The (1-14) fragment of parathyroid hormone (PTH) activates intact and amino-terminally truncated PTH-1 receptors.

Recent mutagenesis and cross-linking studies suggest that residues in the carboxyl-terminal portion of PTH(1-34) interact with the amino-terminal extracellular domain of the receptor and thereby contribute strongly to binding energy; and that residues in the amino-terminal portion of the ligand interact with the receptor region containing the transmembrane helices and extracellular loops and thereby induce second messenger signaling. We investigated the latter component of this hypothesis using the short amino-terminal fragment PTH(1-14) and a truncated rat PTH-1 receptor (r delta Nt) that lacks most of the amino-terminal extracellular domain. The binding of PTH(1-14) to LLC-PK1 or COS-7 cells transfected with the intact PTH-1 receptor was too weak to detect; however, PTH(1-14) dose-dependently stimulated cAMP formation in these cells over the dose range of 1-100 microM. PTH(1-14) also stimulated cAMP formation in COS-7 cells transiently transfected with r delta Nt, and its potency with this receptor was nearly equal to that seen with the intact receptor. In contrast, PTH(1-34) was approximately 100-fold weaker in potency with r delta Nt than it was with the intact receptor. Alanine scanning of PTH(1-14) revealed that for both the intact and truncated receptors, the 1-9 segment of PTH forms a critical receptor activation domain. Taken together, these results demonstrate that the amino-terminal portion of PTH(1-34) interacts with the juxtamembrane regions of the PTH-1 receptor and that these interactions are sufficient for initiating signal transduction.     

High-risk pregnancy in rhesus monkeys (Macaca mulatta): a case of ectopic, abdominal pregnancy with birth of a live, term infant, and a case of gestational diabetes complicated by pre-eclampsia

Background  Cases of abdominal pregnancy, in the form of intra-abdominal mummified fetuses, have been described in nonhuman primates. Gestational diabetes and pre-eclampsia are common pregnancy complications in women.
Methods  Two timed-bred rhesus monkeys had high-risk pregnancies, an abdominal pregnancy with delivery of a live term infant, and a case of gestational diabetes that later developed pre-eclampsia.
Results  The monkey that had abdominal pregnancy later died from septic peritonitis. The monkey had a colonic adenocarcinoma that may have allowed leakage of intestinal contents into the abdomen. Her infant was fostered to another female and survived. The monkey with gestational diabetes and pre-eclampsia was treated with a regimen similar to that used in women, and a live infant was delivered at day 157 of gestation by Caesarian section.
Conclusion  These cases underscore the value of timed-breeding and the similarities between pregnancy complications in women and in nonhuman primates.     

Secretion of IGF-1 by ovine granulosa cells: effects of growth hormone and follicle stimulating hormone

Insulin-like growth factor-1 (IGF-1) is implicated in follicle development and is considered to mediate the actions of growth hormone (GH) and gonadotrophins at the ovarian level. However, the expression and secretion of IGF-1 by the ovary are controversial, partly because of species and cell-type specificity. The present study investigated whether IGF-1 is produced by ovine granulosa cells and whether its production is regulated by GH and follicle stimulating hormone (FSH). Follicles (>/=4.0 mm) were obtained from ewes during seasonal anoestrus. Granulosa cells were cultured for a total period of 96 h in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 medium supplemented with BSA (0.1%, w:v), transferrin (0.5 microg/ml) and testosterone (100 ng/ml). In the first set of experiments, cells were incubated in the presence of bovine calf serum (BCS) (2.5%) for the initial 48 h of culture. The cells were then cultured for the next 48 h in medium without BCS, but containing either GH (0, 2, 20, and 200 ng/ml) or FSH (0, 20, 200, and 2000 ng/ml). The medium was assayed for oestradiol (E), progesterone (P) and IGF-1. There were six wells per treatment and the experiment was carried out four times. Control granulosa cells maintained both IGF-1 and E secretion, with only low levels of progesterone output. In all experiments, both GH and FSH produced significant (P<0.001) dose-related increases in E, IGF-1 and P secretion into the medium. The maximum responses to GH (20 or 200 ng/ml) were 402% for E and 528% for IGF-1 compared with controls. The maximum responses to FSH (200 or 2000 ng/ml) were 460% for E and 514% for IGF-1. The objective of the second set of experiments was to determine the effect of the progestogenic status of cells on IGF-1 production. Granulosa cells were cultured both in the presence and absence of BCS (2.5% in the medium) during the initial 48 h of culture. For the next 48 h, cells were cultured in serum-free medium. Addition of BCS to the medium during the initial 48 h of culture stimulated progesterone production. However, it did not affect either IGF-1 or oestradiol secretion between 49 and 96 h of culture, or the cell numbers at the end of culture. In conclusion, (1) IGF-1 is secreted by granulosa cells irrespective of their progestogenic status and (2) concomitant increases in E and IGF-1 production by granulosa cells as a result of GH and/or FSH treatment suggest a role for GH and FSH in the regulation of ovarian function.     

