Localization of domains within the Drosophila Ref(2)P protein involved in the intracellular control of sigma rhabdovirus multiplication.

The ref(2)P gene of Drosophila melanogaster interferes with sigma rhabdovirus multiplication. This gene is highly variable, and the different alleles are considered permissive or restrictive according to their effects on virus replication. In all cases, the mechanisms involve intracellular interactions between the sigma virus and Ref(2)P proteins. We showed that the N-terminal domain of the Ref(2)P protein was required for its activity in vivo. The protein was inactive in the null p(od)2 mutant when its first 82 amino acids were deleted. The p delta n gene was constructed so that the first 91 amino acids coded for by the restrictive alleles could be expressed in vivo. It was active in a transformed line. This sequence was sufficient to impart a restrictive phenotype to an adult D. melanogaster fly after it was injected with the virus. However, the truncated protein expressed by p delta n did not have an effect on the hereditary transmission of the sigma virus to the offspring of the infected flies, even though it contained the restriction site. The native Ref(2)P protein has been previously shown to have conformation-dependent epitopes common with some of those of the viral N protein. We demonstrated the following. (i) These epitopes were found in a domain of the Ref(2)P protein distinct from the site involved in restriction. (ii) They were modified in the N protein of the haP7 sigma virus mutant selected as being adapted to the restrictive alleles of the ref(2)P gene; only one mutation in the N gene, leading to an amino acid substitution, distinguished the haP7 mutant from the original virus. (iii) The virus strains partially or totally adapted to the effects of the full restrictive protein expressed by pp were always found to multiply to a lesser extent in the presence of the protein expressed by p delta n. These data suggest that two distinct domains of the Ref(2)P protein are involved in the control of sigma virus multiplication.     

7-Valent Pneumococcal Conjugate Vaccination in England and Wales: Is It Still Beneficial Despite High Levels of Serotype Replacement?

Background

The UK introduced the 7-valent pneumococcal conjugate vaccine (PCV7) into the national vaccination program in September 2006. Previous modelling assumed that the likely impact of PCV7 on invasive pneumococcal disease (IPD) would be similar to the US experience with PCV7. However, recent surveillance data show a more rapid replacement of PCV7 IPD cases by non-PCV7 IPD cases than was seen in the US.

Methods and Findings

A previous model of pneumococcal vaccination was re-parameterised using data on vaccine coverage and IPD from England and Wales between 2006 and 2009. Disease incidence was adjusted for the increasing trend in reported IPD cases prior to vaccination. Using this data we estimated that individuals carrying PCV7 serotypes have much higher protection (96%;95% CI 72%-100%) against acquisition of NVT carriage than the 15% previously estimated from US data, which leads to greater replacement. However, even with this level of replacement, the annual number of IPD cases may be 560 (95% CI, -100 to 1230) lower ten years after vaccine introduction compared to what it may have been without vaccination. A particularly marked fall of 39% in children under 15 years by 2015/6 is predicted.

Conclusion

Our model suggests that PCV7 vaccination could result in a decrease in overall invasive pneumococcal disease, particularly in children, even in an environment of rapid replacement with non-PCV7 serotypes within 5 years of vaccine introduction at high coverage.     

Simultaneous detection and functional response of testosterone and estradiol receptors in osteoblast plasma membranes

Osteoblasts derived from the periosteal surfaces of two-three-week-old male broiler chicken tibias were cultured for eight days. The cells were then loaded with fura-2/AM ester to detect surges in intracellular Ca(2+). Treatment with 10(-7) M testosterone (T) or 17beta-estradiol (E) elicited a rapid (within seconds) response that was substantially reduced by introducing the calcium chelating agent EGTA or the calcium-channel blocker verapamil. The hormones were equally effective when covalently linked to bovine serum albumin (BSA), a procedure that ensures the hormone does not enter the cells. The rapid response to surface-bound steroids indicates that the responses were invoked through plasma-membrane receptors. The source of Ca(2+) was shown to be through entry from external sources, as well as from intracellular stores. Flow cytometry of fluorescein-tagged T-BSA and E-BSA revealed that osteoblasts derived from male chickens had similar and substantial levels of both receptors.     

Dynein participates in chromosome segregation in fission yeast

Background information. In eukaryotic cells, proper formation of the spindle is necessary for successful cell division. For faithful segregation of sister chromatids, each sister kinetochore must attach to microtubules that extend to opposite poles (chromosome bi‐orientation). At the metaphase—anaphase transition, cohesion between sister chromatids is removed, and each sister chromatid is pulled to opposite poles of the cell by microtubule‐dependent forces. Results. We have studied the role of the minus‐end‐directed motor protein dynein by analysing kinetochore dynamics in fission yeast cells deleted for the dynein heavy chain (Dhc1) or the light chain (Dlc1). In these mutants, we found an increased frequency of cells showing defects in chromosome segregation, which leads to the appearance of lagging chromosomes and an increased rate of chromosome loss. By following simultaneously kinetochore dynamics and localization of the checkpoint protein Mad2, we provide evidence that dynein function is not necessary for spindle‐assembly checkpoint inactivation. Instead, we have demonstrated that loss of dynein function alters chromosome segregation and activates the Mad2‐dependent spindle‐assembly checkpoint. Conclusions. These results show an unexpected role for dynein in the control of chromosome segregation in fission yeast, most probably operating during the process of bi‐orientation during early mitosis.     

