Therapeutic warfarin use and the extrahepatic functions of vitamin K-dependent proteins.

The impact of warfarin therapy on the functions of extrahepatic vitamin K-dependent proteins (VKDP) is less clearly understood and less widely recognised in clinical practice than that on the hepatic counterparts (clotting factors II, VII, IX and X). Warfarin inhibits osteocalcin, an abundant extrahepatic VKDP involved in the mineralisation and maturation of bone and thus, primarily by this mechanism, may have an adverse effect on bone health. Whilst some studies do link warfarin use to an increase in osteoporosis and fracture risk others have not. Warfarin also inhibits the extrahepatic VKDP matrix gla protein (MGP) which acts to prevent ectopic calcification of the vasculature. Studies have consistently found a correlation between warfarin use and vascular calcification with inhibition of MGP believed to be the main cause. Inhibition of MGP also appears to explain warfarin's well established teratogenic effect. Further adverse effects may also arise from warfarin's inhibition of other known extrahepatic VKDPs. The available evidence is intriguing, and suggests that the impact of warfarin on the extrahepatic functions of vitamin K-dependent proteins warrants further careful consideration.

Aerial perches and free-range laying hens: the effect of access to aerial perches and of individual bird parameters on keel bone injuries in commercial free-range laying hens.

The aim of this trial was to determine the effect of aerial perches on keel bone injuries and tibia bone characteristics in free-range laying hens. The relationship between keel bone injuries and individual bird parameters, such as weight, girth, wing:girth ratio, feather coverage, and tibia bone characteristics, was also assessed. Five commercial free-range houses, each containing between 7,000 and 8,000 birds, were used. The houses and range areas were divided in half; in half of the house, birds had access to aerial perches (P) and in the other half, they did not (NP). On 13 occasions between 17 and 70 wk of age, 20 birds per treatment were randomly selected from the slatted area and palpated for keel bone injury. At 72 wk of age, 30 birds per treatment in each of 4 houses were selected at random, weighed, and then euthanized. Girth and wing area and feather coverage were measured. The keel and left tibia bones were removed and keel bones were scored for injury. Tibia bones were weighed and diameter, length, breaking strength, and ash content recorded. Results indicated that access to aerial perches did not affect tibia bone measures (P > 0.05). Average palpated keel bone score increased with age of the hens (P < 0.001) but was not significantly affected by perch treatment (P > 0.05). There was a significant interaction between treatment and farm on keel bone injuries measured at dissection (P < 0.05), with the probability of birds having high keel-damage scores increasing in the perched treatment in some farms but not others. In general, as the keel bone injury score measured at dissection increased, the breaking strength (P < 0.001) and ash content (P < 0.05) of the tibia bone decreased. It is suggested that individual variation in bone strength contributes to differences in susceptibility to keel injury. No relationship existed between keel-injury score measured at dissection and individual parameters, such as weight, girth, or wing:girth ratio (P > 0.05), although feather coverage tended to decline with increasing keel damage (P < 0.06).

Cloning and functional expression of a rat brain corticotropin releasing factor (CRF) receptor.

Corticotropin releasing factor (CRF), a key neuroregulator of the hypothalamic-pituitary-adrenal cortical axis, also displays a broad range of effects on the endocrine, central nervous and immune systems. Having recently characterized the human pituitary CRF receptor by expression cloning of cDNA from a human Cushing's corticotropic adenoma, we report here the structure of the cDNA for a rat brain CRF receptor (rCRF-R) which was cloned by hybridization from a rat brain cDNA library. The sequence of the rCRF-R encodes a 415 amino acid protein comprising seven membrane spanning domains. The rCRF-R is 97% identical at the amino acid level to the human pituitary tumor CRF receptor, differing by only 12 amino acids. When expressed in COSM6 cells, the rCRF-R binds CRF with high affinity (Kd = 1.7 (0.8-3.8)nM). The receptor transduces a CRF stimulated accumulation of intracellular cAMP which is inhibited by the CRF antagonist, alpha helCRF(9-41). These results suggest that the brain expresses a CRF receptor similar to that in the pituitary.

Activin and inhibin binding to the soluble extracellular domain of activin receptor II.

