Inactivation of de novo DNA methyltransferase activity by high concentrations of double-stranded DNA

The activity of eukaryotic DNA methyltransferase diminishes with time when the enzyme is incubated with high concentrations (200–300 μg/ml) of unmethylated double-stranded Micrococcus luteus DNA. Under similar conditions, single-stranded DNA induces only a limited decrease of enzyme activity. The inactivation process is apparently due to a slowly progressive interaction of the enzyme with double-stranded DNA that is independent of the presence of S-adenosyl-l-methionine. The inhibited enzyme cannot be reactivated either by high salt dissociation of the DNA-enzyme complex or by extensive digestion of the DNA. Among synthetic polydeoxyribonucleotides both poly(dG-dC) · poly(dG-dC) and poly(dA-dT) · poly(dA-dT), but not poly(dI-dC) · poly(dI-dC), cause inactivation of DNA methyltransferase. This inactivation process may be of interest in regulating the ‘de novo’ activity of the enzyme.     

NH2-terminal inactivation peptide binding to C-type-inactivated Kv channels

In many voltage-gated K(+) channels, N-type inactivation significantly accelerates the onset of C-type inactivation, but effects on recovery from inactivation are small or absent. We have exploited the Na(+) permeability of C-type-inactivated K(+) channels to characterize a strong interaction between the inactivation peptide of Kv1.4 and the C-type-inactivated state of Kv1.4 and Kv1.5. The presence of the Kv1.4 inactivation peptide results in a slower decay of the Na(+) tail currents normally observed through C-type-inactivated channels, an effective blockade of the peak Na(+) tail current, and also a delay of the peak tail current. These effects are mimicked by addition of quaternary ammonium ions to the pipette-filling solution. These observations support a common mechanism of action of the inactivation peptide and intracellular quaternary ammonium ions, and also demonstrate that the Kv channel inner vestibule is cytosolically exposed before and after the onset of C-type inactivation. We have also examined the process of N-type inactivation under conditions where C-type inactivation is removed, to compare the interaction of the inactivation peptide with open and C-type-inactivated channels. In C-type-deficient forms of Kv1.4 or Kv1.5 channels, the Kv1.4 inactivation ball behaves like an open channel blocker, and the resultant slowing of deactivation tail currents is considerably weaker than observed in C-type-inactivated channels. We present a kinetic model that duplicates the effects of the inactivation peptide on the slow Na(+) tail of C-type-inactivated channels. Stable binding between the inactivation peptide and the C-type-inactivated state results in slower current decay, and a reduction of the Na(+) tail current magnitude, due to slower transition of channels through the Na(+)-permeable states traversed during recovery from inactivation.     

Altered inactivation of Ca2+ current and Ca2+ release in mouse muscle fibers deficient in the DHP receptor gamma1 subunit

Functional impacts of the skeletal muscle-specific Ca2+ channel subunit gamma1 have previously been studied using coexpression with the cardiac alpha1C polypeptide in nonmuscle cells and primary-cultured myotubes of gamma1-deficient mice. Data from single adult muscle fibers of gamma-/- mice are not yet available. In the present study, we performed voltage clamp experiments on enzymatically isolated mature muscle fibers of the m. interosseus obtained from gamma+/+ and gamma-/- mice. We measured L-type Ca2+ inward currents and intracellular Ca2+ transients during 100-ms step depolarizations from a holding potential of -80 mV. Ratiometric Ca2+ transients were analyzed with a removal model fit approach to calculate the flux of Ca2+ from the sarcoplasmic reticulum. Ca2+ current density, Ca2+ release flux, and the voltage dependence of activation of both Ca2+ current and Ca2+ release were not significantly different. By varying the holding potential and recording Ca2+ current and Ca2+ release flux induced by 100-ms test depolarizations to +20 mV, we studied quasi-steady-state properties of slow voltage-dependent inactivation. For the Ca2+ current, these experiments showed a right-shifted voltage dependence of inactivation. Importantly, we could demonstrate that a very similar shift occurred also in the inactivation curve of Ca2+ release. Voltages of half maximal inactivation were altered by 16 (current) and 14 mV (release), respectively. Muscle fiber bundles, activated by elevated potassium concentration (120 mM), developed about threefold larger contracture force in gamma-/- compared with gamma+/+. This difference was independent of the presence of extracellular Ca2+ and likely results from the lower sensitivity to voltage-dependent inactivation of Ca2+ release. These results demonstrate a specific alteration of voltage-dependent inactivation of both Ca2+ entry and Ca2+ release by the gamma1 subunit of the dihydropyridine receptor in mature muscle fibers of the mouse.     

Assessment of iodine‐treated filter media for removal and inactivation of MS2 bacteriophage aerosols

Aims: To investigate the performance of an iodine‐releasing filter medium for use as a protective device against airborne pathogens. Methods and Results: The filter’s physical and viable removal efficiencies (VRE) were investigated with challenges of MS2 bacteriophage aerosols, and the infectivity of MS2 collected on the filter was analysed. To test a proposed inactivation mechanism, media containing thiosulfate or bovine serum albumin (BSA) were put in impingers to quench and consume I₂ released from the filter. In direct plating experiments, treated filters presented significantly higher VREs than did untreated filters; however, collection in excess BSA decreased VRE by half and in thiosulfate the apparent VRE decreased drastically. No significant difference in infectivity of retained viruses on treated and untreated filters was observed at the same environmental condition. Conclusions: Evidence presented herein for competition by dissolved I₂ in infectivity assays supports a mechanism of induced displacement and capture of I_2. It also requires that dissociation of iodine from the filter and capture of iodine by MS2 aerosols as they pass through the filter be factored in the design of the assessment methodology. The filter’s strong retention capability minimizes reaerosolization but also makes it difficult to discriminate the antimicrobial effect at the surface. Significance and Impact of the Study: This study shows the direct plating assay method to be sensitive to interference by iodine‐releasing materials. This requires reevaluation of earlier reports of VRE measurements.     

