Galphai1 and Galphai3 differentially interact with, and regulate, the G protein-activated K+ channel

G protein-activated K(+) channels (GIRKs; Kir3) are activated by direct binding of Gbetagamma subunits released from heterotrimeric G proteins. In native tissues, only pertussis toxin-sensitive G proteins of the G(i/o) family, preferably Galpha(i3) and Galpha(i2), are donors of Gbetagamma for GIRK. How this specificity is achieved is not known. Here, using a pull-down method, we confirmed the presence of Galpha(i3-GDP) binding site in the N terminus of GIRK1 and identified novel binding sites in the N terminus of GIRK2 and in the C termini of GIRK1 and GIRK2. The non-hydrolyzable GTP analog, guanosine 5'-3-O-(thio)triphosphate, reduced the binding of Galpha(i3) by a factor of 2-4. Galpha(i1-GDP) bound to GIRK1 and GIRK2 much weaker than Galpha(i3-GDP). Titrated expression of components of signaling pathway in Xenopus oocytes and their activation by m2 muscarinic receptors revealed that G(i3) activates GIRK more efficiently than G(i1), as indicated by larger and faster agonist-evoked currents. Activation of GIRK by purified Gbetagamma in excised membrane patches was strongly augmented by coexpression of Galpha(i3) and less by Galpha(i1). Differences in physical interactions of GIRK with GDP-bound Galpha subunits, or Galphabetagamma heterotrimers, may dictate different extents of Galphabetagamma anchoring, influence the efficiency of GIRK activation by Gbetagamma, and play a role in determining signaling specificity.     

Hidden messages in the nef gene of human immunodeficiency virus type 1 suggest a novel RNA secondary structure

The coexistence of multiple codes in the genome of human immunodeficiency virus type 1 (HIV-1) was analyzed. We explored factors constraining the variability of the virus genome primarily in relation to conserved RNA secondary structures overlapping coding sequences, and used a simple combination of algorithms for RNA secondary structure prediction based on the nearest-neighbor thermodynamic rules and a statistical approach. In our previous study, we applied this combination to a non- redundant data set of env nucleotide sequences, confirmed the conservative secondary structure of the rev-responsive element (RRE) and found a new RNA structure in the first conserved (C1) region of the env gene. In this study, we analyzed the variability of putative RNA secondary structures inside the nef gene of HIV-1 by applying these algorithms to a non-redundant data set of 104 nef sequences retrieved from the Los Alamos HIV database, and predicted the existence of a novel functional RNA secondary structure in the β3/β4 regions of nef. The predicted RNA fold in the β3/β4 region of nef appears in two forms with different loop sizes. The loop of the first fold consists of seven nucleotides (positions 494–500), with consensus UCAAGCU appearing in 79% of sequences. The other has a five-base loop (positions 495–499) with consensus CAAGC. The difference in size between these two loops may reflect the difference between respective counterparts in the hairpin recognition. This may also have an adaptive biological significance.     

A novel method of resistance for influenza against a channel-blocking antiviral drug

Effective antivirals are few and far between, and as such, the appearance of resistance toward such treatments is an obvious medical concern. In this article, we analyze the mechanism by which influenza attains resistance toward amantadine, a blocker of the viral M2 H(+) channel. Binding analyses of amantadine to M2 peptides from different viral strains showed that the virus has developed two alternate routes to avoid blockage of its channel: (1) a conventional route, in which the channel no longer binds the blocker and, hence, the blocker cannot exert its inhibitory function; and (2) a novel mechanism, in which binding of the blocker is retained, yet the function of the protein is unaffected. Pore diameter profiles revealed the molecular mechanism by which the virus may attain this novel type of resistance: an increase in the size of the channel. Thus, despite the drug binding the channel, it may not be able to block the pore, since the channel diameter has increased. Our findings may have broad ramifications in the design of new antivirals, and of novel blockers against malfunctioning human channels implicated in disease.     

