Systemic herpes-like virus in catfish Ictalurus melas (Italy) differs from Ictalurid herpesvirus 1 (North America)

A herpesvirus was isolated during 2 occurrences of mass mortality among adult catfish Ictalurus melas raised in different farms in northern Italy. The agent replicated in the channel catfish ovary (CCO) cell line from channel catfish I. punctatus, inducing a cytopathic effect similar to that caused by Ictalurid herpesvirus 1 (also referred to as channel catfish herpesvirus, CCV). The new herpesvirus, designated I. melas herpesvirus (IcmHV) did not react with polyclonal rabbit or monoclonal antibodies directed to CCV in either neutralization or indirect immunofluorescence assays. The virions of IcmHV possessed a hexagonal nucleocapsid of 107 nm in diameter surrounded by an envelope with a diameter of 227 nm (n = 20) typical for members of the family Herpesviridae. Virions of IcmHV purified from infected CCO cells contained 17 polypeptides ranging in size from 17.5 to 175 kDa and most differed in molecular weight from those found for CCV. The IcmHV was also distinct from CCV when compared by restriction fragment length polymorphisms (RFLP) of genomic DNA following digestions with the endonucleases Kpn I and Sac I. Lastly, the virulence of IcmHV for channel catfish fry and juveniles, respectively, was demonstrated by experimental infections induced by bath exposure or intraperitoneal injection that resulted in 78 to 96% cumulative mortality in groups of exposed fish. Preventing the introduction of this agent into geographic regions where significant channel catfish production occurs should be a high priority.     

Combination of two activating mutations in one HOG1 gene forms hyperactive enzymes that induce growth arrest

Mitogen-activated protein kinases (MAPKs) play key roles in differentiation, growth, proliferation, and apoptosis. Although MAPKs have been extensively studied, the precise function, specific substrates, and target genes of each MAPK are not known. These issues could be addressed by sole activation of a given MAPK, e.g., through the use of constitutively active MAPK enzymes. We have recently reported the isolation of eight hyperactive mutants of the Saccharomyces cerevisiae MAPK Hog1, each of which bears a distinct single point mutation. These mutants acquired high intrinsic catalytic activity but did not impose the full biological potential of the Hog1 pathway. Here we describe our attempt to obtain a MAPK that is more active than the previous mutants both catalytically and biologically. We combined two different activating point mutations in the same gene and found that two of the resulting double mutants acquired unusual properties. These alleles, HOG1(D170A,F318L) and HOG1(D170A,F318S), induced a severe growth inhibition and had to be studied through an inducible expression system. This growth inhibition correlated with very high spontaneous (in the absence of any stimulation) catalytic activity and strong induction of Hog1 target genes. Furthermore, analysis of the phosphorylation status of these active alleles shows that their acquired intrinsic activity is independent of either phospho-Thr174 or phospho-Tyr176. Through fluorescence-activated cell sorting analysis, we show that the effect on cell growth inhibition is not a result of cell death. This study provides the first example of a MAPK that is intrinsically activated by mutations and induces a strong biological effect.     

Population differences in the human functional olfactory repertoire

Olfactory receptors (OR) constitute the molecular basis for the sense of smell. They are encoded by a large multigene family that in humans includes approximately 400 functional genes and approximately 600 putative pseudogenes, distributed on all but two chromosomes. To examine the ethnogeographic variability in the functional chemosensory repertoire, we resequenced 32 OR loci reported to contain a single coding region disruption in seven Caucasians and seven Pygmies. Thirteen of the 32 OR loci were found to have an interrupted coding region in all 28 alleles sampled, seven had an intact form in all the individuals examined, and 12 were polymorphic, segregating both the intact and the null variants. Among the latter loci, the frequency of the null allele was higher in Caucasians than in Pygmies, suggesting that African populations may have a larger repertoire of functional OR genes. Interestingly, when analyzing the entire OR coding regions, we find an excess of high-frequency derived alleles at many loci in the Caucasian sample but less so in the Pygmy sample. Our observations are unlikely to be accounted for by simple demographic models but may be explained by positive selection acting on OR loci in Caucasians.     

Experiments towards the synthesis of the ergot alkaloids and related structures. Part 8. The total synthesis of 4-methyl-2,3,4,4a,5,6-hexahydrobenzo[f]quinoline-2-carbonitrile hydrochloride hemihydrate,an immediate precursor of the despyrrole analogues of lysergic acid and its amides

