Deletion of the gag region from FBR murine osteosarcoma virus does not affect its enhanced transforming activity.

Both FBJ murine osteosarcoma virus (FBJ-MSV) and FBR-MSV induce transformation in tissue culture and osteogenic sarcomas in mice. In tissue culture, however, FBR-MSV induces larger foci with a shorter latency than those induced by FBJ-MSV. Transformation is dependent on expression of the fos oncogene in FBJ-MSV and of a gag-fos fusion protein in FBR-MSV. We have determined that the gag sequences can be deleted from FBR-MSV without affecting the high transforming activity of this virus in comparison to FBJ-MSV. The resultant virus, designated FBJ/R-MSV, has a protein coding region that is half the size of that of FBR-MSV and about one-third smaller than that of FBJ-MSV. Thus, FBJ/R-MSV will provide a useful tool for studying the transforming activity of the fos oncogene.     

Effects of cholera toxin on cellular and paracellular sodium fluxes in rabbit ileum

The diarrhea observed in patients which cholera is known to be related to secretion of water and electrolytes into the intestinal lumen. However, the exact mechanisms involved in these secretory processes have remained unclear. Although it is clear that purified toxin acts on epithelial cell metabolism, its activity on Na⁺ transport across intestinal mucosa is equivocal: reported either to prevent net Na⁺ absorption or to cause net secretion of Na⁺ from serosa to mucosa. Since total transmural Na⁺ fluxes across “leaky” epithelia involve very significant movement via a paracellular shunt pathway, we studied the effects of cholera toxin on the cellular and paracellular pathways of Na⁺ movement. Unidirectional Na⁺ fluxes were examined as functions of applied potential in control tissues and in tissues from the same animal treated with purified cholera toxin. Treatment of rabbit ileum in vitro with toxin stimulated the cellular component of serosa-to-mucosa Na⁺ flux (from $\text{2.41 ± 0.49 μ}\text{equiv./h}$ per cm² under control conditions to $\text{4.71 ± 0.43 μ}\text{equiv./h}$ per cm² after treatment with toxin, $\text{P < 0.01}$). The effect of cholera toxin on Na⁺ movement through the cells from mucosa to serosa appeared to be insignificant. Finally, a marked decrease in the Na⁺ permeability ($\text{P < 0.01}$) and no detectable significant changes in transference number for Na⁺ of the paracellular shunt pathway were observed following treatment with cholera toxin. These results provide direct evidence for the hypothesis that purified cholera toxin stimulates active sodium secretion but has minimal effect on sodium absorption.     

The biological activity of ubiquitinated BoNT/B light chain in vitro and in human SHSY‐5Y neuronal cells

BoNT/B light chain is a zinc‐dependent endopeptidase. After entering its target, the neuronal cell, BoNT/B is responsible for synaptobrevin‐2 (VAMP‐2) cleavage. This results in reduced neurotransmitter (acetylcholine) release from synaptic vesicles, yielding muscular paralysis. Since the toxin persists in neuronal cells for an extended period, regeneration of VAMP‐2 is prevented. We evaluated therapeutic targets to overcome botulinum persistence because early removal would rescue the neuronal cell. The ubiquitination/proteasome cellular pathway is responsible for removing “old” or undesirable proteins. Therefore, we assessed ubiquitination of BoNT/B light chain in vitro, and characterized the effects of ubiquitination modulating drugs, PMA (phorbol 12‐myristate 13‐acetate) and expoxomicin, on ubiquitination of BoNT/B light chain in neuronal cells. Both drugs altered BoNT/B light chain ubiquitination. Ubiquitination in vitro and in cells decreased the biological activity of BoNT/B light chain. These results further elucidate BoNT protein degradation pathways in intoxicated neuronal cells and mechanisms to enhance toxin removal. J. Cell. Biochem. 108: 660–667, 2009. Published 2009 Wiley‐Liss, Inc.     

Eosinophils preferentially use bromide to generate halogenating agents

Human eosinophils preferentially utilize bromide to generate a brominating agent, even at physiological halide concentrations, where chloride (140 mM) is over 1000-fold greater than bromide (20-100 microM). Under the same conditions, neutrophils use chloride to generate a chlorinating agent. The total amount of active halogen trapped by 1,3,5-trimethoxybenzene from eosinophils increases by over 2-fold as the added bromide concentration increases from 0 to 100 microM, with approximately 40 nmol of halogen trapped per million cells at the highest bromide level. At least 25-35% of the oxygen consumed by stimulated eosinophils is directed toward the generation of halogenating species. Since the relative halogenating behavior of eosinophil peroxidase and neutrophil myeloperoxidase in this bromide range is essentially identical to that of the cells, the specificity of eosinophils toward bromide is intrinsic to eosinophil peroxidase and not to any special cellular properties. These results suggest that human eosinophils use bromide in vivo and that a deficiency of bromide may influence their ability to produce halogenating agents.     

Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in aphids of the genus Sitobion (Hemiptera: Aphididae)

Polymerase chain reaction (PCR) products corresponding to 803 bp of the cytochrome oxidase subunits I and II region of mitochondrial DNA (mtDNA COI-II) were deduced to consist of multiple haplotypes in three Sitobion species. We investigated the molecular basis of these observations. PCR products were cloned, and six clones from one individual per species were sequenced. In each individual, one sequence was found commonly, but also two or three divergent sequences were seen. The divergent sequences were shown to be nonmitochondrial by sequencing from purified mtDNA and Southern blotting experiments. All seven nonmitochondrial clones sequenced to completion were unique. Nonmitochondrial sequences have a high proportion of unique sites, and very few characters are shared between nonmitochondrial clones to the exclusion of mtDNA. From these data, we infer that fragments of mtDNA have been transposed separately (probably into aphid chromosomes), at a frequency only known to be equalled in humans. The transposition phenomenon appears to occur infrequently or not at all in closely related genera and other aphids investigated. Patterns of nucleotide substitution in mtDNA inferred over a parsimony tree are very different from those in transposed sequences. Compared with mtDNA, nonmitochondrial sequences have less codon position bias, more even exchanges between A, G, C and T, and a higher proportion of nonsynonymous replacements. Although these data are consistent with the transposed sequences being under less constraint than mtDNA, changes in the nonmitochondrial sequences are not random: there remains significant position bias, and probable excesses of synonymous replacements and of conservative inferred amino acid replacements. We conclude that a proportion of the inferred change in the nonmitochondrial sequences occurred before transposition. We believe that Sitobion aphids (and other species exhibiting mtDNA transposition) may be important for studying the molecular evolution of mtDNA and pseudogenes. However, our data highlight the need to establish the true evolutionary relationships between sequences in comparative investigations.      

Mapping of monoclonal antibodies to the Sendai virus P protein and the location of its phosphates.

The epitopes of a panel of five monoclonal antibodies to the Sendai virus P protein were mapped by generating modified forms of the P mRNA via SP6 expression followed by in vitro translation. The epitopes were found to be clustered in the C-terminal region of the protein. Two epitopes were within the last 30 residues, two were within the next 65, and one was between residues 308 and 451 of this 568-residue-long protein. By a combination of partial proteolysis and Western immunoblotting with one of these antibodies, the sites at which phosphates are added in vitro by the virion-associated kinase were mapped to the second quarter of the molecule from the N terminus.     

On the systematics of some marine haploporids (Trematoda) with the description of a new species of Megasolena Linton, 1910

Megasolena mikra sp. nov. is described from the queen angelfish, Holacanthus ciliaris (Linnaeus), off Florida, USA. The new species can be differentiated from all other species of Megasolena Linton, 1910 except Megasolena littoralis Muñoz, George-Nascimento, and Bray, 2017 in possessing testes that are smaller in diameter than the ovary. The new species can be differentiated from M. littoralis in lacking tegumental spines and possessing oral sucker papillae. Molecular data are provided for two species each of Cadenatella Dollfus, 1946, Hapladena Linton, 1910, and Megasolena Linton, 1910. Bayesian inference analysis of concatenated internal transcribed spacer region-2 (ITS2) and partial 28S rDNA sequences of 50 haploporoids revealed 1) a monophyletic Atractotrematidae Yamaguti, 1939 sister to the rest of the haploporoids tested; 2) a paraphyletic Megasoleninae Manter, 1935 – if Hapladena is included; and 3) a monophyletic Cadenatellinae Gibson and Bray, 1982 sister to the ‘mugilid’ haploporids. The ‘mugilid’ haploporids formed a monophyletic clade consisting of the subfamilies Chalcinotrematinae Overstreet and Curran, 2005, Forticulcitinae Blasco-Costa, Balbuena, Kostadinova, and Olson, 2009, Haploporinae Nicoll, 1914, and Waretrematinae Srivastava, 1937. Based on our analysis we restrict the Megasoleninae to include Megasolena, Vitellibaculum Montgomery, 1957, and Metamegasolena Yamaguti, 1970, all of which have species with two testes. To accommodate the former megasolenine taxa with a single testis, we erect the Hapladeninae subf. nov. for species in Hapladena and tentatively, Myodera Montgomery, 1957. Our results further support that haploporoids had a common marine ancestor with two testes, and that members of the Haploporoidea Nicoll, 1914 underwent diversification following a shift from a primarily marine life history with eupercarian hosts to a more euryhaline one with diadromous hosts (namely mullet).