Genomic characterization and chromosomal mapping of 5 river buffalo skeletal muscle differentiation master genes.

River buffalo (Bubalus bubalis, 2n = 50, BBU) is a species of economic relevance in a number of countries. This species shows a very peculiar biology and a great capacity for environmental adaptation. There has been an increasing economic interest as well as a growing demand for a more detailed knowledge of molecular features in this species. From this perspective we report a genomic, transcriptional and cytogenetic analysis of 5 master genes involved in skeletal muscle development. Of these 5 genes, MYOD1, MYF5, MYF6 and MYOG belong to the basic helix-loop helix protein family while MSTN belongs to the TNF-B protein family. In mammals, these genes are involved in the early stages of skeletal muscle differentiation, development and regeneration. These pivotal biological functions are finely regulated in a tissue- and temporal-specific manner. We used a comparative genomic approach to obtain the buffalo specific sequences of MYOD1 and MYF6. The nucleotide sequence similarity and the protein domain conservation of the newly obtained sequences are analysed with respect to bovine and other mammalian species showing sequence similarity. The presence of a polymorphism in MYOD1 coding sequence is described and its possible effect discussed. Using a quantitative PCR approach, we compared the level of the 5 transcripts in adult and fetal muscle. These genes were physically localised on river buffalo R-banded chromosomes by FISH using bovine genomic BAC-clones. Here, we present a genomic and cytogenetic analysis which could offer a background to better characterise the buffalo genes involved in muscle function and which may be responsible for buffalo-specific meat features.

Expressed STSs and transcription of human Xq28.

STSs, which have been used to build and format clone contigs, have been used here to assemble a transcriptional map across a cytogenetic band. Of fifty one STSs in Xq28, 20 were positive by RT-PCR. Thus, an additional 20 possible ESTs were detected among the STSs, and seven of these also identified cDNAs in at least one library. The transcripts confirm the high expression level of this region, correlated with its GC compositional map and CpG island content.

The negative pregnancy test. An opportunity for preconception care.

OBJECTIVE: To identify women who would likely benefit from preconception care. METHODS: A comprehensive preconception risk survey was administered during a structured interview to 136 women who had a negative pregnancy test visit in a family practice residency ambulatory practice. The survey solicited the presence of self-reported risk variables associated with maternal conditions related to poor obstetric outcome, risk factors for poor obstetric outcome, and risks for developing these conditions. RESULTS: Seventy women (51.5%) reported a medical or reproductive risk that could adversely affect pregnancy. In addition, 68 women (50%) reported a genetic risk; 39 (28.7%) reported a risk for human immunodeficiency virus infection, 35 (25.7%) reported an indication for hepatitis B vaccine, and an equal number reported recent use of illegal substances; 23 (16.9%) reported at least one affirmative answer to the CAGE questionnaire; 79 (58.5%) smoked cigarettes; 74 (54.4%) reported a nutrition risk; 126 (92.6%) reported a psychosocial risk; and 39 (28.7%) reported a perceived barrier to ongoing medical care. Even with the psychosocial risk category excluded, 94% of the women still reported at least one factor requiring further evaluation, counseling, or intervention before pregnancy. CONCLUSIONS: We discovered a significant number of women with obstetric risk factors. A negative pregnancy test visit provides an opportunity for preconception risk assessment and counseling. These results will guide us to further develop practical preconception care protocols.

YAC contig organization and CpG island analysis in Xq28.

One hundred nineteen YACs were assembled into 6 contigs spanning about 7.1 Mb of Xq28. The contigs were formatted with 65 STSs and 136 hybridization probes and were extensive enough to be aligned and oriented by published genetic linkage and somatic cell hybrid panel data. Selected YACs from the entire region were mapped with five rare-cutter restriction enzymes to infer the position of putative CpG islands indicative of gene locations; 48 such sites were identified by the near-coincidence of at least three rare-cutter sites. The analysis defined three subregions of Xq28: 4 Mb of moderate GC and CpG island content from the Xq27 border through the GABRA locus; 1.5 to 2 Mb, extending to the G6PD gene, that is variably and poorly cloned, but contains a high concentration of CpG islands and GC; and about 1.5 Mb between G6PD and the telomere, which is generally low in CpG and GC levels, including a subtelomeric DNA region that shows extensive homology to Yq DNA.

