Analysis of genotype 2 and 3 hepatitis C virus variants in patients treated with telaprevir demonstrates a consistent resistance profile across genotypes.

Study C209 evaluated the activity of telaprevir in treatment-naïve patients with genotypes 2 or 3 (G2, G3) hepatitis C virus (HCV) infection. Telaprevir monotherapy showed potent activity against HCV G2, but limited activity against G3. This analysis was performed to characterize HCV viral variants emerging during telaprevir-based treatment of G2/G3 HCV-infected patients. Patients were randomized to receive 2 weeks of treatment with telaprevir (telaprevir monotherapy), telaprevir plus peginterferon alfa-2a and ribavirin (triple therapy), or placebo plus peginterferon alfa-2a and ribavirin (control), followed by 22-24 weeks of peginterferon/ribavirin alone. Viral breakthrough was defined as an increase >1 log10 in HCV RNA from nadir, or HCV RNA >100 IU/mL in patients previously reaching <25 IU/mL. Twenty-three patients (47%) had G2 and 26 (53%) had G3 HCV. Viral breakthrough occurred during the initial 2-week treatment phase in six G2 patients (66.7%; subtypes 2, 2a and 2b) and three G3 patients (37.5%; all subtype 3a), all in the telaprevir monotherapy arm. Four breakthrough patients (three G2, one G3) subsequently achieved sustained virologic response (SVR). In all patients with breakthrough and available sequence data, mutations associated with reduced susceptibility to telaprevir in genotype 1 (G1) HCV were observed. No novel G2/G3-specific mutations were associated with telaprevir resistance. The telaprevir resistance profile appeared consistent across HCV genotypes 1, 2 and 3. Although viral breakthrough with resistance occurred in patients receiving telaprevir monotherapy, half of these patients achieved an SVR upon addition of peginterferon/ribavirin highlighting the importance of combination therapy.

Definitive assignment of proton selectivity and attoampere unitary current to the M2 ion channel protein of influenza A virus.

The viral ion channel protein M2 supports the transit of influenza virus and its glycoproteins through acidic compartments of the cell. M2 conducts endosomal protons into the virion to initiate uncoating and, by equilibrating the pH at trans-Golgi membranes, preserves the native conformation of acid-sensitive viral hemagglutinin. The exceptionally low conductance of the M2 channel thwarted resolution of single channels by electrophysiological techniques. Assays of liposome-reconstituted M2 yielded the average unitary channel current of the M2 tetramer--1.2 aA (1.2 x 10(-18) A) at neutral pH and 2.7 to 4.1 aA at pH 5.7--which activates the channel. Extrapolation to physiological temperature predicts 4.8 and 40 aA, respectively, and a unitary conductance of 0.03 versus 0.4 fS. This minute activity, below previous estimates, appears sufficient for virus reproduction, but low enough to avert abortive cytotoxicity. The unitary permeability of M2 was within the range reported for other proton channels. To address the ion selectivity of M2, we exploited the coupling of ionic influx and efflux in sealed liposomes. Metal ion fluxes were monitored by proton counterflow, employing a pH probe 1,000 times more sensitive than available Na+ or K+ probes. Even low-pH-activated M2 did not conduct Na+ and K+. The proton selectivity of M2 was estimated to be at least 3 x 10(6) (over sodium or potassium ions), in agreement with electrophysiological studies. The stringent proton selectivity of M2 suggests that the cytopathology of influenza virus does not involve direct perturbation of cellular sodium or potassium gradients.

Urinary N-acetyl-beta-glucosaminidase as an indicator of renal dysfunction in electroplating workers.