Activins and inhibins belong to the transforming growth factor-beta-like superfamily of growth and differentiation factors that exert pleiotropic effects in many target tissues. Heteromeric association of activin with two structurally related receptor serine/threonine kinases, activin receptor types I and II, initiates downstream signaling events. The extracellular domain of type II mouse activin receptor (ActRII ECD) was expressed in the baculovirus system, purified in three steps by lectin affinity, anion exchange, and reverse phase chromatography, and further characterized by mass spectrometry. The reduction in the apparent size of the purified ActRII ECD on SDS-PAGE after treatment with glycosidases provided evidence for N- and O-linked oligosaccharides. Specific receptor/ligand complexes of [125I] activin A to ActRII ECD or [125I]ActRII ECD to activin A were analyzed by cross-linking and immunoprecipitation. Two major radiolabeled bands were observed on SDS-PAGE with mobilities consistent with the expected size of ActRII ECD/betaA or ActRII ECD/betaAbetaA. When inhibin A was cross-linked to [125I]ActRII ECD, a slower migrating complex corresponding to ActRII ECD/betaAalpha was also observed. The apparent dissociation constant (Kd) for activin A binding to ActRII ECD was 2-7 nM. This Kd value is approximately an order of magnitude greater than that of the full-length membrane-associated type II receptor. Treatment of cultured rat anterior pituitary cells with ActRII ECD attenuated FSH secretion in response to exogenous activin A or endogenous activin B. These data indicate that the soluble ActRII ECD has structural determinants that are sufficient for high affinity ligand binding.

Expression of the type II activin receptor gene in the human placenta.

Activin has been suggested to be an autocrine/paracrine regulator in the human placenta. In the present study, we examined the expression of the gene encoding activin type II receptor (ActRII) in this tissue. Using primers corresponding to the published sequence of human ActRII, a 456bp fragment was obtained from cDNAs prepared from the placenta, as well as the ovary and brain, by polymerase chain reaction (PCR). Southern blot hybridization of the PCR products and DNA cloning and sequencing confirmed that the product is the authentic ActRII. Trophoblast cells prepared from both first trimester and term placentae expressed the ActRII gene. When trophoblast cells from term placenta were separated into syncytiotrophoblast- and cytotrophoblast-enriched fractions and incubated for 1-6 days, ActRII gene expression was observed in both cell preparations, with the syncytiotrophoblast-enriched fraction having higher levels of expression at days 1, 3, and 4. These results provide the first direct evidence that the activin type II receptor mRNA is present in human trophoblast cells and strengthen the hypothesis that activin is an autocrine/paracrine regulator of placental function. To our knowledge, this is also the first report that the ActRII gene is expressed in the human brain and ovary.

Cloning and characterization of human urocortin.

Urocortin, a new member of the CRF peptide family which also includes urotensin I and sauvagine, was recently cloned from the rat midbrain. The synthetic replicate of urocortin was found to bind with high affinity to type 1 and type 2 CRF receptors and, based upon its anatomic localization within the brain, was proposed to be a natural ligand for the type 2 CRF receptors. Using a genomic library, we have cloned the human counterpart of rat urocortin and localized it to human chromosome 2. Human and rat urocortin share 95% identity within the mature peptide region. Synthetic human urocortin binds with high affinity to CRF receptor types 1, 2 alpha, and 2 beta, stimulates cAMP accumulation from cells stably transfected with these receptors, and acts in vitro to release ACTH from dispersed rat anterior pituitary cells. In addition, the CRF-binding protein binds human urocortin with high affinity and can prevent urocortin-stimulated ACTH secretion in vitro. The inhibitory effect of the CRF-binding protein on human urocortin can be blocked by biologically inactive CRF fragments, such as CRF(9-33).

Human growth hormone-releasing hormone hGHRH(1-29)-NH2: systematic structure-activity relationship studies.

