Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity.

To define catalytically essential residues of bacteriophage T7 RNA polymerase, we have generated five mutants of the polymerase, D537N, K631M, Y639F, H811Q and D812N, by site-directed mutagenesis and purified them to homogeneity. The choice of specific amino acids for mutagenesis was based upon photoaffinity-labeling studies with 8-azido-ATP and homology comparisons with the Klenow fragment and other DNA/RNA polymerases. Secondary structural analysis by circular dichroism indicates that the protein folding is intact in these mutants. The mutants D537N and D812N are totally inactive. The mutant K631M has 1% activity, confined to short oligonucleotide synthesis. The mutant H811Q has 25% activity for synthesis of both short and long oligonucleotides. The mutant Y639F retains full enzymatic activity although individual kinetic parameters are somewhat different. Kinetic parameters, (kcat)app and (Km)app for the nucleotides, reveal that the mutation of Lys to Met has a much more drastic effect on (kcat)app than on (Km)app, indicating the involvement of K631 primarily in phosphodiester bond formation. The mutation of His to Gln has effects on both (kcat)app and (Km)app; namely, three- to fivefold reduction in (kcat)app and two- to threefold increase in (Km)app, implying that His811 may be involved in both nucleotide binding and phosphodiester bond formation. The ability of the mutant T7 RNA polymerases to bind template has not been greatly impaired. We have shown that amino acids D537 and D812 are essential, that amino acids K631 and H811 play significant roles in catalysis, and that the active site of T7 RNA polymerase is composed of different regions of the polypeptide chain. Possible roles for these catalytically significant residues in the polymerase mechanism are discussed.     

Influence of Nitrate and Sulfate on the Anaerobic Treatment of Pharmaceutical Wastewater

Anaerobic treatment of wastewater from the pharmaceutical industry, which contained about 3.2 g/L of sulfate, was carried out in an Upflow Anaerobic Sludge Blanket (UASB) reactor. After a startup period of 120 days, a chemical oxygen demand (COD) removal efficiency of more than 90 % was obtained along with an organic loading rate (OLR) of 1.5 g COD/(L day). During the same period, the sulfate removal was about 90 %. However, the performance of the reactor was affected when the loading rate was increased to 2.09 g COD/(L day). It was found that the accumulation of sulfides, combined with a decrease in the pH, affected the reactor performance. In batch reactor studies with pharmaceutical wastewater it was observed that methane production began only after the initiation of nitrate consumption. The denitrification process can inhibit sulfate reduction at high nitrate concentrations, but compared to reactors without nitrate, the sulfate reduction process and sulfide formation were quickly initiated at low nitrate concentrations. The methanogenic activity was however affected by the presence of more than 2 g/L of sulfate.     

Germination of concentrated suspensions of spores from Aspergillus niger

Summary Germination of condiospores fromA.niger in very concentrated suspension was required to inoculate solid fermentation media, but a germination self-inhibitor was detected in spores. It was found that the inhibitory effect depended on the composition of the medium and was reduced when glucose was used as a carbon source. The effect of the self-inhibitor was eliminated by washing the spores and using glucose and a protein nitrogen source in the germination medium. By this method it was possible to increase about 100 times (10⁶ to 10⁸) spore concentration, keeping more than 90% germination.     

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

Ethanol-sensitive mutants of Saccharomyces cerevisiae

Saccharomyces cerevisiae mutants unable to grow at ethanol concentrations at which the wild type strain S288C does grow, have been isolated. Some of them show additional phenotypic alterations in colony size, temperature sensitivity and viability in ethanol, which cosegregate with the growth sensitivity in ethanol. 21 selected monogenic ethanol-sensitive mutants define 20 complementation groups, denominated ETA1 to ETA20, which indicates that there is a high number of genes involved in the ethanol tolerance/sensitivity mechanism.Out of 21 selected monogenic mutants, 20 are not altered in the glycolytic pathway since, when maintained in glucosesupplemented medium, they can produce as much ethanol as the wild type and at about the same velocity. Nor do any of the mutants seem to be altered in the lipid biosynthetic pathway since, whether grown in the absence or in the presence of ethanol, their concentration of fatty acids and ergosterol is similar to that of the wild type under the same conditions. Therefore growth sensitivity to ethanol does not seem necessarily to be related to carbohydrate or lipid metabolism.Non-common abbreviations YP yeast extract peptone medium - YPD yeast extract peptone dextrose agar or medium - YPG yeast extract peptone glycerol agar - YPDE yeast extract peptone dextrose ethanol agar or medium - SD yeast nitrogen base dextrose agar - SPO yeast extract potassium acetate glucose agar - PD parental ditype - NPD non-parental ditype - TT tetratype     

Selection of Wine Yeasts for Growth and Fermentation in the Presence of Ethanol and Sucrose

To optimize the conversion of carbohydrates to ethanol, strains of several Saccharomyces species were examined for the ability to grow and ferment in a range of sucrose and ethanol concentrations. A total of 632 wine yeasts, most of them isolated from wineries in Andalusia and Extremadura, southwestern Spain, were subjected to screening and selection. Growth and fermentative capacity in different ethanol and sucrose concentrations varied from one strain to another. There was no correlation between growth and fermentative capacity. The best 35 strains grew in 15% ethanol and fermented in 18% ethanol. Ethanol accumulated, although at a reduced rate, after the cells stopped growing. Most yeast strains were highly fermentative in 50% sucrose. Some of them effectively utilized the carbohydrates of the culture, yielding final ethanol concentrations of > 14%. Of the 35 selected strains, 16 were promising for genetic analysis and breeding because of their capacity to sporulate. These strains were homothallic, and their spores were viable. The meiotic products analyzed so far were also homothallic.     