Alterations of Circadian Rhythms in Mice Induced by Anti-Cancer Drugs

Carboplatinum was injected into four groups of mice for 5 successive days at one out of four daily equidistant hours. Upon the cessation of these injections, granulocyte colony stimulating factor was injected into half of these mice for 2 days at the same times. The stimulating factor was also injected into groups of untreated mice for 2 days at the same times. In each experiment the rhythms of four variables were monitored after the cessation of all injections: peripheral WBC count, bone marrow and sperm stem cells DNA synthesis, and sperm ribosomal RNA content. The results indicated that in the course of repeated drug administrations, variables' rhythm parameters (phases and periods) were altered. These changes have to be considered in chronotherapeutic schedules when variables which serve for toxicity diagnosis or as drug target are rhythmic in nature.     

The protective effect of desferrioxamine on paraquat-treated pea (Pisum sativum)

Paraquat, a widely used herbicide, is photoreduced by photosystem I to the monovalent cation radical, which in turn, can react quickly and efficiently with molecular oxygen to produce superoxide anion radicals. In the presence of redox-active iron (or copper) superoxide radicals can serve as a source for the more active species such as hydroxyl radicals. The present sludv investigated the possible mediatory role of iron in paraquat to xicity. The results demonstrate that desferrioxamme (0–150μM) a highiy specific iron chelator, reduces the loss of proteins (by 34–69%) and lipid peroxidation (by 31–96%) in paraquat treated leaf cuts. Dcsferrioxamine also protects malate dehydrogenase (61–70%) hydroxvpyruvate reductase (54–100%), and Ca²⁺-dependent ATPase (25–34%) against the paraquat-induced loss of their activity. It also induces an increase in glutathione reductase activity (by 188%). These results, together with those from other experiments concerning the effect of desferrioxamine on paraquat uptake by the leaf cuts, suggest that the protection by desferrioxamine arises from its specific iron chelanon properties, and lead to the conclusion that nan-protein-bound and redoxactive forms of iron pluy a role in the manifestation of paraquat toxicity in plants.     

Comparison of dimethyl dioctadecyl ammonium bromide, Freund's complete adjuvant and mineral oil for induction of humoral antibodies, cellular immunity and resistance to Newcastle disease virus in chickens

Abstract Dimethyl diotadecyl ammonium bromide (DDA), a lipophilic quaternary amine, was evaluated in adult chickens for potentiation of immunological responses to subcutaneously administered inactivated Newcastle disease virus (NDV) vaccines. DDA enhanced humoral and cell-mediated immune (CMI) responses to levels which were significantly higher than those induced by the vaccine alone The haemagglutination inhibition antibody titers induced by DDA were slightly lower than those induced by mineral oil although neutralizing antibody titers seemed to be higher. DDA induced strong CMI (DTH and lymphocyte proliferation) responses, more than those induced by Freund's complete adjuvant and mineral oil. Both DDA and mineral oil induced comparable high levels of protection to challenge doses of 200 000 LD₅₀ per chicken. No toxic effects or local tissue damage were observed in any of the inoculated chickens.     

Measurement of the inactivation kinetics of poliovirus by ozone in a fast-flow mixer.

Inactivation kinetics of poliovirus type 1 in ozone demand-free water was investigated by utilizing a fast-flow mixing apparatus. Ozonated water and a solution of ozone demand-free water containing a known quantity of poliovirus type 1 were introduced simultaneously into a mixing chamber, both at a constant rate. This mixture was then passed through a narrow tube of known length and diameter into a neutralizing solution. By altering the rate of introduction and/or tube length, different contact periods between ozone and virus could be determined with an accuracy of 0.01 s. Inactivation of the poliovirus occurred in two steps. During the first step, which lasted for 0.2 to 1.0 s, 95 to 99% of the virus was inactivated, depending on the ozone concentration (which ranged from 0.1 to 2.0 mg/liter). The second step apparently continued for several minutes; in this period the remainder of the virus was inactivated. An obvious dose-response relationship was demonstrated during the first step of the inactivation curve. The pH of the water slightly affected the viral inactivation rate, but these small differences seem to have no practical value.