Mature Peptide Region Research Articles

Polymorphism at the defensin gene in the Anopheles gambiae complex: Testing different selection hypotheses

Genetic variation in defensin, a gene encoding a major effector molecule of insects immune response was analyzed within and between populations of three members of the Anopheles gambiae complex. The species selected included the two anthropophilic species, An. gambiae and An. arabiensis and the most zoophilic species of the complex, An. quadriannulatus. The first species was represented by four populations spanning its extreme genetic and geographical ranges, whereas each of the other two species was represented by a single population. We found (i) reduced overall polymorphism in the mature peptide region and in the total coding region, together with specific reductions in rare and moderately frequent mutations (sites) in the coding region compared with non-coding regions, (ii) markedly reduced rate of non-synonymous diversity compared with synonymous variation in the mature peptide and virtually identical mature peptide across the three species, and (iii) increased divergence between species in the mature peptide together with reduced differentiation between populations of An. gambiae in the same DNA region. These patterns suggest a strong purifying selection on the mature peptide and probably the whole coding region. Because An. quadriannulatus is not exposed to human pathogens, identical mature peptide and similar pattern of polymorphism across species implies that human pathogens played no role as selective agents on this peptide.

Mutational analysis of BMP15 and GDF9 as candidate genes for premature ovarian failure

Mutational screening of the bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) genes in a population with premature ovarian failure (POF) identified no new mutations. However, three single nucleotide polymorphisms in the BMP15 gene, two in the 5' untranslated region (31T>G and 71C>G) and another in exon 1 (387G>A), were found to be common in both POF and control groups.

Discovery of a novel class of conotoxin from Conus litteratus, lt14a, with a unique cysteine pattern

Conus litteratus is a worm-hunting cone snail with a highly sophisticated neuropharmacological defense strategy using small peptides in its venom. By analyzing different clones in the cDNA library of venom ducts from C. litteratus, we identified the peptide lt14a which displays a characteristic signal peptide sequence in its precursor and a unique arrangement of Cys residues (–C–C–C–C–) in its mature peptide region. RT-PCR analysis suggested that lt14a is abundantly expressed throughout the whole venom duct. An intensive analysis in sequence suggested that lt14a is similar to α-conotoxin qc1.1 cloned from Conus quercinus. We conducted the chemical synthesis of lt14a. The synthetic lt14a has a remarkable biological activity to suppress pain and inhibits the neuronal-type nicotinic acetylcholine receptors.

Enhanced Synonymous Site Divergence in Positively Selected VertebrateAntimicrobial Peptide Genes

Nonrandom patterns associated with adaptively evolving genes can shed light on how selection and mutation produce rapid changes in sequences. I examine such patterns in two independent families of antimicrobial peptide genes: those in frogs, which are known to have evolved under positive selection, and those in flatfishes, which I show have also evolved under positive selection. I address two recently proposed hypotheses about the molecular evolution of antimicrobial peptide genes. The first is that the mature peptide region is replicated by an error-prone polymerase that increases the mutation rate and the transversion/transition ratio compared to the signal sequence of the same genes. The second is that mature peptides evolve in a coordinated fashion with their propieces, such that a change in net charge in one molecular region prompts an opposite change in charge in the other region. I test these hypotheses using alternative methods that minimize alignment errors, correct for phylogenetic nonindependence, reduce sequence saturation, and account for differing selection pressures on different regions of the gene. In both gene families I show that divergence at both synonymous and nonsynonymous sites within the mature peptide region is enhanced. However, in neither gene family is there evidence of an increased mutational transversion/transition ratio or coordinated evolution. My observations are consistent with either an elevated mutation rate in an adaptively evolving gene region or widespread selection on "silent" sites. These hypotheses challenge the assumption that mutations are random and can be measured by the synonymous substitution rate.

Molecular Cloning and Characterization of Three β-Defensins from Canine Testes

ABSTRACTMammalian β-defensins are small cationic peptides possessing broad antimicrobial and physiological activities. Because dogs are particularly resilient to sexually transmitted diseases, it has been proposed that their antimicrobial peptide repertoire might provide insight into novel antimicrobial therapeutics and treatment regimens. To investigate this proposal, we cloned the full-length cDNA of three canine β-defensin isoforms (cBD-1,-2, and-3) from canine testicular tissues. Their predicted peptides share identical N-terminal 65-amino-acid residues, including the β-defensin consensus six-cysteine motif. The two longer isoforms,cBD-2and-3, possess 4 and 34 additional amino acids, respectively, at the C terminus. To evaluate the antimicrobial activity of cBD, a 34-amino-acid peptide derived from the shared mature peptide region was synthesized. Canine β-defensin displayed broad antimicrobial activity against gram-positive bacteria (Listeria monocytogenesandStaphylococcus aureus; MICs of 6 and 100 μg/ml, respectively), gram-negative bacteria (Escherichia coli,Klebsiella pneumoniae, andNeisseria gonorrhoeae; MICs of 20 to 50, 20, and 50 μg/ml, respectively), and yeast (Candida albicans; MIC of 5 to 50 μg/ml) and lower activity againstUreaplasma urealyticumandU. canigenitalium(MIC of 200 μg/ml). Antimicrobial potency was significantly reduced at salt concentrations higher than 140 mM. All three canine β-defensins were highly expressed in testis. In situ hybridization indicated thatcBD-1was expressed primarily in Sertoli cells within the seminiferous tubules. In contrast,cBD-2was located primarily within Leydig cells. The longest isoform,cBD-3, was detected in Sertoli cells and to a lesser extent in the interstitium. The tissue-specific expression and broad antimicrobial activity suggest that canine β-defensins play an important role in host defense and other physiological functions of the male reproductive system.