Two complete and two partial structure-activity relationship scans of the active fragment of human growth hormone-releasing hormone, [Nle27]-hGHRH(1-29)-NH2, have identified potent agonists in vitro. Single-point replacement of each amino acid by alanine led to the identification of [Ala8]-, [Ala9]-, [Ala15]- (Felix et al. Peptides 1986 1986, 481), [Ala22]-, and [Ala28, Nle27]-hGHRH(1-29)-NH2 as being 2-6 times more potent than hGHRH(1-40)-OH (standard) in vitro. Nearly complete loss of potency was seen for [Ala1], [Ala3], [Ala5], [Ala6], [Ala10], [Ala11], [Ala13], [Ala14], and [Ala23], whereas [Ala16], [Ala18], [Ala24], [Ala25], [Ala26], and [Ala29] yielded equipotent analogues and [Ala7], [Ala12], [Ala17], [Ala20], [Ala21], and [Ala27] gave weak agonists with potencies 15-40% that of the standard. The multiple-alanine-substituted peptides [MeTyr1,Ala15,22,Nle27]-hGHRH(1-29)-NH2 (29) and [MeTyr1,Ala8,9,15,22,28,Nle 27]-hGHRH(1-29)-NH2 (30) released growth hormone 26 and 11 times, respectively, more effectively than the standard in vitro. Individual substitution of the nine most potent peptides identified from the Ala series with the helix promoter alpha-aminoisobutyric acid (Aib) produced similar results, except for [Aib8] (doubling vs [Ala8]), [Aib9] (having vs [Ala9]), and [Aib15] (10-fold decrease vs [Ala15]). A series of cyclic analogues was synthesized having the general formula cyclo(25-29)[MeTyr1,-Ala15,Xaa25,Nle27,Yaa29+ ++]-GHRH(1-29)-NH2, where Xaa and Yaa represent the bridgehead residues of a side-chain cystine or [i-(i + 4)] lactam ring. The ring size, bridgehead amino acid chirality, and side-chain amide bond location were varied in this partial series in an attempt to maximize potency. Application of lactam constraints in the C-terminus of GHRH(1-29)-NH2 identified cyclo(25-29)[MeTyr1,Ala15,DAsp25,Nle27,Orn29+ ++]-hGHRH(1-29)-NH2 (46) as containing the optimum bridging element (19-membered ring) in this region of the molecule. This analogue (46) was 17 times more potent than the standard. Equally effective was an [i-(i + 3)] constraint yielding the 18-membered ring cyclo(25-28)[MeTyr1,Ala15,Glu25,Nle,27Lys28]- hGHRH-(1-29)-NH2 (51) which was 14 times more potent than the standard. A complete [i-(i + 3)] scan of cyclo(i,i + 3)[MeTyr1,Ala15,Glui,Lys(i + 3),Nle27]-hGHRH(1-29)-NH2 was then produced in order to test the effects of a Glu-to-Lys lactam bridge at all points in the peptide. Of the 26 analogues in the series, 11 had diminished potencies of less than 10% that of the agonist standard, 4 were weak agonists (15-40% relative potency), and 4 analogues were equipotent to the standard. The 7 most potent analogues ranged in potency from 3 to 14 times greater than that of the standard and contained the [i-(i + 3)] cycles between residues 4-7, 5-8, 9-12, 16-19, 21-24, 22-25, and 25-28. The combined results from these systematic studies allowed for an analysis of structural features in the native peptide that are important for receptor activation. Reinforcement of the characteristics of amphiphilicity, helicity, and peptide dipolar effects, using recognized medicinal chemistry approaches including introduction of conformational constraints, has resulted in several potent GHRH analogues.

Alanine series of ovine corticotropin releasing factor (oCRF): a structure-activity relationship study.

Previous structure-activity relationship studies of CRF have shown that residues 1-4 were not necessary for receptor binding or transduction, that residues 4-8 were important for activation, and that residues 12-41 were mostly responsible for binding. Finally it was proposed that CRF assumed an alpha-helical structure when interacting with its receptor. By systematic substitution of each residue (except residues 1-4) in ovine CRF (oCRF) by Ala, we have investigated the role played by individual side chains in receptor recognition and activation. Out of 33 analogues (synthesized using SPPS on an MBHA resin, purified by RPHPLC and characterized by amino acid and mass spectral analyses), a significant loss of biological potency (less than 1% potency of native) was observed for 6 analogues ([Ala6], [Ala8], [Ala10], [Ala12], [Ala14], and [Ala38]); 12 analogues had biological potencies ranging from 1% to 60% and ranked as follows: [Ala35] less than [Ala16] less than [Ala9] less than [Ala19] less than [Ala15] less than [Ala13] less than [Ala7] less than [Ala23] less than [Ala11] less than or equal to [Ala21] less than [Ala27] less than or equal to [Ala18]; 8 analogues were found to be equipotent (greater than 60% and less than 150%) ([Ala5], [Ala17], [Ala26], [Ala29], [Ala30], [Ala34], [Ala36], and [Ala37]; and 7 analogues were found to be approximately 2-5 times more potent than native oCRF ([Ala25] = [Ala40] less than or equal to [Ala39] less than or equal to [Ala33] less than [Ala20] less than [Ala22] less than [Ala32], in an in vitro pituitary cell culture assay. In summary, the Ala substitutions which showed the greatest loss of potency (less than 1% of native oCRF) were those replacing hydrophobic residues while those showing the greatest increase in potency were replacing hydrophilic residues. Of the 22 Ala-containing analogues in the C-terminal half of the molecule, 17 analogues have equal or greater potencies than native oCRF. Substitution of Ala in the N-terminal region (residues 5-19) on the other hand is generally detrimental to biological activity. These results suggest that the side chains of residues 5-19 are very important for receptor binding and activation while, in the C-terminal region, the amino acid side chains may be more responsible for structural conservation than for functional expression.

