Effects of naloxone on basal and vagus nerve-induced secretions of GRP, gastrin, and somatostatin from the isolated perfused rat stomach

The effects of naloxone, an opiate antagonist, on basal and vagus nerve-induced secretions of GRP, gastrin, and somatostatin were examined using the isolated perfused rat stomach prepared with vagal innervation. Naloxone (10(-6) M) significantly inhibited basal somatostatin secretion in the presence and absence of atropine and of hexamethonium, whereas basal GRP and gastrin secretion was not affected by naloxone. Electrical stimulation (10 Hz, lms duration, 10V) of the distal end of the subdiaphragmatic vagal trunks elicited a significant increase in both GRP and gastrin but a decrease in somatostatin. Naloxone (10(-6) M) failed to affect these responses in the presence or absence of atropine. On the other hand, when hexamethonium was infused, naloxone significantly inhibited both the GRP and gastrin responses to electrical vagal stimulation. Somatostatin secretion was unchanged by vagal stimulation during the infusion of hexamethonium with or without naloxone. These findings suggest that basal somatostatin secretion is under the control of an opiate neuron and that opioid peptides might be involved in vagal regulation of GRP and gastrin secretion.     

The glycerol utilization operon of Streptomyces coelicolor: genetic mapping of gyl mutations and the analysis of cloned gylDNA

Summary Glycerol-3-phosphate dehydrogenase (gylB) mutations (which cause glycerol sensitivity), and presumed gylcerol kinase (gylA) and/or regulatory mutations eliminating both glycerol-3-phosphate dehydrogenase and glcerol kinase activities, map close to the argA locus of Streptomyces coelicolor A3(2). Using the plasmid vector pIJ702 and restriction enzymes Bg/II and SstI, extensively overlapping S. coelicolor DNA fragments of 2.74 kb and 2.84 kb were isolated, either of which could restore the wild-type phenotype to gylB and some gylA mutants. Genetic and biochemical analyses of mutants carrying the cloned gylDNA suggested that a functional gyl promoter had not been cloned, and that restoration of the Gyl⁺ phenotype was achieved by recombination between the cloned and chromosomal gyl DNA sequences. After subcloning parts of this DNA into the phage vector C31 KC400, gene disruption analysis was carried out, which confirmed the absence of the gyl promoter, and indicated that a polycistronic mRNA traverses gylA and then gylB.     

Genetic mapping, cloning and physiological aspects of the glucose kinase gene of Streptomyces coelicolor

Summary Glucose kinase in Streptomyces coelicolor has a molecular weight of about 110,000. In crude extracts, the enzyme exhibited apparent K_m values of 0.20 mM for ATP, 0.27 mM for glucose, and 2.2 mM for the glucose analogue 2-deoxyglucose. Mutations (glk) to 2-deoxyglucose-resistance, which greatly reduce glucose kinase activity and result in relief of glucose repression of utilisation of various carbon sources, were mapped between proA and hisA in the S. coelicolor linkage map. Glucose kinase activity, 2-deoxyglucose-sensitivity, glucose utilisation and glucose repression, were all restored to glk mutants by a 3.5 kb DNA fragment cloned from S. coelicolor into a phage vector (C31 KC515), and by larger (10–30 kb) fragments cloned into a low copy number plasmid vector (pIJ916). The glk gene was further localised to a 2.9 kb BclI fragment of the cloned DNA by sub-cloning. Part or all of this fragment was present in each of five primary plasmid clones tested.     

Molecular characterization of recombinant human acidic fibroblast growth factor produced in E. coli: comparative studies with human basic fibroblast growth factor.

Synthetic cDNA coding for human acidic fibroblast growth factor (haFGF) was expressed in E. coli under the control of the T7 promoter. The haFGF produced was purified extensively using heparin-Sepharose and phenyl-Sepharose columns. The mitogenic activity of haFGF on 3T3 and endothelial cells was significantly potentiated in the presence of heparin (10-50 micrograms/ml), while angiogenic activity was observed on chick embryo chorioallantoic membrane without exogenously added heparin. This significant potentiation of mitogenic activity was observed specifically with haFGF, not human basic fibroblast growth factor (hbFGF). Circular dichroism spectra of haFGF was not affected by the presence of heparin. The affinity of haFGF for heparin was examined using heparin affinity HPLC and was precisely confirmed to be relatively lower than that of hbFGF. These results implied that haFGF was potentiated by heparin and that this potentiation did not involve a significant change in the conformation of the haFGF molecule. The affinity of haFGF for copper was also confirmed to be higher than that of hbFGF using a copper affinity HPLC column. In addition, under acidic conditions, haFGF appeared more stable than hbFGF and was further stabilized in the presence of heparin.     