Ice seals as sentinels for algal toxin presence in the Pacific Arctic and subarctic marine ecosystems

Domoic acid (DA) and saxitoxin (STX)-producing algae are present in Alaskan seas, presenting exposure risks to marine mammals that may be increasing due to climate change. To investigate potential increases in exposure risks to four pagophilic ice seal species (Erignathus barbatus, bearded seals; Pusa hispida, ringed seals; Phoca largha, spotted seals; and Histriophoca fasciata, ribbon seals), this study analyzed samples from 998 seals harvested for subsistence purposes in western and northern Alaska during 2005–2019 for DA and STX. Both toxins were detected in bearded, ringed, and spotted seals, though no clinical signs of acute neurotoxicity were reported in harvested seals. Bearded seals had the highest prevalence of each toxin, followed by ringed seals. Bearded seal stomach content samples from the Bering Sea showed a significant increase in DA prevalence with time (logistic regression, p = .004). These findings are consistent with predicted northward expansion of DA-producing algae. A comparison of paired samples taken from the stomachs and colons of 15 seals found that colon content consistently had higher concentrations of both toxins. Collectively, these results suggest that ice seals, particularly bearded seals (benthic foraging specialists), are suitable sentinels for monitoring HAB prevalence in the Pacific Arctic and subarctic.     

Isolation and characterization of the Cryptococcus neoformans MATa pheromone gene

Cryptococcus neoformans is a heterothallic basidiomycete with two mating types, MATa and MATalpha. The mating pathway of this fungus has a number of conserved genes, including a MATalpha-specific pheromone (MFalpha1). A modified differential display strategy was used to identify a gene encoding the MATa pheromone. The gene, designated MFa1, is 42 amino acids in length and contains a conserved farnesylation motif. MFa1 is present in three linked copies that span a 20-kb fragment of MATa-specific DNA and maps to the MAT-containing chromosome. Transformation studies showed that MFa1 induced filament formation only in MATalpha cells, demonstrating that MFa1 is functionally conserved. Sequence analysis of the predicted Mfa1 and Mfalpha1 proteins revealed that, in contrast to other fungi such as Saccharomyces cerevisiae, the C. neoformans pheromone genes are structurally and functionally conserved. However, unlike the MFalpha1 gene, which is found in MATalpha strains of both varieties of C. neoformans, MFa1 is specific for the neoformans variety of C. neoformans.     

Structural and functional determinants in adenovirus type 2 penton base recombinant protein.

Discrete domains involved in structural and functional properties of adenovirus type 2 (Ad2) penton base were investigated with site-directed mutagenesis of the recombinant protein expressed in baculovirus-infected cells. Seventeen substitution mutants were generated and phenotyped for various functions in insect and human cells as follows. (i) Pentamerization of the penton base protein was found to be dependent on three amino acid side chains, the indole ring of Trp119, the hydroxylic group of Tyr553, and the basic group of Lys556. (ii) Arg254, Cys432, and Trp439, the stretch of basic residues at positions 547 to 556, and Arg340 of the RGD motif played a critical role in stable fiber-penton base interactions in vivo. (iii) Nuclear localization of penton base in Sf9 cells was negatively affected in mutants W119H or W165H, and, to a lesser extent, by substitutions in the consensus polybasic signal at positions 547 to 549. (iv) Penton base mutants were also assayed for HeLa cell binding, cell detachment, plasmid DNA internalization, and Ad-mediated gene delivery. The results obtained suggested that the previously identified integrin-binding motifs RGD340 and LDV287 were functionally and/or topologically related to other discrete regions which include Trp119, Trp165, Cys246, Cys432, and Trp439, all of which were involved in penton base-cell surface recognition, endocytosis, and postendocytotic steps of the virus life cycle.     

Variable content of double minute chromosomes is not correlated with degree of phenotype instability in methotrexate-resistant human cell lines.

Several variants resistant to 1.8 x 10(-4) M DL-methotrexate (MTX) have been isolated from the human cell lines HeLa BU25 and VA2-B by exposing them to progressively increasing concentrations of the drug. A striking variability of phenotype and chromosome constitution was observed among the different variants. All resistant cell lines exhibited a greatly increased dihydrofolic acid reductase (DHFR) activity and DHFR content; however, the DHFR activity levels varied considerably among the variants, ranging between about 35 and 275 times the parental level. In the absence of selective pressure, the increased DHFR activity was unstable, and in all cell lines but one was completely lost over a period ranging in different variants between 25 and 200 days. The MTX-resistant cells lines showed anomalies in their chromosome constitution, which involved the occurrence of a duplicated set of chromosomes in most cells of some of the variants and the presence of double minute chromosomes in all cell lines. An analysis of the correlation of loss of double minute chromosomes and loss of DHFR activity in the absence of MTX has given results consistent with the idea that the double-minute chromosomes contain amplified DHFR genes. However, the most significant finding is that, in contrast to what has been reported in the mouse system, the recognizable double-minute chromosomes varied greatly in number in different variants without any relationship to either the level of DHFR activity or the degree of instability of MTX resistance in the absence of selective pressure. These and other observations point to the occurrence in the human MTX-resistant variants of another set of DHFR genes, representing a varied proportion of the total, which is associated with the regular chromosomes, and which may be unstable in the absence of selective pressure.