OBJECTIVES: To investigate chromium-induced renal dysfunction in electroplating workers. METHODS: A cross-sectional study was used to evaluate four biochemical markers of renal function. A total of 178 workers were divided into 3 comparable groups consisting of 34 hard-chrome plating workers, 98 nickel-chrome electroplating workers. and 46 aluminum anode-oxidation workers, who represented the reference group. Ambient and biological monitoring of urinary chromium were performed to measure exposure concentrations. RESULTS: Overall, urinary chromium concentrations were highest among hard-chrome plating workers (geometric mean 2.44 microg/g creatinine), followed by nickel-chrome electroplating workers (0.31 microg/g creatinine) and aluminum workers (0.09 microg/g creatinine). Airborne chromium concentrations were also highest in the hard-chrome plating area (geometric mean 4.20 microg/m3), followed by the nickel-chrome electroplating area (0.58 microg/m3) and the aluminum area (0.43 microg/m3). A positive correlation was found between urinary chromium and airborne concentrations (r=0.54, P < 0.01). Urinary concentrations of N-acetyl-beta-D-glucosaminidase (NAG) were also highest among hard-chrome plating workers (geometric mean 4.9 IU/g creatinine), followed by nickel-chrome workers (3.4 IU/g creatinine) and aluminum workers (2.9 IU/g creatinine). The prevalence of "elevated" NAG (>7 IU/g creatinine) was significantly highest among hard-chrome plating workers (23.5%), then among nickel-chrome workers (7.1%) and aluminum workers (8.7%). Differences in beta2-microglobulin, total protein, and microalbumin were not significant. CONCLUSION: The author's evidence indicates that NAG is an early indicator of renal dysfunction in hard-chrome plating workers.

Different modes of inhibition by adamantane amine derivatives and natural polyamines of the functionally reconstituted influenza virus M2 proton channel protein.

The influenza virus M2 protein, target of the antiviral drugs amantadine and rimantadine, forms a proton channel which functions during virus uncoating and maturation by modifying the pH in virions as well as in trans-Golgi vesicles. We studied the influence of different ionic gradients on the inhibition of the proton translocation activity of isolated, baculovirus-expressed M2 protein reconstituted into liposomes. Two distinct patterns of inhibition were observed. A group of amphiphilic amines including amantadine, cyclooctylamine and rimantadine inhibited M2 effectively in the presence of physiological Na+ concentrations. The 10-fold greater activity of rimantadine over amantadine and the 100-fold stronger effect of cyclooctylamine compared to cyclopentylamine matched the relative activities in influenza virus-infected cells. A completely different inhibitory pattern emerged for the polyamines spermine, spermidine and putrescine. Polyamines have recently been identified as the 'intrinsic' rectifiers of a class of potassium channels and shown to interact with acidic amino acid residues lining and flanking the channel pore. In the presence of a physiological Na+/K+ gradient their minimal inhibitory concentrations for influenza virus M2 protein were 100, 400 and 500 microM, polyamine levels reported to exist in oocytes. In conditions depleted for Na+, polyamines inhibited M2 at concentrations two to three orders of magnitude lower. The data suggest that influenza virus M2 protein possesses a binding site for polyamines, distinct from the amantadine binding site, which is normally masked by Na+ and which could be targeted by selective antiviral inhibitors.

Nasal septum lesions and lung function in workers exposed to chromic acid in electroplating factories.

The objective of this study was to compare workers from chromium, nickel-chromium, and zinc electroplating factories with regards to nasal septum lesions and lung function. Also investigated was the relationship between chromium levels in air and urine. A total of 189 workers from 11 electroplating factories (three chromium, six nickel-chromium, two zinc) were chosen from central Taiwan. All subjects were interviewed by constructed-questionnaire, given a nasal examination by a certified otolaryngologist and a lung function test. In the chromium factories 30.8% of the workers showed evidence of nasal septum perforations and 38.5% showed evidence of nasal septum ulcers. A Mantel extension test for trend showed that workers in the chromium factories were 31.7 times more likely to experience nasal ulcers than nickel-chromium and zinc factory workers. Those who worked in the electroplating tank area were 4.2 times more likely to experience ulcers and those with over 9 years' experience were 30.8 times more likely. A comparison of lung function adjusted for age, gender and smoking habit among workers showed that vital capacity (VC), forced vital capacity (FVC), and forced expiratory volume in 1 s (FEV1) were all significantly decreased among chromium factory workers. Because the results showed that the workers' health is being severely damaged by the harmful environment of chromium electroplating factories, the authors wish to suggest improvements in the workplace are vitally needed to ensure the safety of the workers.

Concentration and size distribution of airborne hexavalent chromium in electroplating factories.

