Mutational analysis of the hepatitis C virus RNA helicase

The carboxyl-terminal three-fourths of the hepatitis C virus (HCV) NS3 protein has been shown to possess an RNA helicase activity, typical of members of the DEAD box family of RNA helicases. In addition, the NS3 protein contains four amino acid motifs conserved in DEAD box proteins. In order to inspect the roles of individual amino acid residues in the four conserved motifs (AXXXXGKS, DECH, TAT, and QRRGRTGR) of the NS3 protein, mutational analysis was used in this study. Thirteen mutant proteins were constructed, and their biochemical activities were examined. Lys1235 in the AXXXXGKS motif was important for basal nucleoside triphosphatase (NTPase) activity in the absence of polynucleotide cofactor. A serine in the X position of the DEXH motif disrupted the NTPase and RNA helicase activities. Alanine substitution at His1318 of the DEXH motif made the protein possess high NTPase activity. In addition, we now report inhibition of NTPase activity of NS3 by polynucleotide cofactor. Gln1486 was indispensable for the enzyme activity, and this residue represents a distinguishing feature between DEAD box and DEXH proteins. There are four Arg residues in the QRRGRTGR motif of the HCV NS3 protein, and the second, Arg1488, was important for RNA binding and enzyme activity, even though it is less well conserved than other Arg residues. Arg1490 and Arg1493 were essential for the enzymatic activity. As the various enzymatic activities were altered by mutation, the enzyme characteristics were also changed.     

Mechanisms for increasing stroke volume during static exercise with fixed heart rate in humans

Ten patients with preserved inotropic function having a dual-chamber (right atrium and right ventricle) pacemaker placed for complete heart block were studied. They performed static one-legged knee extension at 20% of their maximal voluntary contraction for 5 min during three conditions: 1) atrioventricular sensing and pacing mode [normal increase in heart rate (HR; DDD)], 2) HR fixed at the resting value (DOO-Rest; 73 +/- 3 beats/min), and 3) HR fixed at peak exercise rate (DOO-Ex; 107 +/- 4 beats/min). During control exercise (DDD mode), mean arterial pressure (MAP) increased by 25 mmHg with no change in stroke volume (SV) or systemic vascular resistance. During DOO-Rest and DOO-Ex, MAP increased (+25 and +29 mmHg, respectively) because of a SV-dependent increase in cardiac output (+1.3 and +1.8 l/min, respectively). The increase in SV during DOO-Rest utilized a combination of increased contractility and the Frank-Starling mechanism (end-diastolic volume 118-136 ml). However, during DOO-Ex, a greater left ventricular contractility (end-systolic volume 55-38 ml) mediated the increase in SV.     

In vitro and in vivo detection of functional somatostatin receptors in canine insulinomas

Ten dogs with hypoglycemia due to insulinomas were studied to assess the expression of somatostatin receptors (SSTRs) in canine insulinomas and its potential diagnostic value. METHODS: The response of circulating glucose and insulin concentrations to the subcutaneous administration of a somatostatin analog, octreotide, was measured. SSTRs were visualized in vitro by autoradiography. [Iodine-125-Tyr3]-octreotide and [125I-Tyr11]-somatostatin-14 (SRIF-14) were used as radioligands. SPECT was performed 6 hr after the injection of [111In-DTPA-D-Phe1]-octreotide. RESULTS: After subcutaneous injection of 50 micrograms octreotide, plasma glucose concentration rose from 2.3 +/- 0.2 mmol/liter to 3.2 +/- 0.3 mmol/liter at 3.5 hr (p < 0.05) and plasma insulin concentration decreased from 451 +/- 135 pmol/liter to a nadir of 249 +/- 115 pmol/liter at 30 min (p < 0.05). In vitro autoradiography revealed that all primary insulinomas and their metastases had specific SSTRs for both [125I-Tyr3]-octreotide and [126I-Tyr11]-SRIF-14. Scatchard analysis of SSTR binding in the tumor tissue of one dog revealed high-affinity binding sites for [125I-Tyr3]-octreotide (dissociation constant (Kd) 1.7 nM, maximum binding capacity (Bmax) 499 fmol/mg membrane protein). The primary tumor and/or metastases in five of six dogs could be visualized and localized by SPECT with [111In-DTPA-D-Phe1]-octreotide. In the remaining dog, multiple metastases (< 3 mm) were found in the liver at necropsy, apparently too small to be visualized by SPECT. CONCLUSION: The in vitro autoradiography and ligand binding studies indicate that canine insulinomas express one type of SSTR. This is in contrast with findings in humans where, on the basis of ligand binding studies, different subtypes of SSTRs have been identified. The uniformity of SSTRs, their high frequency of expression and the high incidence of metastatic disease make canine insulinomas very suitable for investigation of the value of SRIF analogs in the diagnosis and treatment of metastasized endocrine pancreatic tumors.     

