Cold ethanol fractionation and heat inactivation of hepatitis a virus during manufacture of albumin from human plasma

The purpose of the present study was to examine the efficacy and mechanism of fraction IV cold ethanol fractionation and pasteurization (60°C heat treatment for 10h), involved in the manufacture of albumin from human plasma, in the removal and/or inactivation of the hepatitis A virus (HAV). Samples from the relevant stages of the production process were spiked with HAV and the amount of virus in each fraction then quantified using a 50% tissue culture infectious dose (TCID₅₀). HAV was effectively partitioned from albumin during the fraction IV cold ethanol fractionation with a log reduction factor of 3.43. Pasteurization was also found to be a robust and effective step in inactivating HAV, where the titers were reduced from an initial titer of 7.60 log TCID₅₀ to undetectable levels within 5 h of treatment. The log reduction factor achieved during pasteurization was≽4.76. Therefore, the current results indicate that the production process for albumin has sufficient HAV reducing capacity to achieve a high margin of virus safety.     

Mechanistic studies with N-benzyl-1-aminobenzotriazole-inactivated CYP2B1: differential effects on the metabolism of 7-ethoxy-4-(trifluoromethyl)coumarin, testosterone, and benzphetamine

Mechanistic studies with N-benzyl-1-aminobenzotriazole (BBT)-inactivated cytochrome P450 2B1 were conducted to determine which step(s) in the reaction cycle had been compromised. Stopped-flow studies, formation of the oxy-ferro intermediate, and analysis of products suggested that the reductive process was slower with the BBT-modified enzyme. The reduced rate of reduction alone could not account for the loss in 7-ethoxy-4-(trifluoromethyl)coumarin (EFC) O-deethylation or testosterone hydroxylation activity. Surprisingly, the ability of the BBT-modified enzyme to generate formaldehyde from benzphetamine was much less affected. Benzphetamine metabolite analysis by electrospray ionization-mass spectrometry showed that the BBT-modified enzyme had a slightly greater propensity towards aromatic hydroxylation together with reduced levels of N-demethylation and little change in the N-debenzylation of benzphetamine. Orientation of substrates within the active site of the BBT-inactivated enzyme may be affected such that the more flexible benzphetamine can be metabolized, whereas metabolism of rigid, planar molecules such as EFC and testosterone is hindered.     

Behavior of microbial communities developed in the presence/reduced level of soluble microbial products

Soluble microbial products (SMP) are organics produced by microorganisms as they degrade substrates. The available literature does not reveal how SMP affect and regulate microbial activities. In this study, we monitored variations in pH, dissolved oxygen concentration, soluble biological and chemical oxygen demands (sBOD₅ and sCOD) as a measure of microbial activity in synthetic wastewater. Aerobic degradation tests were carried out under the following conditions: aeration, 1,500 cm³ /min; initial sBOD₅, 515±5 mg/l; initial sCOD, 859±6 mg/l; initial biomass concentration (defined as mixed liquor suspended solids), 1,200±25 mg/l; sludge retention time, 24 h; and temperature, 20±1°C. The study involved non-acclimated biomass (R0 flora), biomass developed in the presence of SMP (R1 flora), and biomass developed in reduced level of SMP (R2 flora). We also determined which of these flora produced more refractory SMP. The results showed that R2 flora utilized the synthetic feed more quickly, and produced less refractory organic matter than R0 and R1 flora. The production of more refractory organics by R0 and R1 flora shows that not all the biomass was active. R1 flora degraded the substrates irregularly, suggesting that some microbes were dependent on the metabolic products of those that could utilize the feed components. These results show that production of SMP also depends on the prior substrates and on the ability of the flora to respond to changes in substrate composition.     

X-inactivation is stably maintained in mouse embryos deficient for histone methyl transferase G9a

One of the two X chromosomes becomes inactivated during early development of female mammals. Recent studies demonstrate that the inactive X chromosome is rich in histone H3 methylated at Lys-9 and Lys-27, suggesting an important role for these modifications in X-inactivation. It has been shown that in the mouse Eed is required for maintenance of X-inactivation in the extraembryonic lineages. Interestingly, Eed associates with Ezh2 to form a complex possessing histone methyltransferase activity predominantly for H3 Lys-27. We previously showed that G9a is one of the histone methyltransferases specific for H3 Lys-9 and is essential for embryonic development. Here we examined X-inactivation in mouse embryos deficient for G9a. Expression of Xist, which is crucial for the initiation of X-inactivation, was properly regulated and the inactivated X chromosome was stably maintained even in the absence of G9a. These results demonstrate that G9a is not essential for X-inactivation.     

