Superovulation and embryo collection in nulliparous Boer goat does immunized against a recombinant ovine alpha-subunit inhibin

With the purpose of eliciting a superovulatory response, 12 adult nulliparous Boer goat does were actively immunized against a recombinant a-subunit of ovine inhibin (roIHN-alpha; two injections of 100 mg 4 weeks apart). Another 12 control Boer goat does were treated with physiological saline and acted as controls. One year later the immunized animals were boostered by the administration of another dose (100 mg) of the immunogen. Following treatment, blood samples were collected twice weekly for the periods of 16 and 12 weeks, respectively, to monitor the inhibin binding ability with the aid of a radio-tracer binding assay. Throughout the experiment, estrus detection was conducted twice daily with the aid of an aproned intact buck. From the first day after treatment to 48 h after standing estrus, ovarian activity was monitored daily by transrectal ultrasonography. On alternate estrous cycles, does were mated and 6 days later flushed transcervically to recover embryos. All goats treated with the roIHN-alpha produced antibodies reactive to the native bovine inhibin tracer-the titre increasing from 2.9 +/- 0.4 to a maximum of 21.9 +/- 2.9% binding after the second injection. The antibody titre gradually subsided over the next 16 weeks. The booster injection restored an elevated antibody titre (11.7 +/- 0.4%), which was maintained until the end of the sampling period 12 weeks later. In the control goats only trace amounts of antibody were recorded throughout the trial. In the roIHN-alpha-immunized goats the number of follicles reaching a diameter of > 4 mm was 14.6 +/- 1.2 per doe. A positive correlation was recorded between the follicle number and antibody titre (r=0.61; P < 0.01). The number of follicles ovulating per doe (6.9 +/- 0.7) followed the same tendency-however, the proportion decreased with increasing follicle numbers. A relatively weak correlation was recorded between the inhibin binding ability and number of ovulations (r=0.27; P < 0.05). In the control goats the majority (92%) of follicles exceeding 4 mm in diameter ovulated (2.5 +/- 0.1 follicles/doe). Embryo collection proved unsatisfactory (42% versus 39% recovery for immunized and control animals, respectively)-presumably because the uterine lumen of the nulliparous does was too narrow to permit effective flushing. In the group of immunized goats the occurrence of short estrous cycles (< 15 days) recorded was 34% versus only 6% in the controls. Overall, immunization of goats against roIHN-alpha led to an almost six-fold increase in number of ovarian follicles, a three-fold increase in ovulations and, despite the low recovery rate, a more than three-fold increase in ova or embryos recovered. It may be concluded that treatment of female goats with roIHN-alpha leads to an inhibin antibody response, accompanied by enhanced ovarian activity. The response was, however, accompanied by a large proportion of retained follicles and a high incidence of short estrous cycles. These problems need to be further investigated before rendering the method fit for application in embryo transfer programs in goats. (C) 2007 Elsevier B.V. All rights reserved.

Functional expression of a pseudohypoaldosteronism type I mutated epithelial Na+ channel lacking the pore-forming region of its alpha subunit.

The autosomal recessive form of type I pseudohypoaldosteronism (PHA-I) is an inherited salt-losing syndrome resulting from diminution-of-function mutations in the 3 subunits of the epithelial Na+ channel (ENaC). A PHA-I stop mutation (alpha(R508stop)) of the ENaC alpha subunit is predicted to lack the second transmembrane domain and the intracellular COOH-terminus, regions of the protein involved in pore function. Nonetheless, we observed a measurable Na+ current in Xenopus laevis oocytes that coexpress the beta and gamma subunits with the truncated alpha subunit. The mutant alpha was coassembled with beta and gamma subunits and was present at the cell surface at a lower density, consistent with the lower Na+ current seen in oocytes with the truncated alpha subunit. The single-channel Na+ conductance for the mutant channel was only slightly decreased, and the appearance of the macroscopic currents was delayed by 48 hours with respect to wild-type. Our data suggest novel roles for the alpha subunit in the assembly and targeting of an active channel to the cell surface, and suggest that channel pores consisting of only the beta and gamma subunits can provide significant residual activity. This activity may be sufficient to explain the absence of a severe pulmonary phenotype in patients with PHA-I.

IL-2/anti-IL-2 antibody complexes show strong biological activity by avoiding interaction with IL-2 receptor alpha subunit CD25.

