RNA Virus Population Diversity,an Optimum for Maximal Fitness and Virulence

The ability of an RNA virus to exist as a population of genetically distinct variants permits the virus to overcome events during infections that would otherwise limit virus multiplication or drive the population to extinction. Viral genetic diversity is created by the ribonucleotide misincorporation frequency of the viral RNA-dependent RNA polymerase (RdRp). We have identified a poliovirus (PV) RdRp derivative (H273R) possessing a mutator phenotype. GMP misincorporation efficiency for H273R RdRp in vitro was increased by 2–3-fold that manifested in a 2–3-fold increase in the diversity of the H273R PV population in cells. Circular sequencing analysis indicated that some mutations were RdRp-independent. Consistent with the population genetics theory, H273R PV was driven to extinction more easily than WT in cell culture. Furthermore, we observed a substantial reduction in H273R PV virulence, measured as the ability to cause paralysis in the cPVR mouse model. Reduced virulence correlated with the inability of H273R PV to sustain replication in tissues/organs in which WT persists. Despite the attenuated phenotype, H273R PV was capable of replicating in mice to levels sufficient to induce a protective immune response, even when the infecting dose used was insufficient to elicit any visual signs of infection. We conclude that optimal RdRp fidelity is a virulence determinant that can be targeted for viral attenuation or antiviral therapies, and we suggest that the RdRp may not be the only source of mutations in a RNA virus genome.     

No evidence for induction or selection of mutant sodium channel expression in the copepod Acartia husdsonica challenged with the toxic dinoflagellate Alexandrium fundyense

Some species in the dinoflagellate genus Alexandrium spp. produce a suite of neurotoxins that block sodium channels, known as paralytic shellfish toxins (PST), which have deleterious effects on grazers. Populations of the ubiquitous copepod grazer Acartia hudsonica that have co‐occurred with toxic Alexandrium spp. are better adapted than naïve populations. The mechanism of adaptation is currently unknown. We hypothesized that a mutation in the sodium channel could account for the grazer adaptation. We tested two hypotheses: (1) Expression of the mutant sodium channel could be induced by exposure to toxic Alexandrium fundyense; (2) in the absence of induction, selection exerted by toxic A. fundyense would favor copepods that predominantly express the mutant isoform. In the copepod A. hudsonica, both isoforms are expressed in all individuals in varying proportions. Thus, in addition to comparing expression ratios of wild‐type to mutant isoforms for individual copepods, we also partitioned copepods into three groups: those that predominantly express the mutant (PMI) isoform, the wild‐type (PWI) isoform, or both isoforms approximately equally (EI). There were no differences in isoform expression between individuals that were fed toxic and nontoxic food after three and 6 days; induction of mutant isoform expression did not occur. Furthermore, the hypothesis that mutant isoform expression responds to toxic food was also rejected. That is, no consistent evidence showed that the wild‐type to mutant isoform ratios decreased, or that the relative proportion of PMI individuals increased, due to the consumption of toxic food over four generations. However, in the selected line that was continuously exposed to toxic food sources, egg production rate increased, which suggested that adaptation occurred but was unrelated to sodium channel isoform expression.     

Division of labor of anthers in heterantherous plants: flexibility of bee pollen collection behavior may serve to keep plants honest

Heteranthery is thought to reflect a division of labor, with some anthers serving a pollinator-feeding function and others serving a pollinating function. Mutualism theory predicts that each participant should try to maximize the benefit it receives from its partner: plants should allocate more pollen to pollination, and pollinators should collect more pollen. Accordingly, plant and pollinator may engage in a ‘tug of war’ with respect to pollen from each anther type, resulting in incomplete division of labor. Here, we explored this idea by conducting a fully factorial manipulation of the availability of pollen in long and short anthers of staminate flowers of Solanum houstonii. We found the following: (1) Bumble bees (Bombus impatiens) preferred to sonicate (collect pollen from) short anthers over long anthers, consistent with a role as feeding and pollinating anthers, respectively; (2) Blocking short anther pores alone increased sonication of long anthers and resulted in collection of pollen from long anthers; (3) Blocking long anther pores alone did not influence sonication of short anthers; (4) The increase in sonication of long anthers, when short anther pores are blocked, was greater when pollen was available in long anthers; (5) Despite shifting sonication effort to long anthers, bees do not move their bodies closer to long anther pores where pollen could be collected more effectively; and (6) analysis of the growth of corbicular loads over time spent buzzing indicates that significant amounts of pollen are collected from long anthers as well as short anthers. We conclude that bees can flexibly increase pollen collection from pollinating anthers, but are constrained from fully exploiting this pollen. This results in checks and balances between plant and bee that may help maintain heteranthery.     