PROGINS Alu insertion and human genomic diversity.

A polymorphic Alu element belonging to the young Ya5 subfamily of Alu repeats located in the progesterone receptor gene has been characterized. Using a polymerase chain reaction (PCR)-based assay, the genetic diversity associated with the PROGINS Alu repeat was determined in a diverse array of human populations. The level of insertion polymorphism associated with PROGINS suggests that it will be a useful marker for the study of human evolution. In addition, we determined the distribution of the PROGINS Alu insertion in two groups of women from greater New Orleans, LA with breast cancer. The PROGINS Alu insertion was not associated with breast cancer in the populations tested.

Molecular cloning and binding properties of the human type II activin receptor.

A full-length cDNA for the type II human activin receptor was cloned by hybridization from a human testis cDNA library. The sequence encodes a 513 amino acid protein that is 99% identical, at the amino acid level, with the mouse type II activin receptor. The type II human activin receptor consists of an extracellular domain that specifically binds activin A with a Kd of 360 pM, a single-membrane spanning domain, and an intracellular kinase domain with predicted serine/threonine specificity.

The structures of the mouse and human urocortin genes (Ucn and UCN).

The mouse and human urocortin genes (Ucn and UCN, respectively) have been isolated, characterized, and found to have very similar structures. Each has two exons, and the entire coding region is located in the second exon, as is the case for the gene of the related peptide, corticotropin-releasing factor. Several putative transcription factor-binding sites were identified in each of the urocortin promoters, including a TATA box, a cyclic AMP response element (CRE), GATA-binding sites, and a C/EBP-binding site as well as a Brn-2-binding site(s). Sequence analyses of the mouse and human genes also revealed the presence of a previously identified gene, Mpv17, in the 5' region upstream of the urocortin gene. Functional studies following transient transfection of urocortin reporter plasmids in PC12 cells revealed that the urocortin promoter is controlled by both positive and negative elements; the CRE is important for basal activity as well as responsiveness to forskolin stimulation.

Molecular and functional characterization of GnRH receptors cloned from rat pituitary and a mouse pituitary tumor cell line.

A cDNA for a GnRH receptor (mtGnRH-R) was obtained from a mouse gonadotropic pituitary cell line (alpha T3-1) by expression cloning. This full-length cDNA was subsequently used as a probe to clone a rat pituitary GnRH receptor (rGnRH-R). The two receptors differ by 13 amino acids and are 100% identical to those recently reported. The analysis of the cloned receptors by photoaffinity-labeling followed by SDS-PAGE reveals a major band of approximately 70 kDa. This is in contrast to the native rat pituitary and mouse alpha T3-1 receptors whose major labeled species migrate with an apparent size of approximately 45 kDa. Functional studies reveal that both receptors, when transiently expressed in COSM6 cells, can bind GnRH with high affinity and transduce the stimulation of IP3 accumulation in response to GnRH.

Identification of a binding site on the type II activin receptor for activin and inhibin.

Type II activin receptors (ActRII and ActRIIB) are single-transmembrane domain serine/threonine kinase receptors that bind activin to initiate the signaling and cellular responses triggered by this hormone. Inhibin also binds type II activin receptors and antagonizes many activin effects. Here we describe alanine scanning mutagenesis of the ActRII extracellular domain. We identify a cluster of three hydrophobic residues (Phe(42), Trp(60), and Phe(83)) that, when individually mutated to alanine in the context of the full-length receptor, cause the disruption of activin and inhibin binding to ActRII. Each of the alanine-substituted ActRII mutants retaining activin binding maintains the ability to form cross-linked complexes with activin and supports activin cross-linking to the type I activin receptor ALK4. Unlike wild-type ActRII, the three mutants unable to bind activin do not cause an increase in activin signaling when transiently expressed in a corticotroph cell line. Together, our results implicate these residues in forming a critical binding surface on ActRII required for functional interactions with both activin and inhibin. This first identification of a transforming growth factor-beta family member binding site may provide a general basis for characterizing binding sites for other members of the superfamily.