Basic residues of the helix six domain of influenza virus M1 involved in nuclear translocation of M1 can be replaced by PTAP and YPDL late assembly domain motifs

Influenza type A virus matrix (M1) protein possesses multiple functional motifs in the helix 6 (H6) domain (amino acids 91 to 105), including nuclear localization signal (NLS) (101-RKLKR-105) involved in translocating M1 from the cytoplasm into the nucleus. To determine the role of the NLS motif in the influenza virus life cycle, we mutated these and the neighboring sequences by site-directed mutagenesis, and influenza virus mutants were generated by reverse genetics. Our results show that infectious viruses were rescued by reverse genetics from all single alanine mutations of amino acids in the H6 domain and the neighboring region except in three positions (K104A and R105A within the NLS motif and E106A in loop 6 outside the NLS motif). Among the rescued mutant viruses, R101A and R105K exhibited reduced growth and small-plaque morphology, and all other mutant viruses showed the wild-type phenotype. On the other hand, three single mutations (K104A, K105A, and E106A) and three double mutations (R101A/K102A, K104A/K105A, and K102A/R105A) failed to generate infectious virus. Deletion (Delta YRKL) or mutation (4A) of YRKL also abolished generation of infectious virus. However, replacement of the YRKL motif with PTAP or YPDL as well as insertion of PTAP after 4A mutation yielded infectious viruses with the wild-type phenotype. Furthermore, mutant M1 proteins (R101A/K102A, Delta YRKL, 4A, PTAP, 4A+PTAP, and YPDL) when expressed alone from cloned cDNAs were only cytoplasmic, whereas the wild-type M1 expressed alone was both nuclear and cytoplasmic as expected. These results show that the nuclear translocation function provided by the positively charged residues within the NLS motif does not play a critical role in influenza virus replication. Furthermore, these sequences of H6 domain can be replaced by late (L) domain motifs and therefore may provide a function similar to that of the L domains of other negative-strand RNA and retroviruses.     

alpha3beta1 integrin-CD151, a component of the cadherin-catenin complex, regulates PTPmu expression and cell-cell adhesion

The beta1 family of integrins has been primarily studied as a set of receptors for the extracellular matrix. In this paper, we define a novel role for alpha3beta1 integrin in association with the tetraspanin CD151 as a component of a cell-cell adhesion complex in epithelial cells that directly stimulates cadherin-mediated adhesion. The integrin-tetraspanin complex affects epithelial cell-cell adhesion at the level of gene expression both by regulating expression of PTPmu and by organizing a multimolecular complex containing PKCbetaII, RACK1, PTPmu, beta-catenin, and E-cadherin. These findings demonstrate how integrin-based signaling can regulate complex biological responses at multiple levels to determine cell morphology and behavior.     

Synergized bifenthrin plus chlorpyrifos-methyl for control of beetles and psocids in sorghum in Australia

The efficacy of bifenthrin (0.5 mg/kg) + piperonyl butoxide (7 mg/kg) + chlorpyrifosmethyl (10 mg/kg) against beetle and psocid pests of sorghum was evaluated in silo-scale trials in southeast Queensland, Australia. The pyrethroid bifenthrin was evaluated as a potential new protectant in combination with the organophosphate chlorpyrifos-methyl, which is already registered for control of several insect pests of stored cereals. Sorghum (approximately 200 metric tons) was treated after both the 1999 and 2000 harvests and sampled at intervals to assess treatment efficacy and residue decline during up to 7 mo of storage. Generally, test strains of the beetles Rhyzopertha dominica (F.), Tribolium castaneum (Herbst), Oryzaephilus surinamensis (L), and Cryptolestes ferrugineus (Stephens) were prevented from producing live progeny for up to 7 mo. The treatment failed against one strain of R. dominica known to be resistant to bioresmethrin and organophosphates. Two malathion-resistant strains of O. surinamensis were marginally controlled with 94-100% fewer adult progeny produced. For psocids, no strains of Liposcelis bostrychophila Badonnel, Liposcelis decolor (Pearman), or Liposcelis paeta Pearman produced live progeny, although the control of a field strain of Liposcelis entomophila (Enderlein) was generally poor. Results show that this treatment should protect sorghum for at least 7 mo against most prevalent strains of grain insect in eastern Australia, although control may be limited in areas in which L. entomophila or pyrethroid-resistant R. dominica are present.     

Drosophila perlecan modulates FGF and hedgehog signals to activate neural stem cell division

Mutations in the Drosophila trol gene cause cell cycle arrest of neuroblasts in the larval brain. Here, we show that trol encodes the Drosophila homolog of Perlecan and regulates neuroblast division by modulating both FGF and Hh signaling. Addition of human FGF-2 to trol mutant brains in culture rescues the trol proliferation phenotype, while addition of a MAPK inhibitor causes cell cycle arrest of the regulated neuroblasts in wildtype brains. Like FGF, Hh activates stem cell division in the larval brain in a Trol-dependent fashion. Coimmunoprecipitation studies are consistent with interactions between Trol and Hh and between mammalian Perlecan and Shh that are not competed with heparin sulfate. Finally, analyses of mutations in trol, hh, and ttv suggest that Trol affects Hh movement. These results indicate that Trol can mediate signaling through both of the FGF and Hedgehog pathways to control the onset of stem cell proliferation in the developing nervous system.     

