Experimental vaccination of rainbow trout against Loma salmonae using a live low-virulence variant of L. salmonae

Prior exposure of rainbow trout Oncorhynchus mykiss juveniles to the low-virulence variant of Loma salmonae, L. salmonae SV, spores resulted in a xenoma intensity in the gill filaments fourteen times lower (0·044 v. 0·641 xenomas per filament; P=0·0001) than that observed in the naive controls, challenged with L. salmonae spores, as determined morphometrically by in situ hybridization at the peak of the disease (between 4 and 6 weeks post exposure). The marked degree of reduction in numbers of xenomas that formed after challenge suggests that use of the low-virulence variants should be further considered as a means to protect fish in regions where the parasite is endemic, to protect them during grow out periods.     

The structures of amentoflavone glycosides isolated from Psilotum nudum

On the basis of new spectroscopic evidence, structures are proposed for three amentoflavone glycosides and an apigenin di-C-glycoside previously isolated from Psilotum nudum. The major glycoside is identified as the 7,4′,4′“-tri-O-β-D-glucopyranoside, minor glycosides as the 4′,4′“-di-O-β-D-glucopyranoside and 7,4′“-di-O-β-D-glucopyranoside, and the apigenin di-C-glycoside as vicenin-2. The amentoflavone glucosides are all new natural products.     

Recombinant Vaccine-Induced Protection against the Highly Pathogenic Simian Immunodeficiency Virus SIVmac251: Dependence on Route of Challenge Exposure

Vaccine protection from infection and/or disease induced by highly pathogenic simian immunodeficiency virus (SIV) strain SIV_mac251 in the rhesus macaque model is a challenging task. Thus far, the only approach that has been reported to protect a fraction of macaques from infection following intravenous challenge with SIV_mac251 was the use of a live attenuated SIV vaccine. In the present study, the gag, pol, and env genes of SIV_K6W were expressed in the NYVAC vector, a genetically engineered derivative of the vaccinia virus Copenhagen strain that displays a highly attenuated phenotype in humans. In addition, the genes for the α and β chains of interleukin-12 (IL-12), as well as the IL-2 gene, were expressed in separate NYVAC vectors and inoculated intramuscularly, in conjunction with or separate from the NYVAC-SIV vaccine, in 40 macaques. The overall cytotoxic T-lymphocyte (CTL) response was greater, at the expense of proliferative and humoral responses, in animals immunized with NYVAC-SIV and NYVAC–IL-12 than in animals immunized with the NYVAC-SIV vaccine alone. At the end of the immunization regimen, half of the animals were challenged with SIV_mac251 by the intravenous route and the other half were exposed to SIV_mac251 intrarectally. Significantly, five of the eleven vaccinees exposed mucosally to SIV_mac251 showed a transient peak of viremia 1 week after viral challenge and subsequently appeared to clear viral infection. In contrast, all 12 animals inoculated intravenously became infected, but 5 to 6 months after viral challenge, 4 animals were able to control viral expression and appeared to progress to disease more slowly than control animals. Protection did not appear to be associated with any of the measured immunological parameters. Further modulation of immune responses by coadministration of NYVAC-cytokine recombinants did not appear to influence the outcome of viral challenge. The fact that the NYVAC-SIV recombinant vaccine appears to be effective per se in the animal model that best mirrors human AIDS supports the idea that the development of a highly attenuated poxvirus-based vaccine candidate can be a valuable approach to significantly decrease the spread of human immunodeficiency virus (HIV) infection by the mucosal route.     

Reliability of the RNA-DNA Filter Hybridization for the Detection of Oncornavirus-Specific DNA Sequences

Denatured DNA from leukemic myeloblasts or uninfected chicken embryos, immobilized on nitrocellulose filters, was hybridized to a vast excess of [(3)H]70S RNA from purified avian myeloblastosis virus. The viral RNA was eluted from the RNA-DNA hybrids, purified, and then rehybridized in solution to an excess of either leukemic or normal chicken embryonic DNA. This study revealed that all the slow and the fast hybridizing viral RNA sequences detectable by liquid hybridization in DNA excess had hybridized to the filter bound DNA. Both techniques also gave similar values for the number of 28S ribosomal RNA genes contained in a chicken cell genome: 210 by the liquid hybridization procedure and 218 by the filter hybridization technique. Therefore, filter hybridization can accurately detect DNA sequences present in relatively few numbers in the genome of higher organisms.     