Studies have shown that chromium from electroplating factories can cause respiratory problems to workers after prolonged exposure. Since Taiwan has a relatively high number of electroplating factories and minimal data regarding chromium's effect on workers, the authors undertook this study focusing on the size distribution of airborne hexavalent chromium and its concentration levels within the factory. Both area and personal sampling were conducted using a particle fractionating sampler. Samples were analyzed using visible spectrophotometry. A comparison of three types of factories-chromium, nickel-chromium, and zinc-showed that the highest concentrations were found in chromium electroplating factories near the electroplating tank. Hexavalent chromium particles obtained from area sampling had mass median diameters between 1.67-6.38 microns and 0.75-4.73 microns for personal sampling. Particles of this high concentration and small size can enter and cause considerable damage to the respiratory tract.

Determination of carbohydrates by high-performance capillary electrophoresis with indirect absorbance detection.

High-performance capillary electrophoresis (HPCE) methods with indirect absorbance detection (IAD) have been developed for the determination of carbohydrates, e.g. glucose, fructose, rhamnose, ribose, maltose, lactose, sucrose and gluconic acid. The suitability and performance of six background electrolytes (BGEs), i.e., 1-naphthylacetic acid (NAA), 2-naphthalenesulfonic acid, 1,3-dihydroxynaphthalene, phenylacetic acid, p-cresol and sorbic acid, for the IAD method were investigated. The effects of the concentration of the BGE, pH and temperature on the CE separation of these analytes were evaluated. NAA was found to be best suited as the carrier buffer and background absorbance provider for the detection at 222 nm. The optimal CE performance was found when employing 2 mM NAA, pH 12.2, at 25 degrees C. In comparison with the previous method that used sorbate as the BGE, the present method utilizing NAA shows a 3-6 fold increase in the separation efficiency and a 2-5 fold improvement in the detection limit. The calculated number of theoretical plates is in the range of 1.0-3.0 x 10(5). The precision of the present method for most sugar analytes, measured by the coefficient of variation (C.V.), typically, is less than 1% for the migration time and better than 3% for the peak height and peak area (n = 6). The detection limit is about 0.1 mM for all analytes, except for ribose for which it is about 0.2 mM. This new method is fast, accurate and can be readily applied to real biological samples for quantitative determination of selected carbohydrates.

Capillary electrophoretic analysis of cyclodextrins and determination of formation constants for inclusion complexes.

Capillary zone electrophoresis (CZE) methods with indirect absorbance detection for analyzing mixtures of alpha-, beta-, and gamma-cyclodextrins (CDs) and their derivatives have been developed. Benzylamine, salicylic, sorbic, or 1-naphthylacetic acid (NAA) was utilized as background electrolyte (BGE) and absorbance provider. Separation of alpha-, beta-, and gamma-CD could be achieved in less than 18 min when the CZE was run in 2 mM NAA or 5 mM sorbate solution (pH 12.2) and detected by indirect absorbance at 222 or 254 nm, respectively. Mixtures of alpha- and beta-CDs, and dimethyl- and trimethyl-derivatives of beta-CD could also be analyzed by CZE, using 50 mM salicylic acid or benzylamine solution (pH 6.0) as BGE with indirect absorbance detection at 230 and 210 nm, respectively. CZE methods for determining the inclusion complex formation constants of various CDs for salicylic acid or benzylamine with either direct or indirect absorbance detection have also been developed. The formation constants of salicylate are in the range from ca. 8 +/- 0.3 mole-1 for the complex with alpha-CD to ca. 99 +/- 2 molarity-1 for the complex with methyl-beta-CD. The detection limits (determined at a signal-to-noise ratio of 3) for the NAA and the salicylate system are ca. 0.1 mM and 1 mM, respectively.

Analysis of the iron-sulfur clusters within the complex I (NADH:ubiquinone oxidoreductase) isolated from potato tuber mitochondria.

The mitochondrial complex I (NADH:ubiquinone oxidoreductase) isolated from potato (Solanum tuberosum) has been investigated for the presence of iron-sulfur clusters. EPR spectroscopic analysis detected signals arising from clusters N1, N2, N3 and N4. Quantitation of the content of iron and sulfur within the isolated complex I showed the preparation to contain 22.6 mol acid-labile sulfide and 30.4 mol iron/mol complex I. The iron-sulfur cluster composition of the plant complex I appears to be similar to the well-known composition found in Neurospora crassa.

