Reproductive hormones and bone mineral density in women runners.

We examined the relationships among reproductive hormone concentrations and bone mineral density (BMD) in 43 women runners classified as eumenorrheic (n = 24), oligomenorrheic (n = 8), or amenorrheic (n = 11). Results were compared with a eumenorrheic nonrunner control group (n = 11). Serum 17 beta-estradiol, progesterone, and dehydroepiandrosterone sulfate concentrations were determined in daily blood samples for 21 days, and integrated concentrations (areas under the curve) were calculated. BMD was assessed at the lumbar spine and proximal femur by dual-photon absorptiometry. As expected, 17 beta-estradiol, progesterone, and lumbar spine BMD were higher in the control and eumenorrheic runner groups than in the oligomenorrheic and amenorrheic runner groups (P less than 0.05). Progesterone concentration was significantly correlated with lumbar spine BMD in the eumenorrheic runners (r = 0.61). None of the steroid hormones was significantly related to BMD in the oligomenorrheic/amenorrheic group. The present data suggest that circulating levels of gonadal steroid hormones affect axial BMD in eumenorrheic runners.     

Galactomannanases Man2 and Man5 from Thermotoga species: growth physiology on galactomannans, gene sequence analysis, and biochemical properties of recombinant enzymes

The enzymatic hydrolysis of mannan-based hemicelluloses is technologically important for applications ranging from pulp and paper processing to food processing to gas and oil well stimulation. In many cases, thermostability and activity at elevated temperatures can be advantageous. To this end, the genes encoding beta-mannosidase (man2) and beta-mannanase (man5) from the hyperthermophilic bacteria Thermotoga neapolitana 5068 and Thermotoga maritima were isolated, cloned, and expressed in Escherichia coli. The amino acid sequences for the mannosidases from these organisms were 77% identical and corresponded to proteins with an M(r) of approximately 92 kDa. The translated nucleotide sequences for the beta-mannanase genes (man5) encoded polypeptides with an M(r) of 76 kDa that exhibited 84% amino acid sequence identity. The recombinant versions of Man2 and Man5 had similar respective biochemical and biophysical properties, which were also comparable to those determined for the native versions of these enzymes in T. neapolitana. The optimal temperature and pH for the recombinant Man2 and Man5 from both organisms were approximately 90 degrees C and 7.0, respectively. The presence of Man2 and Man5 in these two Thermotoga species indicates that galactomannan is a potential growth substrate. This was supported by the fact that beta-mannanase and beta-mannosidase activities were significantly stimulated when T. neapolitana was grown on guar or carob galactomannan. Maximum cell densities increased by at least tenfold when either guar or carob galactomannan was added to the growth medium. For T. neapolitana grown on guar at 83 degrees C, Man5 was secreted into the culture media, whereas Man2 was intracellular. These localizations were consistent with the presence and lack of signal peptides for Man5 and Man2, respectively. The identification of the galactomannan-degrading enzymes in these Thermotoga species adds to the list of biotechnologically important hemicellulases produced by members of this hyperthermophilic genera.     

Novel complexes of guanylate cyclase with heat shock protein 90 and nitric oxide synthase

Soluble guanylate cyclase (sGC) is an important downstream intracellular target of nitric oxide (NO) that is produced by endothelial NO synthase (eNOS) and inducible NO synthase (iNOS). In this study, we demonstrate that sGC exists in a complex with eNOS and heat shock protein 90 (HSP90) in aortic endothelial cells. In addition, we show that in aortic smooth muscle cells, sGC forms a complex with HSP90. Formation of the sGC/eNOS/HSP90 complex is increased in response to eNOS-activating agonists in a manner that depends on HSP90 activity. In vitro binding assays with glutathione S-transferase fusion proteins that contain the alpha- or beta-subunit of sGC show that the sGC beta-subunit interacts directly with HSP90 and indirectly with eNOS. Confocal immunofluorescent studies confirm the subcellular colocalization of sGC and HSP90 in both endothelial and smooth muscle cells. Complex formation of sGC with HSP90 facilitates responses to NO donors in cultured cells (cGMP accumulation) as well as in anesthetized rats (hypotension). These complexes likely function to stabilize sGC as well as to provide directed intracellular transfer of NO from NOS to sGC, thus preventing inactivation of NO by superoxide anion and formation of peroxynitrite, which is a toxic molecule that has been implicated in the pathology of several vascular diseases.     

Exploring Nitrilase Sequence Space for Enantioselective Catalysis

Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10⁶ to 10¹⁰ members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.     

Pharmacological and Genetic Targeting of the PI4KA Enzyme Reveals Its Important Role in Maintaining Plasma Membrane Phosphatidylinositol 4-Phosphate and Phosphatidylinositol 4,5-Bisphosphate Levels

Phosphatidylinositol 4-kinase type IIIα (PI4KA) is a host factor essential for hepatitis C virus replication and hence is a target for drug development. PI4KA has also been linked to endoplasmic reticulum exit sites and generation of plasma membrane phosphoinositides. Here, we developed highly specific and potent inhibitors of PI4KA and conditional knock-out mice to study the importance of this enzyme in vitro and in vivo. Our studies showed that PI4KA is essential for the maintenance of plasma membrane phosphatidylinositol 4,5-bisphosphate pools but only during strong stimulation of receptors coupled to phospholipase C activation. Pharmacological blockade of PI4KA in adult animals leads to sudden death closely correlating with the drug''s ability to induce phosphatidylinositol 4,5-bisphosphate depletion after agonist stimulation. Genetic inactivation of PI4KA also leads to death; however, the cause in this case is due to severe intestinal necrosis. These studies highlight the risks of targeting PI4KA as an anti-hepatitis C virus strategy and also point to important distinctions between genetic and pharmacological studies when selecting host factors as putative therapeutic targets.     