The fusogenic state of mayaro virus induced by low pH and by hydrostatic pressure

Mayaro virus is an enveloped virus that belongs to the Alphavirus genus. To gain insight into the mechanism involved in Mayaro virus membrane fusion, we used hydrostatic pressure and low pH to isolate a fusion-active state of Mayaro glycoproteins. In response to pressure, E1 glycoprotein undergoes structural changes resulting in the formation of a stable conformation. This state was characterized and correlated to that induced by low pH as measured by intrinsic fluorescence, 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, dipotassium salt fluorescence, fluorescence resonance energy transfer, electron microscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In parallel, we used a neutralization assay to show that Mayaro virus in the fusogenic state retained most of the original immunogenic properties and could elicit high titers of neutralizing antibodies.     

Identification of a novel glycosyltransferase involved in LOS biosynthesis of Moraxella catarrhalis

Moraxella catarrhalis is an important human mucosal pathogen that contributes to otitis media in infants and exacerbates conditions such as chronic obstructive pulmonary disease in the elderly. This study describes the identification of a novel gene, lgt5 that encodes a glycosyltransferase involved in the LOS biosynthesis of M. catarrhalis. Analysis of NMR data of LOS-derived oligosaccharide from a Serotype A lgt5 mutant strain of M. catarrhalis indicate that lgt5 encodes an alpha-(1-->4)-galactosyltransferase.     

Solubilization and Characterization of D2-Dopamine Receptors in an Estrone-Induced, Prolactin-Secreting Rat Pituitary Adenoma

D2-dopamine (3,4-dihydroxyphenylethylamine) receptors were successfully solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate from an estrone-induced rat pituitary adenoma. Forty-five percent of initial protein and 48% of initial [3H]spiroperidol binding sites were solubilized. The high affinity as well as the stereoselectivity of the sites was preserved. The order of potency of dopaminergic agonists was found to be typical of D2 receptors. Target size analysis by radiation inactivation indicated a molecular weight of 143,000 +/- 3,000 and of 106,000 +/- 4,000 daltons for membrane-bound and solubilized receptors, respectively. This suggests the loss of a 37,000-dalton subunit during solubilization without significant modification of binding characteristics. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of receptor protein preparation photolabeled with N-(p-azido-m[125I]iodophenethyl)spiroperidol confirmed the existence of a 94,000-dalton peptide which probably constitutes the ligand binding site of the receptor. Thus, our data indicate that chronic estrogen treatment of rats, although inducing a pituitary adenoma, does not modify the pharmacological characteristics of D2 receptors. These data suggest therefore that these adenoma may represent an ideal source of material for further biochemical characterization of D2 receptors.     

Study of the thermal stability of D-amino acid oxidase from Trigonopsis variabilis reveals enzyme inactivation via multiple steps

The thermal stability of a highly purified preparation of D-amino acid oxidase from Trigonopsis variabilis (TvDAO), which does not show microheterogeneity due to the partial oxidation of Cys-108, was studied based on dependence of temperature (20-60°C) and protein concentration (5-100 µmol L^-1). The time courses of loss of enzyme activity in 100 mmol L^-1 potassium phosphate buffer, pH 8.0, are well described by a formal kinetic mechanism in which two parallel denaturation processes, partial thermal unfolding and dissociation of the FAD cofactor, combine to yield the overall inactivation rate. Estimates from global fitting of the data revealed that the first-order rate constant of the unfolding reaction (k _a) increased 10⁴-fold in response to an increase in temperature from 20 to 60°C. The rate constants of FAD release (k _b) and binding (k _-b) as well as the irreversible aggregation of the apo-enzyme (k _agg) were less sensitive to changes in temperature, their activation energy (E _a) being about 52 kJ mol^-1 in comparison with an E _a value of 185 kJ mol^-1 for k _a. The rate-determining step of TvDAO inactivation switched from FAD dissociation to unfolding at high temperatures. The model adequately described the effect of protein concentration on inactivation kinetics. Its predictions regarding the extent of FAD release and aggregation during thermal denaturation were confirmed by experiments. TvDAO is shown to contain two highly reactive cysteines per protein subunit whose modification with 5,5'-dithio-bis (2-nitrobenzoic acid) was accompanied by inactivation. Dithiothreitol (1 mmol L^-1) enhanced up to 10-fold the recovery of enzyme activity during ion exchange chromatography of technical-grade TvDAO. However, it did not stabilize TvDAO at all temperatures and protein concentrations, suggesting that deactivation of cysteines was not responsible for thermal denaturation.     

鱼类培养细胞干扰素的诱导

对紫外线灭活的草鱼出血病病毒（ＧＣＨＶ）－Ｆ９株,在几种鲤科鱼类培养细胞中诱导产生干扰素的能力及影响干扰素产量的各因素进行了研究。纯化的ＧＣＨＶ在紫外线照射５分丧失感染性,但获得了在ＣＡＢ等５种鲤科鱼类培养细胞中高铲诱导产生干扰素的能力。干扰素的诱导需要病毒高复数感染细胞。干扰素主要在诱导后１４小时时内产生。培养上清中的新生牛血清对干扰素的产量有抑制作用。而在ＰＨ６．２－７．８范围内对干扰素产量无