Synergistic effects of ethanol and temperature on yeast mitochondria

At temperatures lower than 37°C, the ethanol inhibition constant (Ki) for growth or fermentation inrho ⁺ cells of theSaccharomyces cerevisiae strain S288C was always higher (1.1M) than inrho ^– mutants (0.7M). At 37°C these differences disappeared, and both strains were equally inhibited by ethanol (Ki=0.7m). Mitochondrial activity can be inhibited by high ethanol concentration and temperature. In fact, the stronger inhibition by ethanol of therho ⁺ strain at 37°C was due to the fact that, under these conditions, this strain loses the advantage conferred by mitochondrial activity since the induction ofrho ^– cells in the population is very high. This does not result in an increase in the frequency ofrho ^– mutants because of the poor viability of these mutants in conditions of high temperature and ethanol. In consequence, S288C strain becomes as strongly inhibited by ethanol as therho ^– mutant strains. Differences in viability were not related to the fatty acids and ergosterol composition of the strain. In the presence of ethanol, bothrho ⁺ andrho ^– strains modified their lipids in the same way, but these changes did not improve their ethanol tolerance. They were not due to differences in adaptation to ethanol either, since after successive transfers in ethanol, growth () and fermentation () rates in therho ^– mutants were increasingly inhibited with time, whereas in the S288C strain inhibition of and by ethanol remained unaltered. Rather,rho ^– mutants are less viable thanrho ⁺ cells because of the inability of the former to respire. At 37°C the Ki increased to 0.9M ethanol either when mitochondrial from highly ethanol-tolerant wine yeasts were transferred torho ^– mutants of the strain S288C or when the mitochondria of strain S288C were preadapted by growing the strain in glycerol instead of glucose before it was cultivated in ethanol.     

Relative apparent synapomorphy analysis (RASA). I: The statistical measurement of phylogenetic signal

We have developed a new approach to the measurement of phylogenetic signal in character state matrices called relative apparent synapomorphy analysis (RASA). RASA provides a deterministic, statistical measure of natural cladistic hierarchy (phylogenetic signal) in character state matrices. The method works by determining whether a measure of the rate of increase of cladistic similarity among pairs of taxa as a function of phenetic similarity is greater than a null equiprobable rate of increase. Our investigation of the utility and limitations of RASA using simulated and bacteriophage T7 data sets indicates that the method has numerous advantages over existing measures of signal. A first advantage is computational efficiency. A second advantage is that RASA employs known methods of statistical inference, providing measurable sensitivity and power. The performance of RASA is examined under various conditions of branching evolution as the number of characters, character states per character, and mutations per branch length are varied. RASA appears to provide an unbiased and reliable measure of phylogenetic signal, and the general approach promises to be useful in the development of new techniques that should increase the rigor and reliability of phylogenetic estimates.      

Use of recombinant interferon-α in Human Immunodeficiency Virus (HIV)-infected individuals

Rationale and objective Interferon alpha (IFN-) has anti-retroviral activity and is a possible HIV infection-limiting factor. The aim of this work is to prevent or delay disease progression in asymptomatic Human Immunodeficiency Virus (HIV) carriers.Design and interventions Recombinant IFN alpha-2b (3×10⁶ IU 3 times weekly) was compared. to no treatment (control) in a randomized trial. Endpoints were: (i) appearance of any CDC group IV symptoms and (ii) disease progression (which excluded shifts to group IVC2 or reversible IVA, or IVB). The trial lasted from October 1987 to February 1992.Setting The trial was performed at the Santiago de las Vegas sanatorium, a specialized institution for the care of HIV-infected and AIDS patients.Population Subjects were anti-HIV-1 seropositive, Western blot-confirmed, asymptomatic (CDC group II), or with generalized lymphadenopathies (CDC group III). The groups had 79 (control) and 71 (IFN) patients.Main results Long-term IFN- treatments significantly reduced the proportion of patients who shifted to any group IV (control: 46/79; IFN: 14/71;p<0.001) or developed AIDS (control: 27/79; IFN: 12/71;p<0.05). IFN also delayed progression to AIDS (95% confidence interval for 0.5 probability of progression) from 67–83 to 116–180 months after infection. The IFN group had significantly less opportunistic infections and non-infectious complications. CD4 cell count and hemoglobin decreased in the control but not in the IFN group. Fewer IFN-treated patients developed positive serum HIV antigen detection.Conclusion IFN alpha treatment during the early stages of infection seems to be beneficial to the patients.Abbreviations CI confidence interval - AIDS Acquired Immunodeficiency syndrome - HIV Human Immunodeficiency Virus - IFN Interferon - CDC Center for Disease Control (USA) - SD standard deviation