Distinct reaction pathway promoted by non-divalent-metal cations in a tertiary stabilized hammerhead ribozyme

Divalent ion sensitivity of hammerhead ribozymes is significantly reduced when the RNA structure includes appropriate tertiary stabilization. Therefore, we investigated the activity of the tertiary stabilized "RzB" hammerhead ribozyme in several nondivalent ions. Ribozyme RzB is active in spermidine and Na(+) alone, although the cleavage rates are reduced by more than 1,000-fold relative to the rates observed in Mg(2+) and in transition metal ions. The trivalent cobalt hexammine (CoHex) ion is often used as an exchange-inert analog of hydrated magnesium ion. Trans-cleavage rates exceeded 8 min(-1) in 20 mM CoHex, which promoted cleavage through outersphere interactions. The stimulation of catalysis afforded by the tertiary structural interactions within RzB does not require Mg(2+), unlike other extended hammerhead ribozymes. Site-specific interaction with at least one Mg(2+) ion is suggested by CoHex competition experiments. In the presence of a constant, low concentration of Mg(2+), low concentrations of CoHex decreased the rate by two to three orders of magnitude relative to the rate in Mg(2+) alone. Cleavage rates increased as CoHex concentrations were raised further, but the final fraction cleaved was lower than what was observed in CoHex or Mg(2+) alone. These observations suggest that Mg(2+) and CoHex compete for binding and that they cause misfolded structures when they are together. The results of this study support the existence of an alternate catalytic mechanism used by nondivalent ions (especially CoHex) that is distinct from the one promoted by divalent metal ions, and they imply that divalent metals influence catalysis through a specific nonstructural role.     

Polyhydroxyalkanoate (PHA) synthesis by class IV PHA synthases employing Ralstonia eutropha PHB(-)4 as host strain

Class IV polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRC(YB4)) or B. megaterium NBRC15308(T) (PhaRC(Bm)) was expressed in Ralstonia eutropha PHB(-)4 to compare the ability to produce PHA and the substrate specificity of PhaRCs. PhaRC(YB4) produced significant amounts of PHA and had broader substrate specificity than PhaRC(Bm).     

Humpback whale abundance in the North Pacific estimated by photographic capture‐recapture with bias correction from simulation studies

We estimated the abundance of humpback whales in the North Pacific by capture‐recapture methods using over 18,000 fluke identification photographs collected in 2004–2006. Our best estimate of abundance was 21,808 (CV = 0.04). We estimated the biases in this value using a simulation model. Births and deaths, which violate the assumption of a closed population, resulted in a bias of +5.2%, exclusion of calves in samples resulted in a bias of ?10.5%, failure to achieve random geographic sampling resulted in a bias of ?0.4%, and missed matches resulted in a bias of +9.3%. Known sex‐biased sampling favoring males in breeding areas did not add significant bias if both sexes are proportionately sampled in the feeding areas. Our best estimate of abundance was 21,063 after accounting for a net bias of +3.5%. This estimate is likely to be lower than the true abundance due to two additional sources of bias: individual heterogeneity in the probability of being sampled (unquantified) and the likely existence of an unknown and unsampled breeding area (?8.7%). Results confirm that the overall humpback whale population in the North Pacific has continued to increase and is now greater than some prior estimates of prewhaling abundance.     

The motility of a human parasite, Toxoplasma gondii, is regulated by a novel lysine methyltransferase

Protozoa in the phylum Apicomplexa are a large group of obligate intracellular parasites. Toxoplasma gondii and other apicomplexan parasites, such as Plasmodium falciparum, cause diseases by reiterating their lytic cycle, comprising host cell invasion, parasite replication, and parasite egress. The successful completion of the lytic cycle requires that the parasite senses changes in its environment and switches between the non-motile (for intracellular replication) and motile (for invasion and egress) states appropriately. Although the signaling pathway that regulates the motile state switch is critical to the pathogenesis of the diseases caused by these parasites, it is not well understood. Here we report a previously unknown mechanism of regulating the motility activation in Toxoplasma, mediated by a protein lysine methyltransferase, AKMT (for Apical complex lysine (K) methyltransferase). AKMT depletion greatly inhibits activation of motility, compromises parasite invasion and egress, and thus severely impairs the lytic cycle. Interestingly, AKMT redistributes from the apical complex to the parasite body rapidly in the presence of egress-stimulating signals that increase [Ca²⁺] in the parasite cytoplasm, suggesting that AKMT regulation of parasite motility might be accomplished by the precise temporal control of its localization in response to environmental changes.     

Validity of self-reported cancer among a Japanese population: recent results from a population-based prospective study in Japan (JPHC Study)

The recent tendency of Japanese towards greater acceptance of being informed that they have cancer, along with the growing understanding and use of informed consent, appears to have improved the accuracy of self-reported cancer. To clarify the recent validity of self-reports, we measured the sensitivity and positive predictive value of self-reported cancer among a Japanese population. Using a 10-year follow-up questionnaire conducted in 2000-2004 and the cancer registry of the JPHC Study cohort (n=93,680), we calculated the sensitivity and positive predictive value of self-reported cancer diagnoses over 10 years. Sensitivity and positive predictive value of total self-reported cancer diagnoses were 53% and 60%, respectively, but varied by site, at 62% and 52% for stomach, 38% and 47% for colorectum, 57% and 46% for lung, 34% and 31% for liver, 82% and 58% for breast, and 59% and 22% for uterus, respectively. Sensitivity was considerably improved from that in the previous report (36%), which tested for 1990-1995, but was still not considered satisfactory. Self-reported diagnoses of cancer do not provide sufficient accuracy for the detection and classification of incident cancers. Our findings may be extrapolated to other Japanese populations.     

