Presence of a unique male-specific extension of C-terminus to the cytochrome c oxidase subunit II protein coded by the male-transmitted mitochondrial genome of Venustaconcha ellipsiformis (Bivalvia: Unionoidea)

Analyses of unionoidean bivalve male-transmitted (M) mtDNA genomes revealed an approximately 555 bp 3' coding extension to cox2. An antibody was generated against this predicted C-terminus extension to determine if the unique cox2 protein is expressed. Western blot and immunohistochemistry analyses demonstrated that the protein was predominantly expressed in testes. Weak expression was detected in other male tissues but the protein was not detected in female tissues. This is the first report documenting the expression of a cox2 protein with a long C-terminus in animals. Its universal presence in unionoidean bivalve testes suggests a functional significance for the protein.     

Acceptor substrate selectivity and kinetic mechanism of Bacillus subtilis TagA

Wall teichoic acids (WTAs) are anionic polymers that coat the cell walls of Gram-positive bacteria. Because they are essential for survival or virulence in many organisms, the enzymes involved in the biosynthesis of WTAs are attractive antibiotic targets. The first committed step in the WTA biosynthetic pathway in Bacillus subtilis is catalyzed by TagA, which transfers N-acetylmannosamine (ManNAc) to the C4 hydroxyl of a membrane-anchored N-acetylglucosaminyl diphospholipid (GlcNAc-pp-undecaprenyl, lipid I) to make ManNAc-beta-(1,4)-GlcNAc-pp-undecaprenyl (lipid II). We have previously shown that TagA utilizes an alternative substrate containing a saturated C(13)H(27) lipid chain. Here we use unnatural substrates and products to establish the lipid preferences of the enzyme and to characterize the kinetic mechanism. We report that TagA is a metal ion-independent glycosyltransferase that follows a steady-state ordered Bi-Bi mechanism in which UDP-ManNAc binds first and UDP is released last. TagA shares homology with a large family of bacterial glycosyltransferases, and the work described here should facilitate structural analysis of the enzyme in complex with its substrates.     

Life in the slow lane revisited: ontogenetic separation between chimpanzees and humans

This study investigates the evolution of human growth by analyzing differences in body mass growth trajectories among three populations: the Ache of eastern Paraguay, the US (NHANES, 1999-2000), and captive chimpanzees. The relative growth statistic "A" from the mammalian growth law is allowed to vary with age and proves useful for comparing growth across different ages, populations, and species. We demonstrate ontogenetic separation between chimpanzees and humans, and show that interspecific differences are robust to variable environmental conditions. The human pattern of slow growth during the lengthened period from weaning to the beginning of the adolescent growth spurt is found among the Ache (low energy availability and high disease load) and also in the US (high energy availability and low disease load). The human growth pattern contrasts with that of the chimpanzee, where absolute growth rates and relative "A" values are faster and less prolonged. We suggest that selection has acted to decrease human growth rates to allow more time for increased cognitive development with lower body-maintenance costs.     

The slow Wallerian degeneration protein, WldS, binds directly to VCP/p97 and partially redistributes it within the nucleus

Slow Wallerian degeneration (Wld(S)) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD(+) synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with Wld(S) targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. Wld(S) lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD(+) synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of Wld(S) protein influence the intranuclear location of both ubiquitin proteasome and NAD(+) synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP.     

The effects of caffeine on athletic agility

Caffeine has been shown to improve sprint time, anaerobic power, and reaction time, all integral aspects of agility. The purpose of this study was to determine whether an acute caffeine dose would enhance agility and anaerobic power. Sixteen subjects participated in a randomized, double-blind experiment and performed the proagility run and the 30-second Wingate test 60 minutes after ingestion of caffeine (6 mg.kg(-1)) or placebo. No significant change was observed in the proagility run after caffeine ingestion compared with placebo. Also, no significant change was observed in peak power, mean power, or percent power decrease. Agility is an integral component of athletic skill and any reasonable method for enhancing agility would benefit active individuals. However, results from this study indicate that a 6 mg.kg(-1) dose of caffeine does not impact agility as measured by the proagility run test or power output as measured by the 30-second Wingate test in recreationally active young adult males who are not habituated to caffeine.     