Exploring the role of amino acid-18 of the leucine binding proteins of E. coli

Two periplasmic binding proteins of E. coli, the leucine specific-binding protein (LS) and leucine-isoleucine-valine binding protein (LIV), have high similarity in their structure and function, but show different substrate specificity. A key difference between these proteins is residue 18 in the binding pocket, a tryptophan residue in the LS and a tyrosine residue in the LIV. To examine the role of this residue in binding specificity, we used fluorescence and (19)F NMR to monitor ligand binding to three mutants: LSW18Y, LSW18F and LIVY18W. We observed leucine binding to all proteins. LS binds L-phenylalanine but the mutation from Trp to Tyr or Phe disallows this ligand and expands the binding repertoire to L-isoleucine and L-valine. The LIVY18W mutant still retains the ability to bind L-isoleucine and also binds L-phenylalanine.     

Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.     

Conformational changes in the human estrogen receptor observed by (19)F NMR

The (19)F NMR spectra of the 5F-Trp labeled glutathione-S-transferase fusion protein with residues 282-595 of the human estrogen receptor show that there is a distinct conformational change in the protein when estradiol is added to the unliganded protein. Our studies show the empty receptor to have more conformational flexibility than the liganded form. This study shows the applicability of (19)F NMR to study conformational change in large protein systems.     

Two binding modes of netropsin are involved in the complex formation with poly(dA-dT).poly(dA-dT) and other alternating DNA duplex polymers

Using CD measurements we show that the interaction of netropsin to poly(dA-dT).poly(dA-dT) involves two binding modes at low ionic strength. The first and second binding modes are distinguished by a defined shift of the CD maximum and the presence of characteristic isodichroic points in the long wavelength range from 313 nm to 325 nm. The first binding mode is independent of ionic strength and is primarily determined by specific interaction to dA.dT base pairs. Employing a netropsin derivative and different salt conditions it is demonstrated that ionic contacts are essential for the second binding mode. Other alternating duplexes and natural DNA also exhibit more or less a second step in the interaction with netropsin observable at high ratio of ligand per nucleotide. The second binding mode is absent for poly(dA).poly(dT). The presence of a two-step binding mechanism is also demonstrated in the complex formation of poly(dA-dT).poly(dA-dT) with the distamycin analog consisting of pentamethylpyrrolecarboxamide. While the binding mode I of netropsin is identical with its localization in the minor groove, for binding mode II we consider two alternative interpretations.     

The Association between Mycobacterium Tuberculosis Genotype and Drug Resistance in Peru

BackgroundThe comparison of Mycobacterium tuberculosis bacterial genotypes with phenotypic, demographic, geospatial and clinical data improves our understanding of how strain lineage influences the development of drug-resistance and the spread of tuberculosis.MethodsTo investigate the association of Mycobacterium tuberculosis bacterial genotype with drug-resistance. Drug susceptibility testing together with genotyping using both 15-loci MIRU-typing and spoligotyping, was performed on 2,139 culture positive isolates, each from a different patient in Lima, Peru. Demographic, geospatial and socio-economic data were collected using questionnaires, global positioning equipment and the latest national census.ResultsThe Latin American Mediterranean (LAM) clade (OR 2.4, p<0.001) was significantly associated with drug-resistance and alone accounted for more than half of all drug resistance in the region. Previously treated patients, prisoners and genetically clustered cases were also significantly associated with drug-resistance (OR''s 2.5, 2.4 and 1.8, p<0.001, p<0.05, p<0.001 respectively).ConclusionsTuberculosis disease caused by the LAM clade was more likely to be drug resistant independent of important clinical, genetic and socio-economic confounding factors. Explanations for this include; the preferential co-evolution of LAM strains in a Latin American population, a LAM strain bacterial genetic background that favors drug-resistance or the "founder effect" from pre-existing LAM strains disproportionately exposed to drugs.