Evaluation of urinary pseudouridine as a tumor marker in lung cancer.

The urinary concentration of pseudouridine, primarily a breakdown product of tRNA, was determined by high-performance liquid chromatography in 33 patients with small cell lung cancer (SCLC), 18 patients with non-small cell lung cancer (NSCLC), 18 patients with benign pulmonary diseases (eg, TB, COPD), and 16 healthy controls. The mean urinary pseudouridine concentration levels of the patients with SCLC, NSCLC, benign pulmonary disease and the healthy controls were 22.7 +/- 11.8, 15.6 +/- 6.0, 13.4 +/- 3.6, 12.1 +/- 3.2 nmol/mumol creatinine (mean +/- SD), respectively. The mean urinary pseudouridine concentration was significantly higher in patients with SCLC than that in patients with NSCLC, benign pulmonary disease and in the healthy controls. There was no significant difference in the urinary pseudouridine levels of patients with NSCLC and healthy controls or patients with benign pulmonary disease. Urinary pseudouridine levels above 18.5 nmol/mumol creatinine (mean value +/- 2SD for healthy controls) were noted in 54.5% of patients with SCLC, including 11 of 17 (64.7%) in extensive stage and seven of 16 (43.8%) in limited stage, and 27.8% of the patients with NSCLC. Of the 12 patients with SCLC who had follow-up urinary samples taken in series during chemotherapy courses, there was considerable urinary pseudouridine level change which paralleled the change in clinical response. Although urinary pseudouridine is not a specific marker for SCLC, it represents the tumor burden and reflects the clinical status. These findings indicate that urinary pseudouridine may be a useful tumor marker in patients with SCLC.

Fluorescent probes for cancer detection.

Several new fluorescent dyes, derivatives of pyrene and of coumarin, were synthesized that have excitation and emission wavelength maxima considerably red-shifted as compared to most pyrene and coumarin compounds. These new fluorescent compounds have high extinction coefficients and high quantum yields, and they also are very environmentally sensitive, which makes them good probes of biological systems. Several of these fluorescent compounds are preferentially taken up and retained by leukemic and other cancer tissue cell lines as compared to normals, particularly 1,3-dihydroxy 6,8-pyrenedisodiumsulfonate and 3-(carboxymethylester)-7-julolidinocoumarin. These compounds may have some usefulness in cancer detection.

Calcium regulates troponin-tropomyosin binding in the reconstituted thin filament.

Steady-state fluorescence anisotropy technique was used to determine the binding constant of troponin for IAEDANS-labeled tropomyosin under various conditions. In the absence of actin, Ca does not affect the binding between troponin and tropomyosin. The presence of actin greatly strengthens troponin-tropomyosin binding in the absence of Ca. However, Ca weakens troponin-tropomyosin binding by about 2.5-fold in the reconstituted filament. It is suggested that the Ca-regulated binding may serve as a molecular switch for the troponin molecule to get "on" and "off" the actin-myosin interaction site regulating muscle contraction-relaxation cycles.

Studies on the spatial arrangement of muscle thin filament proteins using fluorescence energy transfer.

The distance between a pair of fluorophores attached to Cys-36 of beta-tropomyosin and Cys-373 of actin in reconstituted muscle thin filaments was measured by fluorescence energy transfer. Two pairs of donor/acceptor fluorophores, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid/5-iodoacetamidofluorescein and N-(1-pyrene)maleimide/dimethylamino-4-maleimidostilbene, were covalently attached to tropomyosin and actin. The energy transfer efficiencies in various reconstituted systems were determined from decrease in donor lifetime using nanosecond pulse fluorimetry. Based on the 8 to 13% energy transfer efficiency observed for the first donor/acceptor pair labeled on tropomyosin and actin, respectively, and a calculated critical distance of 45 A (assumed kappa 2 = 2/3) the distance between Cys-373 of actin and Cys-36 of beta-tropomyosin was estimated to be 65 A. Fluorescence energy transfer experiments using other donor/acceptor pairs gave similar results. Since Cys-373 of actin is thought to be in the myosin head-binding sites, the minimal distance between tropomyosin and the myosin-binding site on actin is estimated to be about 34.5 A. These results place some constraints on possible spatial arrangements of thin filament proteins.