Identification and characterization of the proBA operon of Streptococcus bovis.

A genomic DNA library of the rumen bacterium Streptococcus bovis was constructed in Escherichia coli, and recombinant plasmids able to complement proA and proB mutations of the host were found. Southern hybridization and restriction analysis showed that a 3.5-kb fragment of S. bovis DNA contained two genes, organized in an operon and coding for enzymes functionally similar to the glutamyl phosphate reductase-glutamyl kinase enzyme complex that in E. coli catalyzes the first step of proline biosynthesis. Complementation of the E. coli mutations was observed with the fragment inserted in both orientations, which suggested that the S. bovis proBA operon was transcribed from its own promoter. Genetic and biochemical data suggested that the proline biosynthetic pathway of S. bovis is similar to the one previously characterized for E. coli.

Angiotensin II receptors in the fowl aorta.

In the domestic fowl, angiotensin II (ANG II) causes an in vivo depressor response and in vitro relaxation of aortic rings which appear to be a direct action of ANG II on the blood vessels. Thus, we determined whether binding sites specific to ANG II exist in the membrane fraction of the fowl aorta. The particulate fraction of aortas from adult female fowl, Gallus gallus, exhibits high specific binding to ANG II ligand. 125I-[Ile5]ANG II (0.5 nM) binding to the above fraction (30 micrograms protein) in 50 mM Tris (pH 7.2), 10 mM MgCl2, and 0.2% bovine serum albumin at 12 degrees (1) is rapid, saturable, and reversible; (2) increases as a function of ligand or membrane concentration, time, and temperature; and (3) optimally fits to a two-site (high and low affinity) model. The equilibrium dissociation constant (0.15 +/- 0.03 nM) and binding site concentration (28.7 +/- 8.1 fmol/mg protein) of the high affinity site as well as association (0.055 nM-1.min-1) and dissociation (0.0122 min-1) rate constants are similar to those of mammalian vascular ANG II receptors. Both 125I-[Ile5]ANG II and 125I-[Val5]ANG II are competitively displaced by unlabeled ANG II. These results suggest that specific ANG II receptors exist in the fowl aorta.

Identification of a human liver cytochrome P-450 homologous to the major isosafrole-inducible cytochrome P-450 in the rat.

The rat 3-methylcholanthrene-inducible family of liver cytochromes P-450 contains two proteins (P-450c and P-450d) that are immunochemically related, possess 68% total sequence homology, and are induced by a number of toxic or carcinogenic compounds. To determine whether equivalent isozymes of hepatic cytochrome P-450 are expressed in humans, as they are in several mammalian species, we performed immunoblot analyses on microsomes prepared from 14 human liver specimens and found that each one contained a 52.5-kDa protein (termed HLd) that reacted with antibodies specific for rat P-450d. In addition, one specimen contained a 54-kDa protein (termed HLc) that reacted with antibodies specific for rat P-450c. HLd was purified through the use of immunoaffinity chromatography and was found to be 56% homologous to rat P-450d and 61% homologous to the equivalent isozyme in the rabbit (P-450 LM4) through their first 18 NH2-terminal amino acids. Finally, levels of immunoreactive HLd varied more than 10-fold among these patients but were unrelated to the patients' drug treatments, smoking habits, or amount of immunoreactive HLp, a human liver cytochrome P-450 related to the glucocorticoid-inducible family of rat cytochromes P-450. We conclude that, in man, there is a cytochrome P-450 family composed of two isozymes (HLc and HLd) that are immunochemically and structurally related to the 3-methylcholanthrene-inducible family observed in several other species.