Metal ion activation of S-adenosylmethionine decarboxylase reflects cation charge density

S-Adenosylmethionine decarboxylase from Escherichia coli is a pyruvoyl cofactor-containing enzyme that requires a metal cation for activity. We have found that the enzyme is activated by cations of varying charge and ionic radius, such as Li+, A13+, Tb3+, and Eu3+, as well as the divalent cations Mg2+, Mn2+, and Ca2+. All of the activating cations provide kcat values within 30-fold of one another, showing that the charge of the cation does not greatly influence the rate-limiting step for decarboxylase turnover. Cation concentrations for half-maximal activation decrease by >100-fold with each increment of increase in the cation charge, ranging from approximately 300 mM with Li+ to approximately 2 microM with trivalent lanthanide ions. The cation affinity is related to the charge/radius ratio of the ion for those ions with exchangeable first coordination sphere ligands. The exchange-inert cation Co(NH3)63+ activates in the presence of excess EDTA (and NH4+ does not activate), indicating that direct metal coordination to the protein or substrate is not required for activation. The binding of metal ions (monitored by changes in the protein tryptophan fluorescence) and enzyme activation are both cooperative with Hill coefficients as large as 4, the active site stoichiometry of this (alphabeta)4 enzyme. The Hill coefficients for Mg2+ binding and activation increase from 1 to approximately 4 as the KCl concentration increases, which is also observed with NaCl or KNO3; neither Na+ nor K+ activates the enzyme. The single tryptophan in the protein is located 16 residues from the carboxyl terminus of the pyruvoyl-containing alpha chain, in a 70-residue segment that is not present in metal ion independent AdoMet decarboxylases from other organisms. The results are consistent with allosteric metal ion activation of the enzyme, congruent with the role of the putrescine activator of the mammalian AdoMet decarboxylase.     

Studies on predation of Prostephanus truncatus (Horn) (Col., Bostrichidae) and Sitophilus zeamais Mots. (Col., Curculionidae) at different densities on maize by Teretriosoma nigrescens Lewis (Col., Histeridae)

Laboratory experiments using whole cobs were conducted to examine the effect of varying densities of the larger grain borer, Prostephanus truncatus and the maize weevil, Sitophilus zeamais on rate of population increase by the histerid beetle, Teretriosoma nigrescens , a predator primarily of P. truncatus . Densities of all species of insects were determined at the end of the experiment, and an electrophoretic analysis of gut content was conducted on larval and adult T. nigrescens sampled during the experiments. Results indicated that T. nigrescens has a strong preference for P. truncatus and densities of T. nigrescens were associated only with densities of P. truncatus . The maize weevil played little role as an alternative prey or in interfering with T. nigrescens reproduction.     

A locus for isolated cleft palate, located on human chromosome 2q32.

We present evidence for the existence of a novel chromosome 2q32 locus involved in the pathogenesis of isolated cleft palate. We have studied two unrelated patients with strikingly similar clinical features, in whom there are apparently balanced, de novo cytogenetic rearrangements involving the same region of chromosome 2q. Both children have cleft palate, facial dysmorphism, and mild learning disability. Their karyotypes were originally reported as 46, XX, t(2;7)(q33;p21) and 46, XX, t(2;11)(q33;p14). However, our molecular cytogenetic analyses localize both translocation breakpoints to a small region between markers D2S311 and D2S116. This suggests that the true location of these breakpoints is 2q32 rather than 2q33. To obtain independent support for the existence of a cleft-palate locus in 2q32, we performed a detailed statistical analysis for all cases in the human cytogenetics database of nonmosaic, single, contiguous autosomal deletions associated with orofacial clefting. This revealed 2q32 to be one of only three chromosomal regions in which haploinsufficiency is significantly associated with isolated cleft palate. In combination, our data provide strong evidence for the location at 2q32 of a gene that is critical to the development of the secondary palate. The close proximity of these two translocation breakpoints should also allow rapid progress toward the positional cloning of this cleft-palate gene.     

Requirement for the second coding exon of Tat in the optimal replication of macrophage-tropic HIV-1

HIV-1 Tat is essential for virus replication and is a potent transactivator of viral gene expression. Evidence suggests that Tat also influences virus infectivity and cytopathicity. Here, we find that the second coding exon of Tat contributes a novel function for the replication/infectivity of macrophage-tropic HIV-1. We show that macrophage-tropic HIV-1 which expresses the full-length two-exon form of Tat replicates better in monocyte-derived macrophages (MDM) than an otherwise isogenic virus which expresses only the one-exon form of Tat. Similarly, two-exon Tat expressing HIV-1 also replicates better than one-exon Tat expressing HIV-1 in two different models of human cells/tissue reconstituted SCID mice.