IL-2 is crucial to T cell homeostasis, especially of CD4(+) T regulatory cells and memory CD8(+) cells, as evidenced by vigorous proliferation of these cells in vivo following injections of superagonist IL-2/anti-IL-2 antibody complexes. The mechanism of IL-2/anti-IL-2 antibody complexes is unknown owing to a lack of understanding of IL-2 homeostasis. We show that IL-2 receptor alpha (CD25) plays a crucial role in IL-2 homeostasis. Thus, prolongation of IL-2 half-life and blocking of CD25 using antibodies or CD25-deficient mice led in combination, but not alone, to vigorous IL-2-mediated T cell proliferation, similar to IL-2/anti-IL-2 antibody complexes. These data suggest an unpredicted role for CD25 in IL-2 homeostasis.

Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-a receptor

Abnormal production of interferon alpha (IFN-a) has been found in certain autoimmune diseases and can be also observed after prolonged therapy with IFN-a. IFN-a can contribute to the pathogenesis of allograft rejection in bone marrow transplants. Therefore, the development of IFN-a inhibitors as a soluble receptor protein may be valuable for the therapeutic control of these diseases. We have expressed two polypeptides encoding amino acids 93-260 (P1) and 261-410 (P2) of the extracellular domain of subunit 1 of the interferon-a receptor (IFNAR 1-EC) in E. coli. The activities of the recombinant polypeptides and of their respective antibodies were evaluated using antiproliferative and antiviral assays. Expression of P1 and P2 polypeptides was achieved by transformation of cloned plasmid pRSET A into E. coli BL21(DE3)pLysS and by IPTG induction. P1 and P2 were purified by serial sonication steps and by gel filtration chromatography with 8 M urea and refolded by dialysis. Under reducing SDS-PAGE conditions, the molecular weight of P1 and P2 was 22 and 17 kDa, respectively. Polyclonal anti-P1 and anti-P2 antibodies were produced in mice. P1 and P2 and their respective polyclonal antibodies were able to block the antiproliferative activity of 6.25 nM IFN-aB on Daudi cells, but did not block IFN-aB activity at higher concentrations (>6.25 nM). On the other hand, the polypeptides and their respective antibodies did not inhibit the antiviral activity of IFN-aB on Hep 2/c cells challenged with encephalomyocarditis virus.

A cell type-specific constitutive point mutant of the common beta-subunit of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 receptors requires the GM-CSF receptor alpha-subunit for activation

The high affinity receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a cytokine-specific alpha-subunit (hGMR alpha) and a common signal-transducing beta-subunit (hpc) that is shared with the interleukin-3 and -5 receptors, We have previously identified a constitutively active extracellular point mutant of hpc, I374N, that can confer factor independence on murine FDC-P1 cells but not BAF-B03 or CTLL-2 cells (Jenkins, B. J., D'Andrea, R. J., and Gonda, T. J. (1995) EMBO J. 14, 4276-4287), This restricted activity suggested the involvement of cell type-specific signaling molecules in the activation of this mutant. We report here that one such molecule is the mouse GMR alpha (mGMR alpha) subunit, since introduction of mGMR alpha, but not hGMR alpha, into BAF-B03 or CTLL-2 cells expressing the I374N mutant conferred factor independence, Experiments utilizing mouse/human chimeric GMR alpha subunits indicated that the species specificity lies in the extracellular domain of GMRa. Importantly, the requirement for mGMR alpha correlated with the ability of I374N (but not wild-type hpc) to constitutively associate with mGMRa. Expression of I374N in human factor-dependent UT7 cells also led to factor-independent proliferation, with concomitant up-regulation of hGMR alpha surface expression. Taken together, these findings suggest a critical role for association with GMR alpha in the constitutive activity of I374N.

Increased ovulation rate in Merino ewes immunized against small synthetic peptide fragments of the inhibin alpha subunit