Human thymidylate synthase gene: isolation of phage clones which cover a functionally active gene and structural analysis of the region upstream from the translation initiation codon

Two genomic DNA fragments partially encoding human thymidylate synthase (TS) [EC 2.1.1.45] were previously cloned in lambda phage from the mouse cell transformant, but had no transforming activity on mouse TS-negative mutant cells. In this study, an additional genomic DNA for human TS was cloned and demonstrated to have the transforming activity in combination with one of the two previously cloned DNAs and to produce human TS mRNA. The two transforming genomic DNAs overlapped and covered a region of 23 kb in total. Using fragments from one of these DNAs, the structure of the 1.2-kb region around the ATG initiator codon of the TS gene was analyzed in relation to regulatory sequences of the gene. Sequence determination demonstrated the presence of an unusual inverted repeat consisting of a triple tandem repeat of a 28-bp sequence and an inverted sequence of the same length. These sequences can form three possible, stable, stem-loop structures, which may be interconvertible. Based on S1 nuclease mapping data and a line of circumstantial evidence, we deduced two major mRNA cap sites within the inverted sequence. Comparison of the human and mouse sequences upstream from the ATG initiator codon revealed many significant blocks of sequence homology, especially in the regions around the deduced cap sites.     

Distribution, seasonal use, and predation of incubation mounds of Orange-footed Scrubfowl on Komodo Island, Indonesia

ABSTRACT.   Megapodes are unique in using only heat from the environment, rather than body heat, to incubate their eggs as well as the precocious independence of their chicks on hatching. Of 22 recognized species of megapodes, 9 are listed as threatened due to factors including habitat loss and fragmentation, and predation on eggs and chicks. Orange-footed Scrubfowl ( Megapodius reinwardt ) are conspicuous components of the Oriental/Austral avifauna that inhabit the monsoon forests of the Lesser Sunda chain of islands in Indonesia. We examined the abundance, patterns of distribution, physical characteristics, seasonal activity, and predation risk of incubation mounds of Orange-footed Scrubfowl on Komodo Island in eastern Indonesia. We surveyed 13 valleys on Komodo Island from April 2002 to January 2005 and located 113 tended and 107 untended incubation mounds. Densities of scrubfowl mounds in our study were similar to that reported by investigators during the 1970s, suggesting little change in the scrubfowl population since then. Most scrubfowl mounds were on sandy or loamy soils in open monsoon forest with little overhead shade, and placement of mounds in such areas may ensure adequate temperatures for egg incubation. Although some mounds were tended during all months, mound use peaked during the late wet season in March. During the dry season (April–November), only a few mounds were tended. Komodo dragons ( Varanus komodoensis ) and wild pigs ( Sus scrofa ) were the primary predators of scrubfowl eggs, with no indication of egg predation by humans. The valley with the largest number of untended mounds in our study also had the largest number of active Komodo dragon nests. This suggests an effect of Komodo dragons on scrubfowl numbers, but additional study is needed.     

Deoxymyoglobin studied by the conformational normal mode analysis. II. The conformational change upon oxygenation

The conformational change taking place in myoglobin concomitantly with the observed geometrical change at the heme-His(F8) linkage upon oxygenation is studied by normal mode analysis, which is based on the quadratic approximation of the conformational energy function. The heme-globin interaction energy increases for this change by 8.114 kcal/mol when both the heme group and the globin molecule are held rigid. When they are permitted flexibility, the interaction energy relaxes by 7.038 kcal/mol, and the difference (1.076 kcal/mol) is distributed as strain energy within the molecule. This increase is the work necessary for the heme group to move against the force exerted by the globin. If the force is assumed to be invariable during this move, the work is small, 0.276 kcal/mol, meaning that the force is strongly variable. Furthermore, this means that the heme group is located near the equilibrium point of the potential energy of the heme-globin interaction. The change in the localized strain energy stored in the force field at the linkage between the heme and the imidazole of HisF8 is estimated to be of the same order of magnitude as the distributed energy. The largest atomic displacements are observed at the region from the F helix to the beginning of the G helix, and secondary large displacements occur at several regions, i.e, the A helix, from the C helix to the CD corner, the E helix, and the C-terminal side of the H helix. All of these regions have strong dynamic interactions with the heme group, either directly or indirectly. Their secondary structures show complex deformations. In other parts, relatively rigid segments undergo rotational and/or bending changes in a way consistent with the large changes described above and close atomic packing within the molecule. The calculated conformational change is decomposed to vibrational normal modes of deoxymyoglobin. The magnitude of the conformational change, measured by the mass-weighted mean-square atomic displacement, is accounted for up to 92.0% by the 151 normal modes with frequencies lower than 40 cm-1. In descending order of contribution, the first six modes, each of which has a frequency lower than 12 cm-1, account for up to 57.4%. This means that the functionally important conformational change can well be expressed in terms of a relatively small number of collective low frequency normal modes.