Application of rearrangements of T cell receptor gene in the diagnosis of mediastinal T-lymphoblastic lymphoma: a case report

Liver veno-occlusive disease is a severe toxic effect observed after bone marrow transplantation. Clinical manifestations are jaundice, painful liver enlargement, and fluid-sodium retention. Histologically there is non-thrombotic obliteration of the centro-lobular veins associated with centro-lobular necrosis. This severe complication of bone marrow transplantation occurs early and is caused by a toxic processing effect. Incidence is variable, 2 to 50% in reported series, depending on patients, type of marrow provessing and on diagnostic criteria (which hinders comparison between studies). According to most studies, veno-occlusive disease regresses spontaneously. Mortality, depending on the severity of the symptoms, varies from 20 to 50%. Pathogenesis remains under debate: the initial event would occur in the sinusoid endothelium creating a state of local hypercoagulability by release of tissue factors favoring deposit of coagulation factors, especially factor VIII, in the subendothelial region of the veinules. There is also a direct toxic effect on centro-lobular hepatocytes which is further aggravated by ischemia and venous stasis. use of heparin to prevent veno-occlusive disease was proposed by the Besan?on group in 1985 after they observed a low incidence (1 case in 65) in patients who were given low doses of heparin to maintain patent central catheters. The same team confirmed in 1992 the low incidence in a large retrospective series of 444 patients given either an autograft (3 cases in 253 patients, i.e. 1.2%), or an allograft (5 cases in 191 patients, i.e. 2.6%). Two single-center studies, one in Seattle and the other at the Saint-Antoine hospital in Paris, published in 1990 and 1991, did not show any difference in patients given heparin or not. Inversely, a randomized study published by Attal in 1992 including 161 patients showed a significant difference in the incidence of veno-occlusive disease between patients given heparin (2.5%) and those who were not given heparin (13.7%; p < 0.01). All these studies show that with low doses (100-150 U/kg) the risk is very very low. The mechanism of action of heparin would appear to be related to its protective effect on the endothelium rather than its hemostasis effect. The vascular protective effect of prostaglandin E1 suggests it might also be useful in preventing veno-occlusive disease.     

Antibody responses to tetanus toxoid and Haemophilus influenzae type b conjugate vaccines following autologous peripheral blood stem cell transplantation (PBSCT)

OBJECTIVE: Although there are many anecdotal reports that psychological intervention is effective in enhancing adjustment to spinal cord injury (SCI), there are little data to support this assertion. To date, reports of few longitudinal-based controlled trials that assessed psychological outcomes for SCI persons have been published. This study was conducted to determine long-term efficacy of cognitive behavior therapy during rehabilitation. DESIGN: The study employed a nonrandomized controlled trial, and measures were taken on three occasions: before, immediately after, and 12 months after treatment. SETTING, OUTCOME MEASURES, AND INTERVENTION: Anxiety, depressive mood, and self-esteem were assessed in 28 SCI persons consecutively selected on admission to hospital, who participated in specialized group cognitive behavior therapy (CBT) during rehabilitation. CONTROLS: The intervention group's responses on the measures were compared with a control group of 41 SCI persons who only received traditional rehabilitation services during their hospitalization. RESULTS: There were no overall group differences on anxiety, depressive mood, and self-esteem, although there was a trend for the treatment group to have greater levels of improvement in depression scores across time in comparison to the control group. However, those in the treatment group who reported high levels of depressive mood before the CBT treatment were significantly less depressed 1 year after injury, compared to similar persons in the control group. CONCLUSIONS: While it appears not everyone who experiences SCI needs CBT, at least in the hospital phase of their rehabilitation, those who report high levels of depressive mood benefited greatly from CBT.     

