Observation of intersubunit movement of the ribosome in solution using FRET

Protein synthesis is believed to be a dynamic process, involving structural rearrangements of the ribosome. Cryo-EM reconstructions of certain elongation factor G (EF-G)-containing complexes have led to the proposal that translocation of tRNA and mRNA through the ribosome, from the A to P to E sites, is accompanied by a rotational movement between the two ribosomal subunits. Here, we have used F?rster resonance energy transfer (FRET) to monitor changes in the relative orientation of the ribosomal subunits in different complexes trapped at intermediate stages of translocation in solution. Binding of EF-G to the ribosome in the presence of the non-hydrolyzable GTP analogue GDPNP or GTP plus fusidic acid causes an increase in the efficiency of energy transfer between fluorophores introduced into proteins S11 in the 30 S subunit and L9 in the 50 S subunit, and a decrease in energy transfer between S6 and L9. Similar anti-correlated changes in energy transfer occur upon binding the GTP-requiring release factor RF3. These changes are consistent with the counter-clockwise rotation of the 30 S subunit relative to the 50 S subunit observed in cryo-EM studies. Reaction of ribosomal complexes containing the peptidyl-tRNA analogues N-Ac-Phe-tRNAPhe, N-Ac-Met-tRNAMet or f-Met-tRNAfMet with puromycin, conditions favoring movement of the resulting deacylated tRNAs into the P/E hybrid state, leads to similar changes in FRET. Conversely, treatment of a ribosomal complex containing deacylated and peptidyl-tRNAs bound in the A/P and P/E states, respectively, with EF-G.GTP causes reversal of the FRET changes. The use of FRET has enabled direct observation of intersubunit movement in solution, provides independent evidence that formation of the hybrid state is coupled to rotation of the 30 S subunit and shows that the intersubunit movement is reversed during the second step of translocation.     

An Asian Origin for Subtype IV BK Virus Based on Phylogenetic Analysis

Similarly to other members of the Polyomaviridae family, BK virus (BKV) is thought to have co-evolved with its human host. BKV has four subtypes that are distinguishable by immunological reactivity, with two (subtypes I and IV) being most prevalent in human populations. Subtype I is the major subtype worldwide, whereas subtype IV is prevalent in East Asia and Europe but rare in Africa. The geographic distribution pattern of subtype IV BKV is in apparent disagreement with the hypothesis that BKV co-evolved with humans, since subtype IV rarely occurs in Africa. To elucidate the origin of subtype IV, 53 complete subtype IV sequences derived from East Asians and Europeans were subjected to a detailed phylogenetic analysis using the maximum-likelihood and neighbor-joining methods. We identified six subgroups (a1, a2, b1, b2, c1, and c2) that formed a tree represented by the formula: “(a1, a2), ((b1, b2), (c1, c2)).” Interestingly, we found a close correlation between subtype IV subgroups and population geography; thus, all subgroups except c2 were prevalent in particular East Asian populations, with c2 occurring in both Europe and Northeast Asia. From these findings, we conclude that subtype IV of BKV now prevalent in modern humans is derived from a virus that infected ancestral Asians. We introduce two hypotheses to explain how ancestral Asians became infected with subtype IV BKV. [Reviewing Editor: Dr. Joshua Plotkin] Yuriko Nishimoto and Huai-Ying Zheng are two authors contributed equally to this article.     

Hsp70/Hsc70 regulates the effect phosphorylation has on stabilizing ataxin-1

Spinocerebellar ataxia type 1 (SCA1) is an inherited neurodegenerative disorder. The mutation causing SCA1 is an expansion in the polyglutamine tract of the ATXN1 protein. Previous work demonstrated that phosphorylation of mutant ATXN1 at serine 776 (S776), a putative Akt phosphorylation site, is critical for pathogenesis. To examine this pathway further, we utilized a cell-transfection system that allowed the targeting of Akt to either the cytoplasm or the nucleus. In contrast to HeLa cells, we found that Akt targeted to the cytoplasm increased the degradation of ATXN1 in Chinese hamster ovary cells. However, Akt targeted to the cytoplasm failed to destabilize ATXN1 if Hsp70/Hsc70 was present. Thus, Hsp70/Hsc70 can regulate ATXN1 levels in concert with phosphorylation of ATXN1 at S776.     

A pi-helix switch selective for porphyrin deprotonation and product release in human ferrochelatase

Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) is the terminal enzyme in heme biosynthesis and catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX (heme). Due to the many critical roles of heme, synthesis of heme is required by the vast majority of organisms. Despite significant investigation of both the microbial and eukaryotic enzyme, details of metal chelation remain unidentified. Here we present the first structure of the wild-type human enzyme, a lead-inhibited intermediate of the wild-type enzyme with bound metallated porphyrin macrocycle, the product bound form of the enzyme, and a higher resolution model for the substrate-bound form of the E343K variant. These data paint a picture of an enzyme that undergoes significant changes in secondary structure during the catalytic cycle. The role that these structural alterations play in overall catalysis and potential protein-protein interactions with other proteins, as well as the possible molecular basis for these changes, is discussed. The atomic details and structural rearrangements presented herein significantly advance our understanding of the substrate binding mode of ferrochelatase and reveal new conformational changes in a structurally conserved pi-helix that is predicted to have a central role in product release.     