Cytomegalovirus seropositivity drives the CD8 T cell repertoire toward greater clonality in healthy elderly individuals

The deterioration in immune function with aging is thought to make a major contribution to the increased morbidity and mortality from infectious disease in old age. One aspect of immune senescence is the reduction in CD8 T cell repertoire as due to the accumulation of oligoclonal, memory T cells and a reduction in the naive T cell pool. CD8 T cell clonal expansions accumulate with age, but their antigenic specificity remains unknown. In this study, we show that in elderly individuals seropositivity for human CMV leads to the development of oligoclonal populations of CMV-specific CTL that can constitute up to one-quarter of the total CD8 T cell population. Furthermore, CMV-specific CTL have a highly polarized membrane phenotype that is typical of effector memory cells (CD28(-), CD57(+), CCR7(-)). TCR analyses show that CMV-specific CTL have highly restricted clonality with greater restriction in the larger expansions. Clonal analysis of the total CD8 T cell repertoire was compared between CMV-seropositive and CMV-seronegative donors. Thirty-three percent more clonal expansions were observed in CMV-seropositive donors in comparison with seronegative individuals. These data implicate CMV as a major factor in driving oligoclonal expansions in old age. Such a dramatic accumulation of virus-specific effector CTL might impair the ability to respond to heterologous infection and may underlie the negative influence of CMV seropositivity on survival in the very elderly.     

A role for the androgen receptor in the endometrial antiproliferative effects of progesterone antagonists

In women and nonhuman primates, treatment with progesterone antagonists suppresses estrogen-dependent mitotic activity in the endometrial glands. This antiproliferative effect is paradoxical, because progesterone antagonists do not bind to the estrogen receptor (ER). While this phenomenon has been termed a "functional noncompetitive antiestrogenic effect," it does not occur in all species or in all regions of the primate reproductive tract, so is best referred to as an "endometrial antiproliferative effect." Recent studies of ours in both women and macaques revealed that the endometrial androgen receptor (AR) was increased by progesterone antagonist treatment. Because androgens are known to suppress estrogen-dependent endometrial proliferation, we hypothesized that the AR was involved in the antiproliferative effects induced by progesterone antagonists. In a test of this hypothesis, we administered the antiandrogen, flutamide, along with progesterone antagonists to ovariectomized, estrogen-treated macaques. Flutamide counteracted the suppressive effects of the progesterone antagonists on endometrial wet weight, thickness, stromal compaction, and mitotic index. Hyaline degeneration of the spiral arteries was also blocked by flutamide. These data implicate the AR as a functional component of the mechanism through which progesterone antagonists induce endometrial antiproliferative effects in the presence of estrogens.     

Influenza virus assembly: effect of influenza virus glycoproteins on the membrane association of M1 protein

Influenza virus matrix protein (M1), a critical protein required for virus assembly and budding, is presumed to interact with viral glycoproteins on the outer side and viral ribonucleoprotein on the inner side. However, because of the inherent membrane-binding ability of M1 protein, it has been difficult to demonstrate the specific interaction of M1 protein with hemagglutinin (HA) or neuraminidase (NA), the influenza virus envelope glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and floatation in sucrose gradients, we observed that the membrane-bound M1 protein expressed alone or coexpressed with heterologous Sendai virus F was totally TX-100 soluble but the membrane-bound M1 protein expressed in the presence of HA and NA was predominantly detergent resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of HA facilitated binding of M1 to detergent-resistant membranes. Analysis of the membrane association of M1 in the early and late phases of the influenza virus infectious cycle revealed that the interaction of M1 with mature glycoproteins which associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of membrane-bound M1. Immunofluorescence analysis by confocal microscopy also demonstrated that, in influenza virus-infected cells, a fraction of M1 protein colocalized with HA and associated with the HA in transit to the plasma membrane via the exocytic pathway. Similar results for colocalization were obtained when M1 and HA were coexpressed and HA transport was blocked by monensin treatment. These studies indicate that both HA and NA interact with influenza virus M1 and that HA associates with M1 via its cytoplasmic tail and transmembrane domain.     

Crystal structure of an intermolecular 2:1 complex between adenine and thymine. Evidence for both Hoogsteen and ‘quasi-Watson–Crick’ interactions

The titled complex, obtained by co-crystallization (EtOH/25 °C), is apparently the only known complex of the free bases. Its crystal structure, as determined by X-ray diffraction at both 90 K and 313 K, showed that one A–T pair involves a Hoogsteen interaction, and the other a Watson–Crick interaction but only with respect to the adenine unit. The absence of a clear-cut Watson–Crick base pair raises intriguing questions about the basis of the DNA double helix.