Quantitation of chemical exchange rates using pulsed-field-gradient diffusion measurements

A new approach to the quantitation of chemical exchange rates is presented, and its utility is illustrated with application to the exchange of protein amide protons with bulk water. The approach consists of a selective-inversion exchange HMQC experiment in which a short spin echo diffusion filter has been inserted into the exchange period. In this way; the kinetics of exchange are encoded directly in an apparent diffusion coefficient which is a function of the position of the diffusion filter in the pulse sequence. A detailed theoretical analysis of this experiment indicates that, in addition to the measurement of simple exchange rates, the experiment is capable of measuring the effect of mediated exchange, e.g. the transfer of magnetization from bulk water to an amide site mediated by an internal bound water molecule or a labile protein side-chain proton in fast exchange with bulk water. Experimental results for rapid water/amide exchange in acyl carrier protein are shown to be quantitatively consistent with the exchange rates measured using a selective-inversion exchange experiment.     

In vitro activities of the oxazolidinone antibiotics U-100592 and U-100766 against Staphylococcus aureus and coagulase-negative Staphylococcus species

U-100592 and U-100766 are closely related antibiotics of the oxazolidinone class. Their in vitro activities were determined against 100 isolates of Staphylococcus aureus and 100 isolates of coagulase-negative Staphylococcus species by broth and agar dilution test methods. The MICs of both compounds by either test method at which 50 and 90% of isolates are inhibited were 2 and 4 micrograms/ml, respectively, for S. aureus and 1 to 2 micrograms/ml for coagulase-negative staphylococci. Time-kill assay with selected strains indicated a primarily bacteriostatic effect against staphylococci.     

Chemical speciation of lead dust associated with primary lead smelting

The research presented in this article assessed geochemical factors relating to dust produced during primary lead smelting. Bulk dust samples and size-selective airborne dust samples were collected from four areas of a primary lead smelter and analyzed by X-ray diffraction and sequential chemical extraction. X-ray diffraction showed that the smelter dusts were composed primarily of sulfides, oxides, sulfates, and silicates of metal ores, with galena being the primary dust component. Sequential extraction revealed the solubility of lead compounds at less than 7% in the exchangeable and mildly acidic steps for the bulk dusts collected from four smelter areas. The later steps of the extraction procedure were more effective in dissolving the lead compounds associated with the bulk dust samples, with 43%, 26%, and 8% of the total lead, in the ore storage, sinter, and blast/dross smelter areas, respectively, being extracted in the residual step. Sequential extraction of coarse airborne dust samples from the ore storage and sinter plant showed that 1.2% and 4.1% of the total lead, respectively, was exchangeable. The finer particle size fractions from these areas of the smelter showed higher percentages of exchangeable lead. Of the course airborne dust from the blast/dross furnace processes, 65% of the total lead was exchangeable. However, the largest percentage of lead from these areas was associated with the finer particle-size fractions. If lead bioavailability is related to its solubility as determined through sequential extraction, the health hazards associated with lead exposure may be appreciably enhanced in the blast and dross furnace processes.     

Macroaspartasemia as a cause of isolated elevation of aspartate aminotransferase--its biochemical and physiological characteristics

The effects of a physical activity intervention on strength, balance, motor coordination, and mobility were tested in a quasi-experiment at rural congregate nutrition sites. Twice-weekly sessions of low intensity movements were conducted for one year. Logistic regression results showed significant differences between intervention (n = 61) and comparison (n = 49) groups on several performance-based measures. Intervention subjects perceived significantly greater improvements in physical functioning over the previous year than did comparison subjects. A qualitative evaluation revealed perceived program benefits of pain reduction, increased flexibility, muscle strengthening, increased walking speed, and improved mental outlook.     

Idiopathic retroperitoneal fibrosis presented as an abdominal mass and nephrotic syndrome

We present a 30-year-old male patient who was initially diagnosed as minimal change nephrotic syndrome, 5 years later, the patient developed a localized form of idiopathic retroperitoneal fibrosis (IRF). An elevated ESR and concomitant nephrotic syndrome in the patient suggested the immunologic nature of IRF, IRF has been reported in association with collagen diseases and rarely with proliferative and nonproliferative glomerulopathies. To our knowledge, the association between minimal change lesion (MC) and IRF has not been reported. Furthermore, the fact that IRF presented itself as an abdominal mass and lacked systemic symptoms was also unusual.