Protein Interaction between Ameloblastin and Proteasome Subunit α Type 3 Can Facilitate Redistribution of Ameloblastin Domains within Forming Enamel

Enamel is a bioceramic tissue composed of thousands of hydroxyapatite crystallites aligned in parallel within boundaries fabricated by a single ameloblast cell. Enamel is the hardest tissue in the vertebrate body; however, it starts development as a self-organizing assembly of matrix proteins that control crystallite habit. Here, we examine ameloblastin, a protein that is initially distributed uniformly across the cell boundary but redistributes to the lateral margins of the extracellular matrix following secretion thus producing cell-defined boundaries within the matrix and the mineral phase. The yeast two-hybrid assay identified that proteasome subunit α type 3 (Psma3) interacts with ameloblastin. Confocal microscopy confirmed Psma3 co-distribution with ameloblastin at the ameloblast secretory end piece. Co-immunoprecipitation assay of mouse ameloblast cell lysates with either ameloblastin or Psma3 antibody identified each reciprocal protein partner. Protein engineering demonstrated that only the ameloblastin C terminus interacts with Psma3. We show that 20S proteasome digestion of ameloblastin in vitro generates an N-terminal cleavage fragment consistent with the in vivo pattern of ameloblastin distribution. These findings suggest a novel pathway participating in control of protein distribution within the extracellular space that serves to regulate the protein-mineral interactions essential to biomineralization.     

Disruption of rolandic gamma-band functional connectivity by seizures is associated with motor impairments in children with epilepsy

Although children with epilepsy exhibit numerous neurological and cognitive deficits, the mechanisms underlying these impairments remain unclear. Synchronization of oscillatory neural activity in the gamma frequency range (>30 Hz) is purported to be a mechanism mediating functional integration within neuronal networks supporting cognition, perception and action. Here, we tested the hypothesis that seizure-induced alterations in gamma synchronization are associated with functional deficits. By calculating synchrony among electrodes and performing graph theoretical analysis, we assessed functional connectivity and local network structure of the hand motor area of children with focal epilepsy from intracranial electroencephalographic recordings. A local decrease in inter-electrode phase synchrony in the gamma bands during ictal periods, relative to interictal periods, within the motor cortex was strongly associated with clinical motor weakness. Gamma-band ictal desychronization was a stronger predictor of deficits than the presence of the seizure-onset zone or lesion within the motor cortex. There was a positive correlation between the magnitude of ictal desychronization and impairment of motor dexterity in the contralateral, but not ipsilateral hand. There was no association between ictal desynchronization within the hand motor area and non-motor deficits. This study uniquely demonstrates that seizure-induced disturbances in cortical functional connectivity are associated with network-specific neurological deficits.     

Histidyl-Transfer Ribonucleic Acid Synthetase in Positive Control of the Histidine Operon in Salmonella typhimurium

Autolysin-like enzymes appear to be responsible for cell separation in Agmenellum quadruplicatum. Mutants that are impaired in cell separation and grow as chains exhibit reduced cell lytic activity. Lysozyme, extracted autolysin, and antibiotics that affect peptidoglycan synthesis phenotypically suppress chain formation. Various aspects of the regulation of the cell separation process were also examined. Studies involving antibiotic inhibitors of macromolecular synthesis and general growth inhibitors provided no evidence for the active regulation of the cell separation process during the latter portion of the division cycle. Evidence was obtained, however, for the partial restriction of peptidogly-can hydrolysis by unknown secondary modifications. The thin electron-dense layer of peptidoglycan along the sides of cells was much more resistant to hydrolysis by egg-white lysozyme than was the septum between daughter cells. The middle portion of the septum was more sensitive than was the layer immediately adjacent to the cytoplasmic membrane. Under conditions that would not osmotically stabilize spheroplasts, lysozyme facilitates rapid cell separation in chain-forming mutants with little leakage of cellular protein or loss of viability.     

Mammary growth patterns in guinea pigs during puberty, pregnancy and lactation

Mammary gland growth patterns were studied in 110 guinea pigs during the growth phase, pregnancy and lactation. Body weight changes were studied and, in addition, mammary indices were wet weight, dry fat-free tissue (DFFT), deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Statistical analyses were mathematical regression models to best fit the actual data. These included linear, quadratic, cubic, and several forms of exponential regression models. Data were separated into growth phase (60 guinea pigs in 10 age groups), pregnancy (20 guinea pigs in 4 groups), and lactation (30 guinea pigs in 6 groups). Data during pregnancy and the first 5 days of lactation were pooled and analyzed also because mammary growth continued beyond pregnancy to Day 5 of lactation. Mammary wet weight increased according to a cubic expression in the growth phase, while mammary DFFT, DNA and RNA were rectilinear through 200 days of age. During pregnancy and the first 5 days of lactation, mammary growth parameters followed the pattern of an exponential equation. Daily rates of increase for mammary DFFT and DNA were twice the rate for mammary wet weight. During lactation, mammary gland indices increased to Day 5 and then decreased gradually from Day 10 to Day 20. The best mathematical models for these change were those which are used to describe lactation curves, but all mammary gland indices decreased later and more gradually than milk production. Comparisons in growth rates of guinea pig mammary glands were made with those published for dairy goats and dairy cows. Rates of mammary DNA changed inversely to lengths of gestation in these 3 species.