Excimer fluorescence of pyrene-tropomyosin adducts.

Studies of the fluorescence of N-(1-pyrene)maleimide and N-(1-pyrenyl)iodoacetamide adducts of rabbit skeletal muscle tropomyosin revealed the presence of excimer fluorescence characterized by a broad emission band at 480 nm with a shoulder at 505 nm. Monomer fluorescence decay exhibited different lifetimes, viz., about 3, 22 and 87 ns for the pyrenemaleimide adduct; about 2.5, 11 and 51 ns for the aminolyzed maleimide adduct; and 2.5, 15 and 74 ns for the pyrenyliodoacetamide adduct. Almost identical excimer fluorescence lifetimes were found for all adducts; about 9, 35, and 65 ns. Excimer fluorescence was sensitive to changes in ionic strength and pH of the medium while monomer fluorescence did not change. The protein denaturants guanidine hydrochloride and urea caused dissociation of the two tropomyosin subunits and partial disappearance of excimer fluorescence, but not as effectively as the hydrophobic surfactant sodium dodecyl sulfate. The sensitivity of excimer fluorescence to changes in the microenvironment make these pyrene derivatives very useful probes for studying conformational changes and binding interaction of tropomyosin with other contractile proteins. The unique location of the excimer probe at tropomyosin Cys-190 and its characteristic long lifetimes could make it useful in time-resolved anisotropy studies and fluorescence energy-transfer experiments.

Fluorescence spectroscopic studies of pyrene-actin adducts.

Reaction kinetic studies of the sulfhydryl-directed fluorescent probes N-(1-pyrene)maleimide (PM) and N-(1-pyrenyl)iodoacetamide with actin from rabbit skeletal muscle showed that there were three accessible sulfhydryl groups in actin. Fluorescence spectral studies showed energy transfer from aromatic amino acid residues to fluorophore reacted at Cys-373, as well as weak excimer fluorescence probably due to doubly labeled molecules at Cys-10 and Cys-373. These results provide further evidence that trytophan and tyrosine residues are located near the probe attached to Cys-373 or Cys-10 and the latter two thiols are in close proximity. In age PM-labeled F-actin, the succinimido ring of PM underwent intramolecular aminolysis, resulting in large emission spectral changes and increased excimer fluorescence. Solvent perturbation studies indicate that the probes were located in a hydrophobic environment; their quantum yield and spectrum properties were very sensitive to changes in the microenvironment. Nanosecond-pulse fluorimetry studies revealed complex fluorescence emission decays with three intrinsic lifetimes in adducts with low molecular weight thiols as well as in labeled proteins. Fluorescence lifetimes were 17, 48 and 111 ns for the pyrenemaleimide adduct of actin, and 3, 14 and 60 ns for the pyrenyliodoacetamide adduct. Supporting evidence is given for the argument that multiple fluorescence lifetimes are an intrinsic property of the pyrene derivatives and are not due to the presence of impurity or heterogeneity in the protein reaction sites. Because of their high sensitivity and long lifetimes, pyrene derivatives are extremely useful.

Circular-dichroic studies on the conformational behaviour of troponin and tropomyosin from bovine cardiac muscle.

Bovine cardiac troponin is similar to rabbit skeletal troponin with respect to secondary structure, amino acid composition and molecular weight of the subunits, but differs slightly with respect to biological activity and surface charges of the subunits. Previous circular-dichroic studies of the subunits and recombination of subunits have indicated significant Ca2+-induced delocalized conformational changes. Present studies of the native troponin complex are not in accord with such changes. Furthermore the formation of the troponin-tropomyosin complex in vitro results in no delocalized conformational changes, nor does it sensitize troponin to Ca2+-induced changes. It is suggested that the troponin complex cannot be dissociated into subunits without significant and irreversible conformational perturbation.