Evidence for genetically influenced caste determination in phylogenetically diverse species of the termite genus Reticulitermes

A number of social insect species have recently been shown to have genetically influenced caste determination (GCD), challenging the conventional view that caste determination should be strictly environmental. To date, GCD has been found in phylogenetically isolated species; examples of GCD being present in multiple species of a genus are lacking. Through crossing experiments of neotenic (juvenile) reproductives, we have recently provided the first evidence for a royal versus worker GCD in the termite Reticulitermes speratus. To elucidate whether this system is more widespread, we performed crossing experiments using three additional Reticulitermes species. Offspring caste and sex ratios were found to be highly similar to those found previously in R. speratus, raising the possibility that GCD was present in an ancestral lineage of Reticulitermes, and subsequently maintained throughout several episodes of speciation.     

Hydrothermal vent gastropods from the same family (Provannidae) harbour epsilon- and gamma-proteobacterial endosymbionts

The discovery of new hydrothermal vent systems in the back-arc basins of the Western Pacific revealed chemosynthesis-based faunal communities distinct from those of other vents. These vents are dominated by two related gastropods (Alviniconcha spp. and Ifremeria nautilei) that harbour symbiotic bacteria in their gills. We used comparative 16S ribosomal RNA (rRNA) gene sequencing and in situ hybridization with rRNA-targeted probes to characterize the bacterial symbionts of Alviniconcha sp. and I. nautilei from the Manus Basin in the Western Pacific. The analyses revealed that these two gastropod species, although affiliated with the same family, harbour phylogenetically distant chemosymbionts, suggesting independent origins of these endosymbioses. The I. nautilei endosymbiont clusters with sulfur-oxidizing bacteria within the gamma-Proteobacteria, as is the case for all previously characterized endosymbionts from a wide diversity of host taxa harbouring thioautotrophic prokaryotes. In contrast, the Alviniconcha endosymbiont is affiliated with sulfur-oxidizing bacteria within the epsilon-Proteobacteria. These results show that bacteria from the epsilon-Proteobacteria are also capable of forming endosymbiotic associations with marine invertebrates from chemosynthetic environments. More generally, the endosymbiotic lifestyle is now shown to be distributed throughout all recognized classes of the Proteobacteria.     

Differential localization of alternatively spliced hypoxanthine-xanthine-guanine phosphoribosyltransferase isoforms in Toxoplasma gondii

A unique feature of the Toxoplasma gondii purine salvage pathway is the expression of two isoforms of the hypoxanthine-xanthine-guanine phosophoribosyltransferase (HXGPRT) of the parasite encoded by a single genetic locus. These isoforms differ in the presence or absence of a 49-amino acid insertion (which is specified by a single differentially spliced exon) but exhibit similar substrate specificity, kinetic characteristics, and temporal expression patterns. To examine possible functional differences between the two HXGPRT isoforms, fluorescent protein fusions were expressed in parasites lacking the endogenous hxgprt gene. Immunoblot analysis of fractionated cell extracts and fluorescence microscopy indicated that HXGPRT-I (which lacks the 49-amino acid insertion) is found in the cytosol, whereas HXGPRT-II (which contains the insertion) localizes to the inner membrane complex (IMC) of the parasite. Simultaneous expression of both isoforms resulted in the formation of hetero-oligomers, which distributed between the cytosol and IMC. Chimeric constructs expressing N-terminal peptides from either isoform I (11 amino acids) or isoform II (60 amino acids) fused to a chloramphenicol acetyl transferase (CAT) reporter demonstrated that the N-terminal domain of isoform II is both necessary and sufficient for membrane association. Metabolic labeling experiments with transgenic parasites showed that isoform II or an isoform II-CAT fusion protein (but not isoform I or isoform I-CAT) incorporate [(3)H]palmitate. Mutation of three adjacent cysteine residues within the isoform II-targeting domain to serines blocked both palmitate incorporation and IMC attachment without affecting enzyme activity, demonstrating that acylation of N-terminal isoform II cysteine residues is responsible for the association of HXGPRT-II with the IMC.     

Mammalian phospholipase Czeta induces oocyte activation from the sperm perinuclear matrix

Mammalian sperm-borne oocyte activating factor (SOAF) induces oocyte activation from a compartment that engages the oocyte cytoplasm, but it is not known how. A SOAF-containing extract (SE) was solubilized from the submembrane perinuclear matrix, a domain that enters the egg. SE initiated activation sufficient for full development. Microinjection coupled to tandem mass spectrometry enabled functional correlation profiling of fractionated SE without a priori assumptions about its chemical nature. Phospholipase C-zeta (PLCzeta) correlated absolutely with activating ability. Immunoblotting confirmed this and showed that the perinuclear matrix is the major site of 72-kDa PLCzeta. Oocyte activation was efficiently induced by 1.25 fg of sperm PLCzeta, corresponding to a fraction of one sperm equivalent (approximately 0.03). Immunofluorescence microscopy localized sperm head PLCzeta to a post-acrosomal region that becomes rapidly exposed to the ooplasm following gamete fusion. This multifaceted approach suggests a mechanism by which PLCzeta originates from an oocyte-penetrating assembly--the sperm perinuclear matrix--to induce mammalian oocyte activation at fertilization.