6.6% and 15.3%, respectively (P = 0.0001). Rural mud hut dwellers, using unchlorinated river water, were at greater risk (17.4%) than rural villagers (5.3%; P = 0.008). A linear relation was found between seroprevalence and age, suggesting sporadic spread. The high prevalence in mud hut dwellers suggests that contaminated water plays a major role in HEV spread in South Africa. Routine chlorination or boiling of river drinking water before consumption may reduce HEV infection.