Isolation of a neuraminidase gene from Actinomyces viscosus T14V.

A genomic library of Actinomyces viscosus T14V DNA in lambda gt11 was screened for expression of neuraminidase activities. Four recombinant clones were detected that gave blue fluorescence upon incubation with a fluorogenic substrate, 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Of these, two were identical, and all of the neuraminidase-positive clones shared a common 3.4-kbp DNA region. Expression of the enzyme activities in Escherichia coli carrying the cloned DNA was independent of the lacZ promoter of the vector. Maxicell analysis revealed that the 3.4-kbp DNA insert directed synthesis of a protein with an apparent molecular mass of 100,000 Da. The protein from cell extracts of E. coli clones migrated as a single band that stained for enzyme activity after electrophoresis in a nondissociating polyacrylamide gel. Moreover, human erythrocytes incubated previously with cell lysates from neuraminidase-positive E. coli were hemagglutinated by Actinomyces spp. The enzyme expressed by E. coli was active on substrates containing alpha-2,3 and alpha-2,6 ketosidic linked sialyl residues. Similar substrate specificities were obtained for both the extracellular and cell-associated neuraminidases from A. viscosus T14V. The 3.4-kbp insert hybridized to DNA fragments in a Southern blot containing A. viscosus T14V chromosomal DNA that had been digested with various restriction endonucleases. Data from hybridization studies show that A. viscosus T14V contains a single copy of the neuraminidase gene.     

Irreversible inactivation of adenylyl cyclase by the "P"-site agonist 2',5'-dideoxy-,3'-p-fluorosulfonylbenzoyl adenosine

2',5'-Dideoxy,3'-p-fluorosulfonylbenzoyl Adenosine (2',5'-dd3'-FSBA) was synthesized and found to be an agonist and affinity label for the "P"-site of adenylyl cyclase. This compound irreversibly inactivated both a crude detergent-dispersed adenylyl cyclase from rat brain and the partially purified enzyme from bovine brain. The irreversible inactivation by 100 to 200 microM 2',5'-dd3'-FSBA was blocked in a concentration-dependent manner by several established P-site inhibitors of adenylyl cyclase, 2',5'-dideoxyadenosine, 2'-d3'-AMP, adenosine, and 2'-deoxyadenosine, but not by inosine, N6-(phenylisopropyl)adenosine, adenine, 2'-d3':5'-cAMP, or 5'-AMP, agents known not to act at the P-site. Moreover, irreversible inactivation by 2',5'-dd3'-FSBA occurred in the presence of ATP at concentrations up to 3 mM, making it unlikely that inactivation was due to an effect on the enzyme's catalytic site. Adenylyl cyclase was also irreversibly inactivated by 5'-FSBA, although modestly (less than 20%) and apparently nonspecifically. Dithiothreitol protected the enzyme from irreversible inactivation by 2',5'-dd3'-FSBA, but reversible inhibition of the enzyme was still observed, although with reduced potency. When 2 mM dithiothreitol was added after a 30-min preincubation with 2',5'-dd3'-FSBA, the rat brain enzyme was partially (approximately 80%) reactivated. The data suggest that 2',5'-dd3'-FSBA may irreversibly inactivate adenylyl cyclase by reacting with a cysteinyl moiety in proximity to the P-site domain of the enzyme. These data together with results of studies of P-site inhibition kinetics published elsewhere (Johnson, R. A., and Shoshani, I. (1990) J. Biol. Chem. 265, 11595-11600) strongly suggest that the P-site and catalytic site are distinct domains on the enzyme. 2',5'-dd3'-FSBA, and especially its radiolabeled analog, should prove to be a useful probe for structural studies of adenylyl cyclase, particularly with regard to the P-site.     

