Regulation of cyclic guanosine monophosphate-dependent protein kinase in human uterine tissues during the menstrual cycle

Contractility of uterine smooth muscle is essential for the cyclic shedding of the endometrial lining and also for expulsion of the fetus during parturition. The nitric oxide (NO)-cGMP signaling pathway is involved in smooth muscle relaxation. The downstream target of this pathway essential for decreasing cytoplasmic calcium and muscle tone is the cGMP-dependent protein kinase (PKG). The present study was undertaken to localize expression of PKG in tissues of the female reproductive tract and to test the hypothesis that uterine smooth muscle PKG levels vary with the human menstrual cycle. Immunohistochemistry was used to localize PKG in myometrium, cervix, and endometrium obtained during proliferative and secretory phases. The PKG was localized to uterine and vascular smooth muscle cells in myometrium, stromal cells in endometrium, and a small percentage of cervical stromal cells. Using Western blot analysis and protein kinase activity assays, the expression of PKG was reduced significantly in progesterone-dominated uteri compared with myometrium from postmenopausal women or women in the proliferative phase. These findings support a role for PKG in the control of uterine and vascular smooth muscle contractility during the menstrual cycle.     

Integrin alpha v beta 3 binds a unique non-RGD site near the C-terminus of human tropoelastin

Tropoelastin is the soluble precursor of the essential resilient connective tissue protein elastin. We examined the binding of integrin alpha(v)beta(3) to tropoelastin. In quantitative colorimetric solid-phase assays, purified alpha(v)beta(3) demonstrated saturable, divalent cation-dependent, single-site binding behavior on tropoelastin with a dissociation constant of 3.8 +/- 0.9 nM in the presence of 1 mM Mn(2+) which increased to 23 +/- 5 nM in the presence of 1 mM Ca(2+). Association with alpha(v)beta(3) was localized to the C-terminal 16 residues of tropoelastin, encompassing the region encoded by exon 36. This region comprises a unique disulfide loop in tropoelastin that is not essential for the interaction. This is the first identification of a specific, single binding site on tropoelastin and the first observation of direct binding of an integrin to a tropoelastin domain.     

The cover of living and dead corals from airborne remote sensing

Trends in coral cover are widely used to indicate the health of coral reefs but are costly to obtain from field survey over large areas. In situ studies of reflected spectra at the coral surface show that living and recently dead colonies can be distinguished. Here, we investigate whether such spectral differences can be detected using an airborne remote sensing instrument. The Compact Airborne Spectrographic Imager (Itres Research Ltd, Canada) was flown in two configurations: 10 spectral bands with 1-m² pixels and 6 spectral bands with 0.25-m² pixels. First, we show that an instrument with 10 spectral bands possesses adequate spectral resolution to distinguish living Porites, living Pocillopora spp., partially dead Porites, recently dead Porites (total colony mortality within 6 months), old dead (>6 months) Porites, Halimeda spp., and coralline red algae when there is no water column to confuse spectra. All substrata were distinguished using fourth-order spectral derivatives around 538 nm and 562 nm. Then, at a shallow site (Tivaru) at Rangiroa Atoll, Tuamotu Archipelago (French Polynesia), we show that live and dead coral can be distinguished from the air to a depth of at least 4 m using first- and fourth-order spectral derivatives between 562–580 nm. However, partially dead and recently dead Porites colonies could not be distinguished from an airborne platform. Spectral differences among substrata are then exploited to predict the cover of reef substrata in ten 25-m² plots at nearby Motu Nuhi (max depth 8 m). The actual cover in these plots was determined in situ using quadrats with a 0.01-m² grid. Considerable disparity occurred between field and image-based measures of substrate cover within individual 25-m² quadrats. At this small scale, disparity, measured as the absolute difference in cover between field and remote-sensing methods, reached 25% in some substrata but was always less than 10% for living coral (99% of which consisted of Porites spp.). At the scale of the reef (all ten 25-m² quadrats), however, disparities in percent cover between imagery and field data were less than 10% for all substrata and extremely low for some classes (e.g. <3% for living Porites, recently dead Porites and Halimeda). The least accurately estimated substrata were sand and coralline red algae, which were overestimated by absolute values 7.9% and 6.6%, respectively. The precision of sampling was similar for field and remote-sensing methods: field methods required 19 plots to detect a 10% difference in coral cover among three reefs with a statistical power of 95%. Remote-sensing methods required 21 plots. However, it took 1 h to acquire imagery over 92,500 m² of reef, which represents 3,700 plots of 25 m² each, compared with 3 days to survey 10 such plots underwater. There were no significant differences in accuracy between 1-m² and 0.25-m² image resolutions, suggesting that the advantage of using smaller pixels is offset by reduced spectral information and an increase in noise (noise was observed to be 1.6–1.8 times greater in 0.25-m² pixels). We show that airborne remote sensing can be used to monitor coral and algal cover over large areas, providing that water is shallow and clear, and that brown fleshy macroalgae are scarce, that depth is known independently (e.g. from sonar survey).     

