SARS-CoV 9b protein diffuses into nucleus, undergoes active Crm1 mediated nucleocytoplasmic export and triggers apoptosis when retained in the nucleus

BACKGROUND: 9b is an accessory protein of the SARS-CoV. It is a small protein of 98 amino acids and its structure has been solved recently. 9b is known to localize in the extra-nuclear region and has been postulated to possess a nuclear export signal (NES), however the role of NES in 9b functioning is not well understood. PRINCIPAL FINDINGS/METHODOLOGY: In this report, we demonstrate that 9b in the absence of any nuclear localization signal (NLS) enters the nucleus by passive transport. Using various cell cycle inhibitors, we have shown that the nuclear entry of 9b is independent of the cell cycle. Further, we found that 9b interacts with the cellular protein Crm1 and gets exported out of the nucleus using an active NES. We have also revealed that this NES activity influences the half-life of 9b and affects host cell death. We found that an export signal deficient SARS-CoV 9b protein induces apoptosis in transiently transfected cells and showed elevated caspase-3 activity. CONCLUSION/SIGNIFICANCE: Here, we showed that nuclear shuttling of 9b and its interaction with Crm1 are essential for the proper degradation of 9b and blocking the nuclear export of this protein induces apoptosis. This phenomenon may be critical in providing a novel role to the 9b accessory protein of SARS-CoV.     

Genetic and environmental influences on Chinese language and reading abilities

This study investigated the etiology of individual differences in Chinese language and reading skills in 312 typically developing Chinese twin pairs aged from 3 to 11 years (228 pairs of monozygotic twins and 84 pairs of dizygotic twins; 166 male pairs and 146 female pairs). Children were individually given tasks of Chinese word reading, receptive vocabulary, phonological memory, tone awareness, syllable and rhyme awareness, rapid automatized naming, morphological awareness and orthographic skills, and Raven's Coloured Progressive Matrices. All analyses controlled for the effects of age. There were moderate to substantial genetic influences on word reading, tone awareness, phonological memory, morphological awareness and rapid automatized naming (estimates ranged from .42 to .73), while shared environment exerted moderate to strong effects on receptive vocabulary, syllable and rhyme awareness and orthographic skills (estimates ranged from .35 to .63). Results were largely unchanged when scores were adjusted for nonverbal reasoning as well as age. Findings of this study are mostly similar to those found for English, a language with very different characteristics, and suggest the universality of genetic and environmental influences across languages.     

Imaging of zebrafish in vivo with second-harmonic generation reveals shortened sarcomeres associated with myopathy induced by statin

We employed second-harmonic generation (SHG) imaging and the zebrafish model to investigate the myopathy caused by statin in vivo with emphasis on the altered microstructures of the muscle sarcomere, the fundamental contractile element of muscles. This approach derives an advantage of SHG imaging to observe the striated skeletal muscle of living zebrafish based on signals produced mainly from the thick myosin filament of sarcomeres without employing exogenous labels, and eliminates concern about the distortion of muscle structures caused by sample preparation in conventional histological examination. The treatment with statin caused a significantly shortened sarcomere relative to an untreated control (1.73±0.09 μm vs 1.91±0.08 μm, P<0.05) while the morphological integrity of the muscle fibers remained largely intact. Mechanistic tests indicated that this microstructural disorder was associated with the biosynthetic pathway of cholesterol, or, specifically, with the impaired production of mevalonate by statins. This microstructural disorder exhibited a strong dependence on both the dosage and the duration of treatment, indicating a possibility to assess the severity of muscle injury according to the altered length of the sarcomeres. In contrast to a conventional assessment of muscle injury using clinical biomarkers in blood, such as creatine kinase that is released from only disrupted myocytes, the ability to determine microstructural modification of sarcomeres allows diagnosis of muscle injury before an onset of conventional clinical symptoms. In light of the increasing prevalence of the incidence of muscle injuries caused by new therapies, our work consolidates the combined use of the zebrafish and SHG imaging as an effective and sensitive means to evaluate the safety profile of new therapeutic targets in vivo.     

Genetic isolation between the Western and Eastern Pacific populations of pronghorn spiny lobster Panulirus penicillatus

The pronghorn spiny lobster, Panulirus penicillatus, is a circumtropical species which has the widest global distribution among all the species of spiny lobster, ranging throughout the entire Indo-Pacific region. Partial nucleotide sequences of mitochondrial DNA COI (1,142-1,207 bp) and 16S rDNA (535-546 bp) regions were determined for adult and phyllosoma larval samples collected from the Eastern Pacific (EP)(Galápagos Islands and its adjacent water), Central Pacific (CP)(Hawaii and Tuamotu) and the Western Pacific (WP)(Japan, Indonesia, Fiji, New Caledonia and Australia). Phylogenetic analyses revealed two distinct large clades corresponding to the geographic origin of samples (EP and CP+WP). No haplotype was shared between the two regional samples, and average nucleotide sequence divergence (Kimura's two parameter distance) between EP and CP+WP samples was 3.8±0.5% for COI and 1.0±0.4% for 16S rDNA, both of which were much larger than those within samples. The present results indicate that the Pacific population of the pronghorn spiny lobster is subdivided into two distinct populations (Eastern Pacific and Central to Western Pacific), with no gene flow between them. Although the pronghorn spiny lobster have long-lived teleplanic larvae, the vast expanse of Pacific Ocean with no islands and no shallow substrate which is known as the East Pacific Barrier appears to have isolated these two populations for a long time (c.a. 1MY).     