Evidence for the role of phosphorylase kinase, protein kinase C, and other Ca2+-sensitive protein kinases in the response of hepatocytes to angiotensin II and vasopressin.

Angiotensin II, catecholamines, and vasopressin can stimulate the phosphorylation of 10 hepatic cytosolic proteins via a Ca2+-linked, cyclic AMP-independent mechanism. To explore the role of known Ca2+-sensitive protein kinases in this response, [32P]PO4(3-)-labeled hepatocytes were stimulated with various agonists, the cytoplasmic proteins were separated on two-dimensional gels, and the resulting autoradiographs were computer analyzed. The role of phosphorylase kinase was examined using hepatocytes from gsd/gsd rats which are deficient in this enzyme. The phosphorylation state of phosphorylase was not increased by glucagon, angiotensin II, or vasopressin in hepatocytes from the gsd/gsd animals. The phosphorylation state of all other substrates was changed by glucagon or the Ca2+-linked hormones to the same extent in gsd/gsd hepatocytes as in normal Wistar controls, suggesting that phosphorylase kinase plays a restricted role in the hormone response. The role of the Ca2+- and phospholipid-sensitive protein kinase (protein kinase C) was examined by stimulating hepatocytes with phorbol esters which are thought to activate protein kinase C by substituting for diacylglycerol. Phorbol esters increased the phosphorylation state of 3 of the 10 substrates affected by angiotensin II or vasopressin, but did not stimulate Ca2+ fluxes in hepatocytes. Treatment of hepatocytes with the Ca2+ ionophore A23187 mimicked the effect of the Ca2+-linked hormones on the phosphorylation of the other 7 substrates. The results demonstrate that at least three Ca2+-sensitive protein kinases are involved in the response of hepatocytes to Ca2+-linked hormones. Since these kinases can be activated independently by phorbol esters or A23187, the results imply that hormones such as vasopressin generate two intracellular messengers, diacylglycerol and Ca2+ ion.

Identification and characterization of the rabbit angiotensin II myocardial receptor.

The octapeptide, angiotensin II, elicits a positive inotropic response in myocardial tissue by activating slow calcium channels. Pharmacological studies suggest that the inotropic action of angiotensin II is receptor mediated. The current investigation was performed to characterize the binding of 125I-angiotensin II to the putative receptor in a plasma membrane-sarcoplasmic reticulum preparation of the rabbit left ventricle. In experiments performed at 18 degrees C, steady state binding occurred at 45 minutes and saturable binding was 80-85% of the total binding. Analysis of the binding data indicated that the 125I-angiotensin II interacted with a single class of sites with a Kd = 4.5 +/- 0.8 nM and exhibited a binding capacity of 53.5 +/- 8 fmol/mg protein. The potency order for the competitive binding of analogues and antagonists of angiotensin II paralleled that observed for in vitro contractile force development in bioassay systems utilizing left atrial tissue. The binding of 125I-angiotensin II was stimulated 2-fold in the presence of the divalent cations of calcium and magnesium (10 mM). Guanine nucleotides modified the binding of 125I-angiotensin II to the rabbit myocardial particulate fraction. Guanine triphosphate and nonhydrolyzable analogues of guanine triphosphate increased the dissociation rate of the bound 125I-angiotensin II and decreased hormone binding to the receptor at equilibrium. In the absence of magnesium, guanine nucleotides had no effect on the dissociation rate of 125I-angiotensin II. 125I-Angiotensin II binding to a rabbit myocardial particulate fraction was found to have high affinity, to be saturable, reversible, specific, and modulated by guanine nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

The hepatic angiotensin II receptor. II. Effect of guanine nucleotides and interaction with cyclic AMP production.