Four experiments were carried out in Merino ewes during a period of 4 years to determine the long-term effects of immunization against different synthetic peptides mimicking the amine terminal of the or subunit of porcine inhibin. Peptides were conjugated to human serum albumin and 100-200 mu g emulsified in Freund's complete adjuvant for the primary immunization. Usually two booster injections were given at monthly intervals with 50-100 mu g conjugated peptide using either incomplete Freund's adjuvant or Montanide : Marcel. In some experiments a further immunization was carried in the next year. Blood samples were taken 10 days after each immunization, during the luteal phase, for estimation of gonadotrophin concentrations and determination of inhibin antibody titres. One day after blood sampling cloprostenol was used to induce luteolysis and laparoscopy was performed in the subsequent oestrous cycle. Immunization of ewes with synthetic peptides 1-32, 1-26, 7-26 and 8-30 resulted in large increases in the ovulation rate (OR). An approximately two-fold increase in OR was observed following the first booster immunization with these peptides and a three- to five-fold increase after the second booster immunization. Immunization with these large peptides resulted in a sustained increase in OR for a period of at least 1 year after the second booster immunization. Of the shorter peptides, peptides 10-26 and 13-26 gave a reasonable ovulatory response, although it was more difficult to obtain a response with peptides 1-16, 8-22, 13-25, 8-19 and 10-19; peptides 7-13 and 1-6 gave no response (but were examined for one breeding season only). The smaller peptides led to lower inhibin antibody titres that were not necessarily associated with increased follicle-stimulating hormone (FSH) or OR. More intensive blood sampling in one experiment showed that following primary immunization against peptide 1-32 there was a transient increase in plasma FSH which did not lead to an increased OR. Moreover, a prolonged period of raised FSH after the first booster was significantly correlated with increased OR. In these animals antibody titres were only slightly increased after primary immunization, but after the first booster immunization higher titres were observed that were significantly correlated with trough FSH values and the subsequent OR. These results are interpreted as showing that (1) to obtain an increase in OR peptides 1-32, 1-26 and 7-26 are suitable as immunogens; (2) smaller peptides are less reliable, often require multiple injections, and the response may be delayed; and (3) an extended period of raised plasma FSH is needed to give a large ovulatory response.

GABA(A) receptor alpha-subunit proteins in human chronic alcoholics

Antibodies were raised against specific peptides from N-terminal regions of the alpha (1) and alpha (3) isoforms of the GABA(A) receptor, and used to assess the relative expression of these proteins in the superior frontal and primary motor cortices of 10 control, nine uncomplicated alcoholic and six cirrhotic alcoholic cases were matched for age and post-mortem delay. The regression of expression on post-mortem delay was not statistically significant for either isoform in either region. In both cortical areas, the regression of a, expression on age differed significantly between alcoholic cases, which showed a decrease, and normal controls, which did not. Age had no effect on alpha (3) expression. The alpha (1) and alpha (3) isoforms were found to be expressed differentially across cortical regions and showed a tendency to be expressed differentially across case groups. In cirrhotic alcoholics, alpha (1) expression was greater in superior frontal than in motor cortex, whereas this regional difference was not significant in controls or uncomplicated alcoholics. In uncomplicated alcoholics, alpha (3) expression was significantly lower in superior frontal than in motor cortex. Expression of alpha (1) was significantly different from that Of alpha (3) in the superior frontal cortex of alcoholics, but not in controls. In motor cortex, there were no significant differences in expression between the isoforms in any case group.

Biochemical characterization of two mutants of human pyruvate dehydrogenase, F205L and T231A of the E1 alpha subunit

Mutations in the E1alpha subunit of the pyruvate dehydrogenase multienzyme complex may result in congenital lactic acidosis, but little is known about the consequences of these mutations at the enzymatic level. Here we characterize two mutants (F205L and T231A) of human pyruvate dehydrogenase in vitro, using the enzyme expressed in Escherichia coli. Wild-type and mutant proteins were purified successfully and their kinetic parameters were measured. F205L shows impaired binding of the thiamin diphosphate cofactor, which may explain why patients carrying this mutation respond to high-dose vitamin B-1 therapy. T231A has very low activity and a greatly elevated K-m for pyruvate, and this combination of effects would be expected to result in severe lactic acidosis. The results lead to a better understanding of the consequences of these mutations on the functional and structural properties of the enzyme, which may lead to improved therapies for patients carrying these mutations.

The fourth extracellular loop of the alpha subunit of Na,K-ATPase. Functional evidence for close proximity with the second extracellular loop.

Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.

The fourth transmembrane segment of the Na,K-ATPase alpha subunit: a systematic mutagenesis study.