Virus-specific CD4+ and CD8+ cytotoxic T-cell responses and long-term T-cell memory in individuals vaccinated against polio

The presence of poliovirus (PV)-specific CD4(+) T cells in individuals vaccinated against polio has been shown, but CD8(+) T-cell responses have not been described. Here, we functionally characterize the CD4(+) T-cell response and show for the first time that dendritic cells and macrophages can stimulate PV-specific CD8(+) T-cell responses in vitro from vaccinees. Both CD4(+) T and CD8(+) T cells secrete gamma interferon in response to PV antigens and are cytotoxic via the perforin/granzyme B-mediated pathway. Furthermore, the T cells also recognize and kill Sabin 1 vaccine-infected targets. The macrophage-stimulated CD4(+) T and CD8(+) T cells most likely represent memory T cells that persist for long periods in vaccinated individuals. Thus, immunity to PV vaccination involves not only an effective neutralizing antibody titer but also long-term CD4(+) and CD8(+) cytotoxic T-cell responses.     

Faster Rubisco is the key to superior nitrogen-use efficiency in NADP-malic enzyme relative to NAD-malic enzyme C4 grasses

In 27 C4 grasses grown under adequate or deficient nitrogen (N) supplies, N-use efficiency at the photosynthetic (assimilation rate per unit leaf N) and whole-plant (dry mass per total leaf N) level was greater in NADP-malic enzyme (ME) than NAD-ME species. This was due to lower N content in NADP-ME than NAD-ME leaves because neither assimilation rates nor plant dry mass differed significantly between the two C4 subtypes. Relative to NAD-ME, NADP-ME leaves had greater in vivo (assimilation rate per Rubisco catalytic sites) and in vitro Rubisco turnover rates (k(cat); 3.8 versus 5.7 s(-1) at 25 degrees C). The two parameters were linearly related. In 2 NAD-ME (Panicum miliaceum and Panicum coloratum) and 2 NADP-ME (Sorghum bicolor and Cenchrus ciliaris) grasses, 30% of leaf N was allocated to thylakoids and 5% to 9% to amino acids and nitrate. Soluble protein represented a smaller fraction of leaf N in NADP-ME (41%) than in NAD-ME (53%) leaves, of which Rubisco accounted for one-seventh. Soluble protein averaged 7 and 10 g (mmol chlorophyll)(-1) in NADP-ME and NAD-ME leaves, respectively. The majority (65%) of leaf N and chlorophyll was found in the mesophyll of NADP-ME and bundle sheath of NAD-ME leaves. The mesophyll-bundle sheath distribution of functional thylakoid complexes (photosystems I and II and cytochrome f) varied among species, with a tendency to be mostly located in the mesophyll. In conclusion, superior N-use efficiency of NADP-ME relative to NAD-ME grasses was achieved with less leaf N, soluble protein, and Rubisco having a faster k(cat).     

Kinetic rates of antibody binding correlate with neutralization sensitivity of variant simian immunodeficiency virus strains

Increasing evidence suggests that an effective AIDS vaccine will need to elicit both broadly reactive humoral and cellular immune responses. Potent and cross-reactive neutralization of simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) by polyclonal and monoclonal antibodies is well documented. However, the mechanisms of antibody-mediated neutralization have not been defined. The current study was designed to determine whether the specificity and quantitative properties of antibody binding to SIV envelope proteins correlate with neutralization. Using a panel of rhesus monoclonal antibodies previously characterized for their ability to bind and neutralize variant SIVs, we compared the kinetic rates and affinity of antibody binding to soluble envelope trimers by using surface plasmon resonance. We identified significant differences in the kinetic rates but not the affinity of monoclonal antibody binding to the neutralization-sensitive SIV/17E-CL and neutralization-resistant SIVmac239 envelope proteins that correlated with the neutralization sensitivities of the corresponding virus strains. These results suggest for the first time that neutralization resistance may be related to quantitative differences in the rates but not the affinity of the antibody-envelope interaction and may provide one mechanism for the inherent resistance of SIVmac239 to neutralization in vitro. Further, we provide evidence that factors in addition to antibody binding, such as epitope specificity, contribute to the mechanisms of neutralization of SIV/17E-CL in vitro. This study will impact the method by which HIV/SIV vaccines are evaluated and will influence the design of candidate AIDS vaccines capable of eliciting effective neutralizing antibody responses.     

A simple chlorophyll fluorescence parameter that correlates with the rate coefficient of photoinactivation of Photosystem II

A method of partitioning the energy in a mixed population of active and photoinactivated Photosystem II (PS II) complexes based on chlorophyll fluorescence measurements is presented. There are four energy fluxes, each with its quantum efficiency: a flux associated with photochemical electron flow in active PS II reaction centres (J_{PS II}), thermal dissipation in photoinactivated, non-functional PS IIs (J_NF), light-regulated thermal dissipation in active PS IIs (J_NPQ) and a combined flux of fluorescence and constitutive, light-independent thermal dissipation (J_f,D). The four quantum efficiencies add up to 1.0, without the need to introduce an ‘excess’ term E, which in other studies has been claimed to be linearly correlated with the rate coefficient of photoinactivation of PS II (k_pi). We examined the correlation of k_pi with various fluxes, and found that the combined flux (J_NPQ + J_f,D= J_pi) is as well correlated with k_pi as is E. This combined flux arises from F_s/F_m^′, the ratio of steady-state to maximum fluorescence during illumination, which represents the quantum efficiency of combined non-photochemical dissipation pathways in active PS IIs. Since F_s/F_m^′ or its equivalent, J_pi, is a likely source of events leading to photoinactivation of PS II, we conclude that F_s/F_m^′ is a simple predictor of k_pi.