Radiation Inactivation Analysis of Progesterone Receptor in Human Uterine Cytosol

It is believed that human progesterone receptor (PR) contains a ligand binding subunit A (83 kDa) or subunit B (120 kDa) and 2 copies of heat shock proteins (hsp90) of molecular weight 90 kDa. To elucidate the mechanism of hormone binding, we employed radiation inactivation to determine its functional size. The functional masses determined in the presence of glycerol, molybdate and potassium chloride were 120 \pm 14, 124 \pm 13 and 130 \pm 20 kDa, respectively. From scatchard plot analysis, the radiation decreased the binding sites and increased the binding affinity of PR with ligand. The functional masses of PR dissolved in the three variant buffers were similar to the molecular weight of PR subunit B. The results implied that PR subunit B could bind with ligand despite hsp90 and hsp90 was not involved in the PR binding to progesterone.     

Mechanism of arachidonic acid modulation of the T-type Ca2+ channel alpha1G

Arachidonic acid (AA) modulates T-type Ca(2+) channels and is therefore a potential regulator of diverse cell functions, including neuronal and cardiac excitability. The underlying mechanism of modulation is unknown. Here we analyze the effects of AA on the T-type Ca(2+) channel alpha(1G) heterologously expressed in HEK-293 cells. AA inhibited alpha(1G) currents within a few minutes, regardless of preceding exposure to inhibitors of AA metabolism (ETYA and 17-ODYA). Current inhibition was also observed in cell-free inside-out patches, indicating a membrane-delimited interaction of AA with the channel. AA action was consistent with a decrease of the open probability without changes in the size of unitary currents. AA shifted the inactivation curve to more negative potentials, increased the speed of macroscopic inactivation, and decreased the extent of recovery from inactivation at -80 mV but not at -110 mV. AA induced a slight increase of activation near the threshold and did not significantly change the deactivation kinetics or the rectification pattern. We observed a tonic current inhibition, regardless of whether the channels were held in resting or inactivated states during AA perfusion, suggesting a state-independent interaction with the channel. Model simulations indicate that AA inhibits T-type currents by switching the channels into a nonavailable conformation and by affecting transitions between inactivated states, which results in the negative shift of the inactivation curve. Slow-inactivating alpha(1G) mutants showed an increased affinity for AA with respect to the wild type, indicating that the structural determinants of fast inactivation are involved in the AA-channel interaction.     

Physiological characterization of a microbial sensor containing the yeast Arxula adeninivorans LS3

The yeast Arxula adeninivorans LS3 is a suitable organism for use as part of a microbial sensor. In combination with an amperometric oxygen electrode the sensor offered a possibility for the physiological characterization of this yeast. About 300-400 measurements could be carried out with a single Arxula sensor. The microbial sensor was remarkably stable for over 35 days, when kept at 37 °C during the operation time and at room temperature overnight. The physiological characteristics of Arxula adeninivorans LS3 obtained with the sensor technique were identical to the data obtained with the conventional techniques. However, the sensor technique makes it additionally possible to quantify the physiological data. So the substrates ribose, citric acid, glycerol, oil and benzoate produced signals lower than 10% in comparison to the glucose signal. Fructose, xylose, sucrose, maltose, gentianose, glucosamine, glutamic acid, tryptophan, butyric acid, lauryl acid and propionic acid reached 10-70%, galactose, alanine, glycine, lysine and methionine signals were similar to the glucose signal whereas acetic acid, ethyl alcohol, capron acid, capryl acid and caproic acid reached the highest signals up to 434%.     