The Na,K-ATPase is a major ion-motive ATPase of the P-type family responsible for many aspects of cellular homeostasis. To determine the structure of the pathway for cations across the transmembrane portion of the Na,K-ATPase, we mutated 24 residues of the fourth transmembrane segment into cysteine and studied their function and accessibility by exposure to the sulfhydryl reagent 2-aminoethyl-methanethiosulfonate. Accessibility was also examined after treatment with palytoxin, which transforms the Na,K-pump into a cation channel. Of the 24 tested cysteine mutants, seven had no or a much reduced transport function. In particular cysteine mutants of the highly conserved "PEG" motif had a strongly reduced activity. However, most of the non-functional mutants could still be transformed by palytoxin as well as all of the functional mutants. Accessibility, determined as a 2-aminoethyl-methanethiosulfonate-induced reduction of the transport activity or as inhibition of the membrane conductance after palytoxin treatment, was observed for the following positions: Phe(323), Ile(322), Gly(326), Ala(330), Pro(333), Glu(334), and Gly(335). In accordance with a structural model of the Na,K-ATPase obtained by homology modeling with the two published structures of sarcoplasmic and endoplasmic reticulum calcium ATPase (Protein Data Bank codes 1EUL and 1IWO), the results suggest the presence of a cation pathway along the side of the fourth transmembrane segment that faces the space between transmembrane segments 5 and 6. The phenylalanine residue in position 323 has a critical position at the outer mouth of the cation pathway. The residues thought to form the cation binding site II ((333)PEGL) are also part of the accessible wall of the cation pathway opened by palytoxin through the Na,K-pump.

Access of extracellular cations to their binding sites in Na,K-ATPase: role of the second extracellular loop of the alpha subunit.

Na,K-ATPase, the main active transport system for monovalent cations in animal cells, is responsible for maintaining Na(+) and K(+) gradients across the plasma membrane. During its transport cycle it binds three cytoplasmic Na(+) ions and releases them on the extracellular side of the membrane, and then binds two extracellular K(+) ions and releases them into the cytoplasm. The fourth, fifth, and sixth transmembrane helices of the alpha subunit of Na,K-ATPase are known to be involved in Na(+) and K(+) binding sites, but the gating mechanisms that control the access of these ions to their binding sites are not yet fully understood. We have focused on the second extracellular loop linking transmembrane segments 3 and 4 and attempted to determine its role in gating. We replaced 13 residues of this loop in the rat alpha1 subunit, from E314 to G326, by cysteine, and then studied the function of these mutants using electrophysiological techniques. We analyzed the results using a structural model obtained by homology with SERCA, and ab initio calculations for the second extracellular loop. Four mutants were markedly modified by the sulfhydryl reagent MTSET, and we investigated them in detail. The substituted cysteines were more readily accessible to MTSET in the E1 conformation for the Y315C, W317C, and I322C mutants. Mutations or derivatization of the substituted cysteines in the second extracellular loop resulted in major increases in the apparent affinity for extracellular K(+), and this was associated with a reduction in the maximum activity. The changes produced by the E314C mutation were reversed by MTSET treatment. In the W317C and I322C mutants, MTSET also induced a moderate shift of the E1/E2 equilibrium towards the E1(Na) conformation under Na/Na exchange conditions. These findings indicate that the second extracellular loop must be functionally linked to the gating mechanism that controls the access of K(+) to its binding site.

Phosphorylation of the Na,K-ATPase alpha-subunit by protein kinase A and C in vitro and in intact cells. Identification of a novel motif for PKC-mediated phosphorylation.

Na,K-ATPase is a potential target for regulatory phosphorylation by protein kinase A and C (PKA and PKC). To identify the phosphorylation sites, we have mutated the alpha 1-subunit of Bufo marinus in a highly conservative PKA and in 20 different PKC consensus sequences. The mutants were expressed in Xenopus oocytes and their phosphorylation capacity tested in homogenates upon stimulation of PKA or PKC. While serine 943 (Ser-943) was identified as a unique target site for PKA, none of the PKC consensus serine or threonine residues are implicated in PKC phosphorylation. Controlled trypsinolysis of phosphorylated alpha-subunits of various purified enzyme preparations and of alpha/beta complexes from oocyte homogenates revealed that PKC phosphorylation was exclusively associated with the N terminus. A fusion protein containing the first 32 amino acids of the Bufo alpha-subunit was phosphorylated in vitro and serine and threonine residues (Thr-15 and Ser-16) in this region were identified by site-directed mutagenesis as the PKC phosphorylation sites. Finally, the Bufo alpha-subunit was phosphorylated by protein kinases in transfected COS-7 cells. In intact cells, PKA stimulation induced phosphorylation exclusively on Ser-943 and PKC stimulation mainly on Thr-15 and Ser-16, which are contained in a novel PKC phosphorylation motif.