Exposure of the N-methoxycarbonyl-bicyclic-keto-acid 5 (improved preparation) to the Barnick beta-keto-acid synthesis yielded an aqueous solution of the sodium salts of the beta-keto-acids 26 and 27 which on heating at 60-65 degrees C furnished the N-methoxycarbonyl-tricyclic-ketone 9 (55%) plus the hydroxy-ketone 28 which on acid treatment raised the yield of 9 to 68%. Reduction (NaBH4) of 9 yielded the alcohol 32 (94%) which was treated with thionyl chloride followed by copper (I) cyanide and sodium iodide in acetonitrile to give the tricyclic-N-methoxycarbonyl nitrile 35 whose relative configuration was obtained by X-ray analysis. Attempts to remove the N-methoxycarbonyl group from 35 were unsuccessful. Conversion of the alcohol 32 to its methoxypropyl ether 41 followed by reaction with ethereal MeLi-LiBr yielded the amino-alcohol 39 (75%) converted to the N-formyl-tricyclic alcohol 42 with formic-acetic anhydride (70%). The alcohol 42 was then converted into the N-formyl nitrile 44 via the chloride 43 as employed in the earlier synthesis of the nitrile 35. Removal of the N-formyl group from the nitrile 44 was achieved by refluxing methanolic hydrochloric acid to give the required amino-nitrile hydrochloride 46 (91%) whose structure was confirmed by X-ray analysis. Reaction of the free base with methyl iodide in ethyl acetate in the presence of calcium carbonate furnished the N-methyl base 48 isolated as its hydrochloride, hemihydrate 49 (59%). The overall yield of 49 via this eleven-step synthesis was 3.4%.     

Observation of calcium-dependent unidirectional rotational motion in recombinant photosynthetic F1-ATPase molecules

ATP hydrolysis and synthesis by the F(0)F(1)-ATP synthase are coupled to proton translocation across the membrane in the presence of magnesium. Calcium is known, however, to disrupt this coupling in the photosynthetic enzyme in a unique way: it does not support ATP synthesis, and CaATP hydrolysis is decoupled from any proton translocation, but the membrane does not become leaky to protons. Understanding the molecular basis of these calcium-dependent effects can shed light on the as yet unclear mechanism of coupling between proton transport and rotational catalysis. We show here, using an actin filament gamma-rotation assay, that CaATP is capable of sustaining rotational motion in a highly active hybrid photosynthetic F(1)-ATPase consisting of alpha and beta subunits from Rhodospirillum rubrum and gamma subunit from spinach chloroplasts (alpha(R)(3)beta(R)(3)gamma(C)). The rotation was found to be similar to that induced by MgATP in Escherichia coli F(1)-ATPase molecules. Our results suggest a possible long range pathway that enables the bound CaATP to induce full rotational motion of gamma but might block transmission of this rotational motion into proton translocation by the F(0) part of the ATP synthase.     

Structure and mechanistic analysis of the anti-human immunodeficiency virus type 1 antibody 2F5 in complex with its gp41 epitope

The membrane-proximal region of the ectodomain of the gp41 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is the target of three of the five broadly neutralizing anti-HIV-1 antibodies thus far isolated. We have determined crystal structures of the antigen-binding fragment for one of these antibodies, 2F5, in complex with 7-mer, 11-mer, and 17-mer peptides of the gp41 membrane-proximal region, at 2.0-, 2.1-, and 2.2-A resolutions, respectively. The structures reveal an extended gp41 conformation, which stretches over 30 A in length. Contacts are made with five complementarity-determining regions of the antibody as well as with nonpolymorphic regions. Only one exclusive charged face of the gp41 epitope is bound by 2F5, while the nonbound face, which is hydrophobic, may be hidden due to occlusion by other portions of the ectodomain. The structures reveal that the 2F5 antibody is uniquely built to bind to an epitope that is proximal to a membrane surface and in a manner mostly unaffected by large-scale steric hindrance. Biochemical studies with proteoliposomes confirm the importance of lipid membrane and hydrophobic context in the binding of 2F5 as well as in the binding of 4E10, another broadly neutralizing antibody that recognizes the membrane-proximal region of gp41. Based on these structural and biochemical results, immunization strategies for eliciting 2F5- and 4E10-like broadly neutralizing anti-HIV-1 antibodies are proposed.     

Absence of the TAP2 human recombination hotspot in chimpanzees

Recent experiments using sperm typing have demonstrated that, in several regions of the human genome, recombination does not occur uniformly but instead is concentrated in “hotspots” of 1–2 kb. Moreover, the crossover asymmetry observed in a subset of these has led to the suggestion that hotspots may be short-lived on an evolutionary time scale. To test this possibility, we focused on a region known to contain a recombination hotspot in humans, TAP2, and asked whether chimpanzees, the closest living evolutionary relatives of humans, harbor a hotspot in a similar location. Specifically, we used a new statistical approach to estimate recombination rate variation from patterns of linkage disequilibrium in a sample of 24 western chimpanzees (Pan troglodytes verus). This method has been shown to produce reliable results on simulated data and on human data from the TAP2 region. Strikingly, however, it finds very little support for recombination rate variation at TAP2 in the western chimpanzee data. Moreover, simulations suggest that there should be stronger support if there were a hotspot similar to the one characterized in humans. Thus, it appears that the human TAP2 recombination hotspot is not shared by western chimpanzees. These findings demonstrate that fine-scale recombination rates can change between very closely related species and raise the possibility that rates differ among human populations, with important implications for linkage-disequilibrium based association studies.