Influence of synthetic sheep urine on the microbial biomass,activity and community structure in two pastures in the Scottish uplands

The impact of urine on the microbial biomass, activity and community structure was compared in the soil beneath two pastures in the Scottish uplands; Fasset, a natural Agrostis capillaris–Festuca ovina–Galium saxatile grassland and Strathfinella, a semi-natural grassland, improved with fertiliser addition. Community level physiological profiles (CLPP) were used to characterise the microbial communities. The utilisation of sugars, oligosaccharides, alcohols, carboxylic acids, long chain aliphatic acids, acidic, basic and neutral amino acids, amide N, phenolic acids and long chain aliphatic acids was used to compare the soils and the impact of synthetic urine addition. In the untreated soils, the utilisation of all the substrates decreased from the first week in May through to October. Averaged over all times and urine treatment, the potential utilisation of all substrates except for phenolic acids, long chain aliphatic acids and carboxylic acids was greater in the improved and more intensively grazed Strathfinella site. When averaged over all sample times, urine increased the utilisation of sugars, oligosaccharides, basic amino acids and amide N and the increases were greater in the unimproved, less intensively grazed, Fasset soil than that at Strathfinella. The effect of urine tended to be greatest during the period between 2 and 5 weeks after urine addition when utilisation of alcohols, acidic and neutral amino acids was also increased. Microbial biomass C in the control soils was 155.9 and 112.7 g C m⁻² at Fasset and Strathfinella, respectively. Values did not change significantly with time and were unchanged by the addition of urine. However, urine addition significantly increased basal respiration rates at Fasset and decreased them at Strathfinella. Urine also increased bacterial numbers in both soils, but had no consistent effect on fungi or yeasts. The significance of these findings for studies of soil microbial community structure and activity in grazed upland grasslands is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Microbial models of soil metabolism: biotransformations of danofloxacin

Danofloxacin is a new synthetic fluoroquinolone antibacterial agent under development for exclusive use in veterinary medicine. Such use could lead to deposition of low levels of danofloxacin residues in the environment in manure from treated livestock. This study was conducted to evaluate the potential for indigenous soil microorganisms to metabolize danofloxacin. Cultures of 72 soil microorganisms representing a diverse panel of bacteria, fungi and yeast were incubated with danofloxacin mesylate substrate and samples analyzed periodically by high performance liquid chromatography for loss of danofloxacin and formation of metabolites. Some samples were further analyzed by liquid chromatography-mass spectrometry and mass spectrometry to confirm metabolite identification. Twelve organisms, representing eight different genera, biotransformed danofloxacin to metabolites detectable by the chromatographic methods employed. Two Mycobacterium species, two Pseudomonas species, and isolates of Nocardia sp, Rhizopus arrhizus and Streptomyces griseus all formed N-desmethyldanofloxacin. The formation of the 7-amino danofloxacin derivative, 1-cyclopropyl-6-fluoro-7-amino-4-oxo-1,4-dihydroquinoline-3-carboxylic acid by cultures of Candida lipopytica, Pseudomonas fluorescens, two Mycobacterium species and three Penicillium species demonstrates the propensities of these cultures to completely degrade the piperazine ring. At least two additional and unidentified metabolite peaks were observed in chromatograms of Aspergillus nidulans and Penicillium sp cultures. Radiolabled [2-¹⁴C]danofloxacin added to cultures of the fungus Curvularia lunata was apparently mineralized, with approximately 31% of the radiolabel recovered as volatile metabolites after 24 h of incubation, indicating the susceptibility of the quinolone ring to microbial metabolic degradation. Received 09 December 1996/ Accepted in revised form 09 April 1997     

The rare occurrence of plant tissue culture contamination by Methylobacterium mesophilicum

A pink-pigmented, facultative methylotrophic (PPFM) bacterium, Methylobacterium mesophilicum, which is found on the leaf surface of most plants, has been reported to be a covert contaminant of tissue cultures initiated from Glycine max (soybean) leaves and seeds by Holland and Polacco (1992). The bacteria can be detected as pink colonies when leaves are pressed or tissue culture homogenates are plated on a medium with methanol as the sole carbon source. Since the presence of contaminating bacteria can confound any biochemical results obtained with such cultures (Holland and Polacco 1992), we wanted to determine the extent of the contamination of our tissue cultures of soybean and other species. No PPFMs were detected in any soybean culture we have, and previous results describing the biochemical characteristics of ureide utilization by one of our soybean suspension cultures (27C) also indicates that PPFM bacteria were not present. Analysis of about 200 other strains of 11 different species maintained in this lab showed that only three of about 160 callus cultures, recently initiated from Datura innoxia leaves, contained PPFMs. The D. innoxia leaves did have PPFMs on their surface but in most cases they did not survive the surface disinfestation and culture regimes. Thus PPFM bacterial contamination should not be a serious problem in most plant tissue cultures.Abbreviations AMS ammonium mineral salts medium - PPFM pink-pigmented facultative methylotrophic bacteria