Guanine nucleotides were observed to modify the binding of 125I-angiotensin II to rat hepatic plasma membrane receptors. GTP and its nonhydrolyzable analogues greatly increased the dissociation rate of bound 125I-angiotensin II and altered hormone binding to the receptor under equilibrium conditions. In the absence of GTP, 125I-angiotensin II labeled both high affinity sites (Kd1 = 0.46 nM, N1 = 650 fmol/mg) and low affinity sites (Kd2 = 4.1 nM, N2 = 1740 fmol/mg). In the presence of guanine nucleotides, the affinities of the two sites were unchanged, but the number of high affinity sites decreased markedly to 52 fmol/mg. In analogous experiments using the angiotensin II antagonist, 125I-sarcosine1,Ala8-angiotensin II (125I-saralasin), guanine nucleotides minimally affected the interaction of 125I-saralasin with its receptor, increasing the dissociation rate 1.9-fold and the Kd 1.4-fold. The guanine nucleotide inhibition of agonist binding required a cation such as Na+ or Mg2+, with a maximal effect occurring at about 1 mM Mg2+. In liver plasma membranes prepared in EDTA, angiotensin II inhibited basal and glucagon-stimulated adenylate cyclase activities by 30% and 10%, respectively. Angiotensin II also caused a 40% inhibition of glucagon-stimulated cyclic AMP accumulation in intact hepatocytes, with a half-maximal effect occurring at 1 nM. The inhibition by angiotensin II of adenylate cyclase in membranes and of cAMP levels in intact cells could be reversed by the antagonist sarcosine1,Ile8-angiotensin II. Vasopressin caused a smaller 26% inhibition of glucagon-stimulated cyclic AMP accumulation. The ability of angiotensin II to inhibit cyclic AMP synthesis may provide an explanation for the observed effects of guanine nucleotides on 125I-angiotensin II binding to plasma membranes.

The effect of prolactin on human aldosterone-producing adenomas in vitro.

There is evidence for an unidentified aldosterone-stimulating factor of pituitary origin. We measured the effect of ovine PRL (oPRL) on aldosterone secretion by isolated cell suspensions of human aldosterone-producing adenomas (APAs) and compared it to the effects of angiotensins, ACTH, and potassium (K+). In the first APA, the aldosteronotropic action of large doses of oPRL was double that of angiotensin II (AII); the response to ACTH was triple that to AII, while K+ had a small stimulatory effect. Results with the second APA showed that physiological concentrations of oPRL caused a response nearly double that to AII, but, once again, less than the response to ACTH; K+ was inert. ACTH contamination of the oPRL preparation was too minute to account for these findings. We conclude that oPRL possesses aldosterone-stimulating activity in APAs greater than that of angiotensins and potassium, but less that that of ACTH. These data suggest a role for PRL in aldosterone secretion by aldosterone-producing adenomas.

Steroids as potential modulators of angiotensin receptors in bovine adrenal glomerulosa and kidney.

To test the hypothesis that there is feedback inhibition of adrenal angiotensin receptors by substances released in response to the peptides, we measured binding of labeled angiotensins in the presence of various steroids. Approximately half of the 70 steroids tested inhibited binding of labeled angiotensin II and III to intact and broken cells from bovine adrenal glomerulosa and kidney, but the concentrations required for inhibition were relatively high. The most potent inhibitors were 3 alpha, 5 beta tetrahydroaldosterone and tetrahydrodeoxycorticosterone (ID50 = 8 x 10-5 M). Kinetic analysis showed that inhibition was mostly competitive. among steroids whose reduced congeners were tested, potency increased in the sequence: parent steroid less than 5 alpha dihydroderivative less than 5 beta dihydro derivative less than 3 alpha, 5 beta tetrahydro-derivative. Tetrahydrodeoxycorticosterone inhibited aldosteronogenesis by intact cells at concentrations that inhibited angiotensin binding. Steroids differentially inhibited binding of labeled angiotensins in II and III, and discriminated between receptors in adrenal glomerulosa and kidney. The results provide additional evidence for heterogeneity of angiotensin receptors, and lead to the prediction that any normal or pathological inhibition of angiotensin receptors by steroids will be mediated by reduced derivatives.