A novel dominant mutation of the Nav1.4 alpha-subunit domain I leading to sodium channel myotonia.

BACKGROUND: Mutations in SCN4A may lead to myotonia. METHODS: Presentation of a large family with myotonia, including molecular studies and patch clamp experiments using human embryonic kidney 293 cells expressing wild-type and mutated channels. RESULTS: In a large family with historic data on seven generations and a clear phenotype, including myotonia at movement onset, with worsening by cold temperature, pregnancy, mental stress, and especially after rest after intense physical activity, but without weakness, the phenotype was linked with the muscle sodium channel gene (SCN4A) locus, in which a novel p.I141V mutation was found. This modification is located within the first transmembrane segment of domain I of the Na(v)1.4 alpha subunit, a region where no mutation has been reported so far. Patch clamp experiments revealed a mutation-induced hyperpolarizing shift (-12.9 mV) of the voltage dependence of activation, leading to a significant increase (approximately twofold) of the window current amplitude. In addition, the mutation shifted the voltage dependence of slow inactivation by -8.7 mV and accelerated the entry to this state. CONCLUSIONS: We propose that the gain-of-function alteration in activation leads to the observed myotonic phenotype, whereas the enhanced slow inactivation may prevent depolarization-induced paralysis.

Evidence for catabolic pathway of propionate metabolism in CNS: expression pattern of methylmalonyl-CoA mutase and propionyl-CoA carboxylase alpha-subunit in developing and adult rat brain.

Methylmalonyl-CoA mutase (MCM) and propionyl-CoA carboxylase (PCC) are the key enzymes of the catabolic pathway of propionate metabolism and are mainly expressed in liver, kidney and heart. Deficiency of these enzymes leads to two classical organic acidurias: methylmalonic and propionic aciduria. Patients with these diseases suffer from a whole spectrum of neurological manifestations that are limiting their quality of life. Current treatment does not seem to effectively prevent neurological deterioration and pathophysiological mechanisms are poorly understood. In this article we show evidence for the expression of the catabolic pathway of propionate metabolism in the developing and adult rat CNS. Both, MCM and PCC enzymes are co-expressed in neurons and found in all regions of the CNS. Disease-specific metabolites such as methylmalonate, propionyl-CoA and 2-methylcitrate could thus be formed autonomously in the CNS and contribute to the pathophysiological mechanisms of neurotoxicity. In rat embryos (E15.5 and E18.5), MCM and PCC show a much higher expression level in the entire CNS than in the liver, suggesting a different, but important function of this pathway during brain development.

Novel Cone Transducin Alpha Subunit Mutation In Tunisian Patients And Genotype-phenotype Correlation In Complete Achromatopsia

Purpose: Complete achromatopsia is a rare autosomal recessive disease due to CNGA3, CNGB3, GNAT2 and PDE6C mutations. We studied a large consanguineous Tunisian family including twelve individuals.Methods: Ophthalmic evaluation included a full clinical examination, color vision testing, optical coherence tomography and electroretinography. Linkage analysis using microsatellite markers flanking CNGA3, CNGB3, GNAT2 and PDE6C genes was performed. Mutations were screened by direct sequencing.Results: In all affected subjects, acuity ranged from 20/50 to 20/200. Fundus examination was normal except for two patients who had respectively 4 mm and 5 mm diameters of peripheral congenital hypertrophy. Likewise retinal layers exploration by OCT revealed no change in the thickness of the central retina. Color Vision with 100 Hue Farnsworth test described a profound color impairment along all three axes of color vision. The haplotype analysis of GNAT2 markers revealed that all affected offspring were homozygous by descent for the four polymorphic markers. The maximum lod score value, 4.33, confirmed the evidence for linkage to the GNAT2 gene.A homozygous novel nonsense mutation R313X was identified segregating with an identical GNAT2 haplotype in all affected subjects. This mutation could interrupt interaction with photoactivated rhodopsin, resulting in a failure of visual transduction. In fact, ERG showed a clearly abolished photopic b-wave and flicker responses with no residual cone function justifying the severe GNAT2 achromatopsia phenotype.Conclusions: This is the first report of the clinical and genetic investigation of complete achromatopsia in North Africa and of the largest family with recessive achromatopsia involving GNAT2, thus providing a unique opportunity for genotype phenotype correlation for this extremely rare condition.