Fluorescent Fusion Proteins of Soluble Guanylyl Cyclase Indicate Proximity of the Heme Nitric Oxide Domain and Catalytic Domain

BACKGROUND: To examine the structural organisation of heterodimeric soluble guanylyl cyclase (sGC) Förster resonance energy transfer (FRET) was measured between fluorescent proteins fused to the amino- and carboxy-terminal ends of the sGC beta1 and alpha subunits. METHODOLOGY/PRINCIPAL FINDINGS: Cyan fluorescent protein (CFP) was used as FRET donor and yellow fluorescent protein (YFP) as FRET acceptor. After generation of recombinant baculovirus, fluorescent-tagged sGC subunits were co-expressed in Sf9 cells. Fluorescent variants of sGC were analyzed in vitro in cytosolic fractions by sensitized emission FRET. Co-expression of the amino-terminally tagged alpha subunits with the carboxy-terminally tagged beta1 subunit resulted in an enzyme complex that showed a FRET efficiency of 10% similar to fluorescent proteins separated by a helix of only 48 amino acids. Because these findings indicated that the amino-terminus of the alpha subunits is close to the carboxy-terminus of the beta1 subunit we constructed fusion proteins where both subunits are connected by a fluorescent protein. The resulting constructs were not only fluorescent, they also showed preserved enzyme activity and regulation by NO. CONCLUSIONS/SIGNIFICANCE: Based on the ability of an amino-terminal fragment of the beta1 subunit to inhibit activity of an heterodimer consisting only of the catalytic domains (alphacatbetacat), Winger and Marletta (Biochemistry 2005, 44:4083-90) have proposed a direct interaction of the amino-terminal region of beta1 with the catalytic domains. In support of such a concept of "trans" regulation of sGC activity by the H-NOX domains our results indicate that the domains within sGC are organized in a way that allows for direct interaction of the amino-terminal regulatory domains with the carboxy-terminal catalytic region. In addition, we constructed "fluorescent-conjoined" sGC's by fusion of the alpha amino-terminus to the beta1 carboxy-terminus leading to a monomeric, fluorescent and functional enzyme complex. To our knowledge this represents the first example where a fluorescent protein links two different subunits of a higher ordered complex to yield a stoichometrically fixed functionally active monomer

Leptospirosis in American Samoa – Estimating and Mapping Risk Using Environmental Data

Leptospirosis is the most common bacterial infection transmitted from animals to humans. Infected animals excrete the bacteria in their urine, and humans can become infected through contact with animals or a contaminated environment such as water and soil. Environmental factors are important in determining the risk of human infection, and differ between ecological settings. The wide range of risk factors include high rainfall and flooding; poor sanitation and hygiene; urbanisation and overcrowding; contact with animals (including rodents, livestock, pets, and wildlife); outdoor recreation and ecotourism; and environmental degradation. Predictive risk maps have been produced for many infectious diseases to identify high-risk areas for transmission and guide allocation of public health resources. Maps are particularly useful where disease surveillance and epidemiological data are poor. The objectives of this study were to estimate leptospirosis seroprevalence at geographic locations based on environmental factors, produce a predictive disease risk map for American Samoa, and assess the accuracy of the maps in predicting infection risk. This study demonstrated the value of geographic information systems and disease mapping for identifying environmental risk factors for leptospirosis, and enhancing our understanding of disease transmission. Similar principles could be used to investigate the epidemiology of leptospirosis in other areas

Clocinnamox inhibits the intravenous self-administration of opioid agonists in rhesus monkeys: comparison with effects on opioid agonist-mediated antinociception

 The effects of CCAM, an insurmountable mu opioid receptor antagonist, were studied on the intravenous self-administration and thermoantinociception of alfentanil and nalbuphine, high- and low-efficacy opioid agonists, respectively, in rhesus monkeys. A single dose of 0.1 mg/kg CCAM IV reduced alfentanil’s reinforcing potency in an FR30 TO 45s schedule 10-fold within a 24-h period. The maximum response rates remained essentially unchanged. At 1 mg/kg, CCAM caused a 300-fold shift of the alfentanil dose-response curve and also depressed the maximum response rates. CCAM also blocked insurmountably responding for nalbuphine, which was essentially abolished in two of three animals after a dose of 0.1 mg/kg CCAM and in all animals after 1 mg/kg. The acute insurmountable antagonism of alfentanil and nalbuphine self-administration by CCAM was used to determine the (relative initial) efficacy values of both agonists. Efficacy values, tau, were 391 for alfentanil and 196 for nalbuphine; the apparent in vivo dissociation constants, K A , were 0.16 mg/kg per injection (i.e., 350 nmol/kg per injection) for alfentanil and 0.14 mg/kg (370 nmol/kg per injection) for nalbuphine. In comparison, in a rhesus monkey 50°C warm-water tail withdrawal assay, the tau values were 11 for alfentanil and 0.92 for nalbuphine, and the K A values were 0.2 mg/kg (440 nmol/kg) for alfentanil and 0.15 mg/kg (400 nmol/kg) for nalbuphine. Therefore, it seems that the higher potency of alfentanil and nalbuphine in self-administration as compared to thermal antinociception in rhesus monkeys is predominantly due to a larger efficacy of the same agonist in self-administration (i.e., a larger receptor pool) rather than differences in apparent in vivo affinity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41957/1/213-129-3-233_71290233.pd