Ultrastructural description of Breviata anathema, n. gen., n. sp., the organism previously studied as "Mastigamoeba invertens"

An understanding of large-scale eukaryotic evolution is beginning to crystallise, as molecular and morphological data demonstrate that eukaryotes fall into six major groups. However, there are several taxa of which the affinities are yet to be resolved, and for which there are only either molecular or morphological data. One of these is the amoeboid flagellate Mastigamoeba invertens. This organism was originally misidentified and studied as a pelobiont using molecular data. We present its first light microscopical and ultrastructural characterisation. We demonstrate that it does not show affinities to the amoebozoan pelobionts, because unlike the pelobionts, it has a double basal body and two flagellar roots, a classical Golgi stack, and a large branching double membrane-bound organelle. Phylogenetic analyses of small subunit ribosomal RNA suggest an affinity with the apusomonads, when a covariotide correction for rate heterogeneity is used. We suggest that previous molecular results have been subject to artefacts from an insufficient correction for rate heterogeneity. We propose a new name for the taxon, Breviata anathema; and the unranked, apomorphy-based name "Breviates" for Breviata and its close relatives.     

Cyclin-dependent kinase inhibitors enhance the resolution of inflammation by promoting inflammatory cell apoptosis

Apoptosis is essential for clearance of potentially injurious inflammatory cells and subsequent efficient resolution of inflammation. Here we report that human neutrophils contain functionally active cyclin-dependent kinases (CDKs), and that structurally diverse CDK inhibitors induce caspase-dependent apoptosis and override powerful anti-apoptosis signals from survival factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF). We show that the CDK inhibitor R-roscovitine (Seliciclib or CYC202) markedly enhances resolution of established neutrophil-dependent inflammation in carrageenan-elicited acute pleurisy, bleomycin-induced lung injury, and passively induced arthritis in mice. In the pleurisy model, the caspase inhibitor zVAD-fmk prevents R-roscovitine-enhanced resolution of inflammation, indicating that this CDK inhibitor augments inflammatory cell apoptosis. We also provide evidence that R-roscovitine promotes apoptosis by reducing concentrations of the anti-apoptotic protein Mcl-1. Thus, CDK inhibitors enhance the resolution of established inflammation by promoting apoptosis of inflammatory cells, thereby demonstrating a hitherto unrecognized potential for the treatment of inflammatory disorders.     

Protein phosphatase activity is necessary for myofibrillogenesis

During myofibrillogenesis, myosin light-chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin II, enabling patterned assembly of myosin thick filaments. A protein phosphatase (PP) has been shown to mediate RLC dephosphorylation in adult smooth and striated muscle. A role for PP activity in regulating myofibrillogenesis during embryonic development, however, has not been investigated. Tautomycin (TM) was used to inhibit both PP1 and PP2A activities, whereas okadaic acid (OA) and fostriecin (FOS) were used to inhibit PP2A. TM affected both actin and myosin assembly at 5nM; the IC50 value was 20 and 8.5nM, respectively. In contrast, OA applied at 10 times above its reported Ki for PP2A caused no significant disruption. There was also no disruption when FOS was applied at a concentration 30 times above its reported Ki for PP2A. Thus, our results suggest a primary role for PP1 isoforms during myofibrillogenesis. Although rho kinase (RK) regulates PP activity in embryonic smooth and cardiac muscle, application of the RK inhibitor Y27632 did not affect actin or myosin assembly in skeletal myocytes. Collectively, our pharmacological results suggest that PP1 is involved in dynamic regulation of RLC phosphorylation. To specifically test involvement of the myosin-targeted isoform (PP1M), we used a morpholino antisense approach to knock down the myosin targeting (M) subunit of PP1. Embryos injected with morpholino targeted to the 110-kDa M targeting subunit had fewer somites, and myosin organization was significantly perturbed. The combined pharmacological and molecular results suggest a dynamic equilibrium between MLCK and PP1M activities is required for proper myofibrillogenesis.     

Predictors of belief that genetic test information about hemochromatosis should be shared with family members

We queried 101,951 white, Hispanic, black, Asian, American Indian (i.e., American Indian or Alaska Native in the United States and North American Indian, Metis, or Inuit in Canada) and Pacific Islander (including Native Hawaiian) adults who agreed to be genotypically and phenotypically screened for hemochromatosis as part of the Hemochromatosis and Iron Overload Screening (HEIRS) study about their views on sharing genetic test information with family members. Multiple logistic regression (adjusting for study site, age group, race/ethnicity, preferred language, gender, education group, income group, SF-36 General Health and Mental Health subscales, perceived benefits and limitations of genetic testing, and belief that genetic testing is a good idea) evaluated independent predictors of responding "Strongly Agree" or "Agree" versus "Disagree" or "Strongly Disagree" to the statement "Information about a person's genetic risk should be shared with family members". Agreement that genetic risk information should be shared with family members was high (93% in the overall sample of 78,952 who answered this question), but differed among racial/ethnic groups. Hispanics were significantly less likely to agree that genetic test information should be shared with family members (i.e., 88% versus 92% or more among all other ethnicities). The relationship of perceived limitations and benefits of testing, gender, and age group to the belief that information should be shared differed among racial/ethnic groups, with Spanish-preferring Hispanics being the most different from other subgroups.