Effect of climatic warming on the Pacific walrus, and potential modification of its helminth fauna

The decreasing extent of sea-ice in the arctic basin as a consequence of climatic warming is modifying the behavior and diets of pagophilic pinnipeds, including the Pacific walrus, Odobenus rosmarus divergens Illiger, the species emphasized here. Mammals such as the walrus and bearded seal, Erignathus barbatus (Erxleben), cannot remain associated with the sea-ice, and continue to feed on their usual diet of benthic invertebrates inhabiting coastal waters to a depth of approximately 100 m, when the northwestward retreating ice reaches deep waters beyond the margins of the continental shelf. With reduction of their customary substrate (ice), the walrus has become more pelagic and preys more often on ringed seals, Phoca hispida Schreber. Dietary changes, with modifications of helminth faunas, may be induced by various factors. Increased consumption of mammals or their remains by walruses may lead to a higher prevalence of trichinellosis in them and to more frequent occurrence in indigenous peoples inhabiting the arctic coasts. To assess predicted effects on the composition of helminth fauna of the walrus, we recommend systematic surveys of their helminths as part of research on effects of climatic warming.     

Role of K(ATP) channels in protection against neuronal excitatory insults

ATP-sensitive K⁺ (K_ATP) channels that are gated by intracellular ATP/ADP concentrations are a unique subtype of potassium channels and play an essential role in coupling intracellular metabolic events to electrical activity. Opening of K_ATP channels during energy deficits in the CNS induces efflux of potassium ions and in turn hyperpolarizes neurons. Thus, activation of K_ATP channels is thought to be able to counteract excitatory insults and protect against neuronal death. In this review, we bring together recent studies about what kinds of molecules are needed to build and regulate arrays of K_ATP channel functions in the CNS neurons. We propose a model to explain how K_ATP channel activation regulates glutamate release from the pre-synaptic terminals and how this regulation protects against ischemic neuronal injury and epilepsy.     

Rotavirus infection alters peripheral T-cell homeostasis in children with acute diarrhea

The patterns of gene expression and the phenotypes of lymphocytes in peripheral blood mononuclear cells (PBMC) from children with diarrhea caused by rotavirus and healthy children were compared by using DNA microarray, quantitative PCR, and flow cytometry. We observed increased expression of a number of genes encoding proinflammatory cytokines and interferon or interferon-stimulated proteins and demonstrated activation of some genes involved in the differentiation, maturation, activation, and survival of B lymphocytes in PBMC of patients with rotavirus infection. In contrast, we observed a consistent pattern of lower mRNA levels for an array of genes involved in the various stages of T-cell development and demonstrated a reduction in total lymphocyte populations and in the proportions of CD4 and CD8 T lymphocytes from PBMC of patients. This decreased frequency of T lymphocytes was transient, since the proportions of T lymphocytes recovered to almost normal levels in convalescent-phase PBMC from most patients. Finally, rotavirus infection induced the activation and expression of the early activation markers CD83 and CD69 on a fraction of CD19 B cells and the remaining CD4 and CD8 T lymphocytes in acute-phase PBMC of patients; the expression of CD83 continued to be elevated and was predominantly exhibited on CD4 T lymphocytes in convalescent-phase PBMC. On the basis of these findings at the molecular, phenotypic, and physiologic levels in acute-phase PBMC, we conclude that rotavirus infection induces robust proinflammatory and antiviral responses and B-cell activation but alters peripheral T-cell homeostasis in children.     

Evidence for the existence of the loop E motif of Potato spindle tuber viroid in vivo

RNA motifs comprising nucleotides that interact through non-Watson-Crick base pairing play critical roles in RNA functions, often by serving as the sites for RNA-RNA, RNA-protein, or RNA small ligand interactions. The structures of viral and viroid RNA motifs are studied commonly by in vitro, computational, and mutagenesis approaches. Demonstration of the in vivo existence of a motif will help establish its biological significance and promote mechanistic studies on its functions. By using UV cross-linking and primer extension, we have obtained direct evidence for the in vivo existence of the loop E motif of Potato spindle tuber viroid. We present our findings and discuss their biological implications.     

A structured viroid RNA serves as a substrate for dicer-like cleavage to produce biologically active small RNAs but is resistant to RNA-induced silencing complex-mediated degradation

RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21- to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.     

Distribution and adult activity of Popillia quadriguttata (Coleoptera: Scarabaeidae) on golf courses in Korea

Japanese beetle traps baited with the Popillia japonica Newman (Coleoptera: Scarabaeidae) pheromone lure and a eugenol feeding attractant were placed at five golf courses in Korea to determine how well they work for detecting activity of a closely related species, Popillia quadriguttata (F.) (Coleoptera: Scarabaeidae), a turf pest in Korea. The traps also were used to determine the time of day and time of year that P. quadriguttata is most active. Nineteen scarab species of 13 genera were attracted to the Japanese beetle traps with P. quadriguttata clearly being the most abundant (383 beetles per trap), followed by Adoretus tenuimaculatus Waterhouse (10 per trap), Popilliaflavosellata Fairmaire (seven per trap), Exomala orientalis Waterhouse (four per trap), and Maladera japonica (two per trap). Other scarab species were trapped at a rate of <1.0 per trap. Popillia quadriguttata adults were active over a 5-wk period in late June and early July. At Yongwon Golf Club in 2002, peak adult activity was during the last week of June in visual counts and approximately 1 wk later in the Japanese beetle traps. In Korea, P. quadriguttata adults are most active between 12:00 p.m. and 4:00 p.m. This information should be helpful to golf course superintendents in Korea and to entomologists interested in finding natural enemies of P. quadriguttata to evaluate as potential biocontrol organisms for the very closely related species, the Japanese beetle.