INDUCTION OF PROPHASE IN INTERPHASE NUCLEI BY FUSION WITH METAPHASE CELLS

Fusion of an interphase cell with a metaphase cell results in profound changes in the interphase chromatin that have been called "chromosome pulverization" or "premature chromosome condensation" In addition to the usual light microscopy, the nature of the changes has been investigated in the present study with electron microscopy and biochemical techniques Metaphase and interphase cells were mixed and fused at 37°C by means of ultraviolet-inactivated Sendai virus. After cell fusion, morphological changes in interphase nuclei occurred only in binucleate cells which contained one intact set of metaphase chromosomes Irrespective of the nuclear stage at the time of cell fusion, the morphologic changes that occurred 5–20 min later simulated very closely a sequence of events that characterizes the normal G₂-prophase transition. Radioautography revealed that, late in the process, substantial amounts of RNA and probably protein were transferred from the interphase nucleus into the cytoplasm of fused cells. Thus, the findings indicate the existence in metaphase cells of factor(s) which are capable of initiating biochemical and morphological events in interphase nuclei intrinsic to the normal mitotic process.     

Sex determination of bovine embryos using the polymerase chain reaction

Abstract

One or two cell biopsies were obtained from 6‐7 days old bovine embryos. The sex of the embryos was determined with two different bovine Y‐chromosome‐specific primer pairs by using the polymerase chain reaction. These results were confirmed by karyotyping as well as in situ hybridization with an independent bovine Y‐chromosome‐specific sequence. The polymerase chain reaction was found to be a quick and accurate method of sex diagnosis of bovine preimplantation embryos.     

Identification of a new small molecule chemotype of Melanin Concentrating Hormone Receptor-1 antagonists using pharmacophore-based virtual screening

MCH receptor is a G protein-coupled receptor with two subtypes R1 and R2. Many studies have demonstrated the role of MCH-R1 in feeding and energy homeostasis. It has been proven that oral administration of small molecule MCH-R1 antagonists significantly reduces food intake and causes a dose-dependent weight loss. In this study, two ligand-based pharmacophores were developed and validated based on recently published MCH-R1 antagonists with diverse structures. Successful pharmacophores had one hydrogen bond acceptor, one positive ionizable, one ring aromatic and two or three hydrophobic groups. These 3D-QSAR models were used for virtual screening of the ZINC chemical database resulting in the identification of nine compounds with more than 50% displacement of radiolabeled MCH at a 20 μM concentration. Moreover, four of these compounds showed antagonistic activities in Aequorin functional assay, including MH-3 which is the first MCH-R1 antagonist based on a diazaspiro[4.5]decane scaffold. The most active compounds were also docked into our previously published MCH-R1 homology model to gain insights into their binding determinants. These compounds could represent a viable starting scaffold for the design of potent MCH-R1 antagonists with improved pharmacokinetic properties as an effective treatment for obesity.     

BIOGEOGRAPHICAL AND TOPOGRAPHICAL VARIATION IN THE PREY OF THE BLACK EAGLE IN THE CAPE PROVINCE,SOUTH AFRICA

Boshoff, A.F., Palmer, N.G., Avery, G., Davies, R.A.G. & Jarvis, M.J.F. 1991. Biogeoraphical and toporaphical variation in the prey of the Black Eagle in the Cape Province, South Africa. Ostrich 62: 58–72.

Prey remains collected at or near Black Eagle Aquilu verreauxii nest sites in the Cape Province, South Africa, were analysed according to frequency of occurrence of prey scies in the samples. A total of 5748 prey individuals, collected from 73 sites, was analysed according to tree biome groups and four nest site types. The Rock Hyrax Procuvia capensis is the dominant prey species, but the eagles' diet sctrum vanestypes. The according to its availability. Indices of species richness and diversity of the prey are inverser correlated with the proportion of the prey contributed by P. capenis, which in turn is determined by topography and vegetation. Biome has a greater influence on the indices than has nest site type. The age structure of the P. capensis prey remains closely reflects the juvenile: sub- adult: adult ratios in the biomes and at the nest site tpes. hfedium-sized (aprox. 1–4,5 kg) pre is usually taken. Juvenile domestic small-stock (lambs ani goat kids) comprised onb 3,4% of the over, total.