A processing product of the Plasmodium falciparum reticulocyte binding protein RH1 shows a close association with AMA1 during junction formation

Plasmodium falciparum responsible for the most virulent form of malaria invades human erythrocytes through multiple ligand‐receptor interactions. The P. falciparum reticulocyte binding protein homologues (PfRHs) are expressed at the apical end of merozoites and form interactions with distinct erythrocyte surface receptors that are important for invasion. Here using a range of monoclonal antibodies (mAbs) against different regions of PfRH1 we have investigated the role of PfRH processing during merozoite invasion. We show that PfRH1 gets differentially processed during merozoite maturation and invasion and provide evidence that the different PfRH1 processing products have distinct functions during invasion. Using in‐situ Proximity Ligation and FRET assays that allow probing of interactions at the nanometre level we show that a subset of PfRH1 products form close association with micronemal proteins Apical Membrane Antigen 1 (AMA1) in the moving junction suggesting a critical role in facilitating junction formation and active invasion. Our data provides evidence that time dependent processing of PfRH proteins is a mechanism by which the parasite is able to regulate distinct functional activities of these large processes. The identification of a specific close association with AMA1 in the junction now may also provide new avenues to target these interactions to prevent merozoite invasion.     

Phospholipid transfer protein (PLTP) mRNA expression is stimulated by developing embryos in the oviduct

In mammal, fertilization and early preimplantation embryo development occurs in the oviduct. Evidence is accumulating that the oviductal epithelia secrete various biomolecules to the lumen during the secretory phase of the estrus cycle to enhance embryo development. This secretory activity of the oviduct is under the regulation of steroid hormones. Observations also suggested that the gametes and embryos modulate the physiology and gene-expressing pattern of the oviduct. However, the underlying molecular changes remain elusive. We hypothesize that the developing embryos interact with the surrounding environment and affect the gene expression patterns of the oviduct, thereby modulating the oviductal secretory activity conducive to the preimplantation embryo development. To test this hypothesis, suppression subtractive hybridization (SSH) was used to compare the gene expressions in mouse oviduct containing transferred in vitro cultured preimplantation embryos with that of oviduct containing oocytes during the preimplantation period. We reported here the identification and characterization of phospholipids transfer protein (PLTP), which is highly expressed in the embryo-containing oviduct and localized at the oviductal epithelium by in situ hybridization. PLTP contains signal peptide putative for secretory function. More importantly, PLTP mRNA increases in the oviductal epithelia of pregnant, but not pseudo-pregnant mice when assayed by real-time PCR. Taken together, our data suggested that PLTP may play important role(s) during in vivo preimplantation embryo development. This molecule would be a target to delineate the mechanisms and the roles of oviductal secretory proteins on early embryonic development.     

Human beta-endorphin. Immunoreactivity of synthetic analogs with various chain lengths.

Immunoreactivity of synthetic human beta-endorphin analogs with various chain lengths has been examined using a specific radioimmunoassay. It was found that beta-endorphin-(1--21) and analogs of shortened chain exhibit no immunoreactivity, whereas beta-endorphin-(1--15) possesses significant in vitro opiate activity. It appears that immunoreactivity of beta-endorphin resides in the COOH-terminal segment of residues (22--31). The data also show the lack of correlation between opiate and immunological activities of beta-endorphin.     

Modeling interactions between voltage-gated Ca2+ channels and KCa1.1 channels

High voltage-activated (HVA) Cav channels form complexes with KCa1.1 channels, allowing reliable activation of KCa1.1 current through a nanodomain interaction. We recently found that low voltage-activated Cav3 calcium channels also create KCa1.1-Cav3 complexes. While coimmunoprecipitation studies again supported a nanodomain interaction, the sensitivity to calcium chelating agents was instead consistent with a microdomain interaction. A computational model of the KCa1.1-Cav3 complex suggested that multiple Cav3 channels were necessary to activate KCa1.1 channels, potentially causing the KCa1.1-Cav3 complex to be more susceptible to calcium chelators. Here, we expanded the model and compared it to a KCa1.1-Cav2.2 model to examine the role of Cav channel conductance and kinetics on KCa1.1 activation. As found for direct recordings, the voltage-dependent and kinetic properties of Cav3 channels were reflected in the activation of KCa1.1 current, including transient activation from lower voltages than other KCa1.1-Cav complexes. Substantial activation of KCa1.1 channels required the concerted activity of several Cav3.2 channels. Combined with the effect of EGTA, these results suggest that the Ca²⁺ domains of several KCa1.1-Cav3 complexes need to cooperate to generate sufficient [Ca²⁺]_i, despite the physical association between KCa1.1 and Cav3 channels. By comparison, Cav2.2 channels were twice as effective at activating KCa1.1 channels and a single KCa1.1-Cav2.2 complex would be self-sufficient. However, even though Cav3 channels generate small, transient currents, the regulation of KCa1.1 activity by Cav3 channels is possible if multiple complexes cooperate through microdomain interactions.     