Choline acetyltransferase structure reveals distribution of mutations that cause motor disorders

Choline acetyltransferase (ChAT) synthesizes acetylcholine in neurons and other cell types. Decreases in ChAT activity are associated with a number of disease states, and mutations in ChAT cause congenital neuromuscular disorders. The crystal structure of ChAT reported here shows the enzyme divided into two domains with the active site in a solvent accessible tunnel at the domain interface. A low-resolution view of the complex with one substrate, coenzyme A, defines its binding site and suggests an additional interaction not found in the related carnitine acetyltransferase. Also, the preference for choline over carnitine as an acetyl acceptor is seen to result from both electrostatic and steric blocks to carnitine binding at the active site. While half of the mutations that cause motor disorders are positioned to affect enzyme activity directly, the remaining changes are surprisingly distant from the active site and must exert indirect effects. The structure indicates how ChAT is regulated by phosphorylation and reveals an unusual pattern of basic surface patches that may mediate membrane association or macromolecular interactions.     

Aryl urea analogs with broad-spectrum antibacterial activity

The preparation and evaluation of novel aryl urea analogs as broad-spectrum antibacterial agents is described. Numerous compounds showed low micromolar minimum inhibitory concentrations (MIC) against both Gram-positive and Gram-negative bacteria. Selected analogs also exhibited in vivo efficacy in a lethal murine model of bacterial septicemia.     

Calpain-mediated proteolysis of talin regulates adhesion dynamics

Dynamic regulation of adhesion complexes is required for cell migration and has therefore emerged as a key issue in the study of cell motility. Recent progress has been made in defining some of the molecular mechanisms by which adhesion disassembly is regulated, including the contributions of adhesion adaptor proteins and tyrosine kinases. However, little is known about the potential contribution of proteolytic mechanisms to the regulation of adhesion complex dynamics. Here, we show that proteolysis of talin by the intracellular calcium-dependent protease calpain is critical for focal adhesion disassembly. We have generated a single point mutation in talin that renders it resistant to proteolysis by calpain. Quantification of adhesion assembly and disassembly rates demonstrates that calpain-mediated talin proteolysis is a rate-limiting step during adhesion turnover. Furthermore, we demonstrate that disassembly of other adhesion components, including paxillin, vinculin and zyxin, is also dependent on the ability of calpain to cleave talin, suggesting a general role for talin proteolysis in regulating adhesion turnover. Together, these findings identify calpain-mediated proteolysis of talin as a mechanism by which adhesion dynamics are regulated.     