Moderate Alcohol Use and Cardiovascular Disease from Mendelian Randomization

Background

Observational studies show moderate alcohol use negatively associated with ischemic heart disease (IHD) and cardiovascular disease (CVD). However, healthier attributes among moderate users compared to never users may confound the apparent association. A potentially less biased way to examine the association is Mendelian randomization, using alcohol metabolizing genes which influence alcohol use.

Methods

We used instrumental variable analysis with aldehyde dehydrogenase 2 (ALDH2) genotypes (AA/GA/GG) as instrumental variables for alcohol use to examine the association of alcohol use (10 g ethanol/day) with CVD risk factors (blood pressure, lipids and glucose) and morbidity (self-reported IHD and CVD) among men in the Guangzhou Biobank Cohort Study.

Results

ALDH2 genotypes were a credible instrument for alcohol use (F-statistic 74.6). Alcohol was positively associated with HDL-cholesterol (0.05 mmol/L per alcohol unit, 95% confidence interval (CI) 0.02 to 0.08) and diastolic blood pressure (1.15 mmHg, 95% CI 0.23 to 2.07) but not with systolic blood pressure (1.00 mmHg, 95% CI -0.74 to 2.74), LDL-cholesterol (0.03 mmol/L, 95% CI -0.03 to 0.08), log transformed triglycerides (0.03 mmol/L, 95% CI -0.01 to 0.08) or log transformed fasting glucose (0.01 mmol/L, 95% CI -0.006 to 0.03), self-reported CVD (odds ratio (OR) 0.98, 95% CI 0.76 to 1.27) or self-reported IHD (OR 1.10, 95% CI 0.83 to 1.45).

Conclusion

Low to moderate alcohol use among men had the expected effects on most CVD risk factors but not fasting glucose. Larger studies are needed to confirm the null associations with IHD, CVD and fasting glucose.     

The evolution of floral nectaries in Disa (Orchidaceae: Disinae): recapitulation or diversifying innovation?

Background and Aims

The Orchidaceae have a history of recurring convergent evolution in floral function as nectar production has evolved repeatedly from an ancestral nectarless state. However, orchids exhibit considerable diversity in nectary type, position and morphology, indicating that this convergence arose from alternative adaptive solutions. Using the genus Disa, this study asks whether repeated evolution of floral nectaries involved recapitulation of the same nectary type or diversifying innovation. Epidermis morphology of closely related nectar-producing and nectarless species is also compared in order to identify histological changes that accompanied the gain or loss of nectar production.

Methods

The micromorphology of nectaries and positionally equivalent tissues in nectarless species was examined with light and scanning electron microscopy. This information was subjected to phylogenetic analyses to reconstruct nectary evolution and compare characteristics of nectar-producing and nectarless species.

Key Results

Two nectary types evolved in Disa. Nectar exudation by modified stomata in floral spurs evolved twice, whereas exudation by a secretory epidermis evolved six times in different perianth segments. The spur epidermis of nectarless species exhibited considerable micromorphological variation, including strongly textured surfaces and non-secreting stomata in some species. Epidermis morphology of nectar-producing species did not differ consistently from that of rewardless species at the magnifications used in this study, suggesting that transitions from rewardlessness to nectar production are not necessarily accompanied by visible morphological changes but only require sub-cellular modification.

Conclusions

Independent nectary evolution in Disa involved both repeated recapitulation of secretory epidermis, which is present in the sister genus Brownleea, and innovation of stomatal nectaries. These contrasting nectary types and positional diversity within types imply weak genetic, developmental or physiological constraints in ancestral, nectarless Disa. Such functional convergence generated by morphologically diverse solutions probably also underlies the extensive diversity of nectary types and positions in the Orchidaceae.     

Paediatric brain tissue properties measured with magnetic resonance elastography

Biomechanics and Modeling in Mechanobiology - The aim of this study is to characterise the stiffness of white and grey matter in paediatric subjects using magnetic resonance elastography (MRE) and...