Cytokines and the acute phase response in post-treatment reactive encephalopathy of Trypanosoma brucei brucei infected mice

Stimulation of the acute phase response during infection of mice with Trypanosoma brucei brucei (T. b. brucei) was investigated in an experimental model of the post-treatment reactive encephalopathy (PTRE), a common side-effect of anti-trypanosome therapy. Plasma levels of the acute phase proteins (APP), haptoglobin (Hp) and serum amyloid P (SAP) increased by day 7 post-infection, but by day 20 had fallen to an intermediate level. This was accompanied by induction of the cytokines, interleukin (IL)-6 and tumour necrosis factor-alpha (TNFalpha) in both liver and brain. Treatment of mice on day 21 with a subcurative dose of diminazene aceturate (Berenil), a procedure known to induce a mild PTRE, cleared the parasite from the circulation with plasma APP and liver expression of mRNA for IL-6 and TNFalpha returning to the levels in the controls. Cytokine mRNA for both IL-6 and TNFalpha was detected in the brains of animals with developing PTRE although TNFalpha was not significantly greater than in the control group. A further subcurative dose of Berenil, leading to a more severe PTRE, was associated with elevated serum concentrations of Hp and SAP, increased TNFalpha mRNA in the liver and detectable IL-6 and TNFalpha mRNA in the brain. mRNA for IL-1alpha was expressed in brain and liver samples from all animals. A severe PTRE caused a systemic acute phase response which was not apparent with a mild PTRE. The pattern of cytokine mRNA induction was similar following both drug treatments. However, the difference in APP production could be caused by a breakdown in the blood-brain barrier during severe PTRE allowing cytokine synthesised in the brain to enter the circulation and maintain a systemic response.     

Hyperconservation of the putative antigen recognition site of the MHC class I-b molecule TL in the subfamily Murinae: evidence that thymus leukemia antigen is an ancient mammalian gene

"Classical" MHC class I (I-a) genes are extraordinarily polymorphic, but "nonclassical" MHC class I (I-b) genes are monomorphic or oligomorphic. Although diversifying (positive) Darwinian selection is thought to explain the origin and maintenance of MHC class I-a polymorphisms, genetic mechanisms underlying MHC class I-b evolution are uncertain. In one extreme model, MHC class I-b loci are derived by gene duplication from MHC class I-a alleles but rapidly drift into functional obsolescence and are eventually deleted. In this model, extant MHC class I-b genes are relatively young, tend to be dysfunctional or pseudogenic, and orthologies are restricted to close taxa. An alternative model proposed that the mouse MHC class I-b gene thymus leukemia Ag (TL) arose approximately 100 million years ago, near the time of the mammalian radiation. To determine the mode of evolution of TL, we cloned TL from genomic DNA of 11 species of subfamily Murinae: Every sample we tested contained TL, suggesting this molecule has been maintained throughout murine evolution. The sequence similarity of TL orthologs ranged from 85-99% and was inversely proportional to taxonomic distance. The sequences showed high conservation throughout the entire extracellular domains with exceptional conservation in the putative Ag recognition site. Our results strengthen the hypotheses that TL has evolved a specialized function and represents an ancient MHC class I-b gene.     

Mathematical modeling of myoglobin facilitated transport of oxygen in devices containing myoglobin-expressing cells

Low pO(2) is perhaps the most significant factor in artificial pancreas failure. In these environments, not only is the beta cell production of insulin reduced, but the cell death rate is also significantly higher. Mathematical models are developed to test the feasibility of facilitated oxygen transport in enhancing O(2) flux to genetically engineered cells in a bioartificial device such as a pancreas. For this device, it is proposed that beta cells be genetically engineered to express myoglobin throughout the cell. In addition, the significance of including myoglobin throughout the alginate matrix present to provide immuno-protection for the transplanted cells is considered. The mathematical analysis predicts that myoglobin facilitated oxygen transport has the potential of increasing the oxygen concentration at the centre of a cluster of cells (islet) with an effective radius of 100 microm by 50%. These theoretical models for myoglobin facilitated oxygen transport with homogeneous Michaelis-Menten consumption also indicate that including myoglobin in the alginate gel would beneficially improve the flux of oxygen to the transplanted cells.