Euphorbia lathyris에서 분비되는 Latex 65kD 단백질의 특성규명

Euphorbia lathyris의 유관세포로부터 분비되는 수용성 latex단백질을 10% SDS-polyacrylamide 전기영동으로 분리하여 특징적으로 잘 나타나고 있는 ELp65, ELp55, ELp43, ELp32 그리고 ELp23 등의 주요단백질을 확인하였다. 이들 latex 주요단백질들 중에서 ELp65는 ammonium sulfate 침전, gel permeation chromatography 그리고 ion exchange chromatography 등의 방법으로 순수 분리하였는데 ELp65의 N-terminal amino acid 서열분석에 의하면 이는 토마토의 p69a subtilisin-like pretense의 mature peptide의 앞부분과 강한 유사성을 지니고 있었으며 식물방어와 관련된 기능이 제시되었다. PCR증폭에 의하여 클로닝된 토마토의 p69a DNA를 probe로 이용하여 Southern blot hybridization을 수행한 결과 E. lathyris genome은 토마토의 subtilisin-like proteases와 유사한 정보를 지닐 수도 있는 3-5 유전자들로 구성된 gene family가 분석되었다. Soluble latex protein fraction excreted from Euporbia lathyris laticifer was resolved by 10% SDS-polyacrylamide gel electrophoresis to identify distinctively displayed latex major protein bands including ELp65, ELp55, ELp43, ELp32 and ELp23. Among them, ELp65 was purified by ammonium sulfate precipitation, gel permeation chromatography and ion exchange chromatography. Its N-terminal amino acid sequencing revealed its homology to the leading region of mature peptide of tomato p69a subtilisin-like protease, suggesting a certain role involved in plant defense system. In the analysis of Southern blot hybridization using PCR-amplified tomato p69a probe DNA, E. lathyris genome was suggested to have a gene family consisting of 3-5 gene members putatively encoding subtilisin-like proteases.

Mutational analysis of the mature peptide region of inhibin genes in Indian women with ovarian failure.

Clinically, premature ovarian failure (POF) is defined as unexplained amenorrhoea (> 6 months) with a high FSH level (> 40 IU) before the age of 40 years. POF is a heterogeneous genetic disease with unknown aetiology. Inhibin and activin regulate the FSH level by their opposing actions and thus have been considered as strong candidate genes in the aetiology of POF. We have screened inhibin genes in patients with POF (n = 80), primary amenorrhoea (n = 33) and secondary amenorrhoea (n = 4). INHbetaB and INHbetaA genes do not show any association with ovarian failure. We found the Ala257Thr missense mutation in INHalpha gene with high statistical significance in POF (nine out of 80, 11.2%) (Fisher's exact test, P = 0.0005), primary amenorrhoea (three out of 33, 9.1%) (Fisher's exact test, P = 0.014) and secondary amenorrhoea (two out of four, 50%) (Fisher's exact test, P = 0.001) with complete absence of this mutation in controls (none out of 100). The INHalpha gene is a strong candidate gene for ovarian failure. Mutations in INHbetaB and INHbetaA genes are not associated with ovarian failure.

821. A Potential Therapeutic Angiogenesis Using a Naked DNA Containing Angiogenin cDNA

Top of pageAbstract A promising therapeutic angiogenesis using gene transfer of angiogenic regulators has been extensively investigated and developed for the treatment of various diseases including cardiovascular diseases such as coronary and peripheral artery disease, ischemic disease, restenosis, vein-graft failure, as well as ulcers. Many clinical trials and animal studies have shown that administration of angiogenic factors can increase permeability and nutrient perfusion to induce the formation of supplemental collateral blood vessel. In those studies, the best angiogenic factors are vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). However, other angiogenic factors showing better angiogenic activity need to be screened. Although angiogenin has been known that it interacts with endothelial and smooth muscle cells to induce a wide range of cellular responses including cell migration, invasion, proliferation, and neovascularization, a possibility of therapeutic approach using angiogenin remains unclear. Here we investigated a potential therapeutic angiogenesis using a naked DNA containing angiogenin cDNA. Angiogenin cDNA that contains either of whole open reading frame including signal peptide (SPANG1) or only mature peptide region (ANG1) was inserted into the linearized pEGFP-N1 vector (Clontech) to generate two different subclones, so called pSPANG1-EGFP and pANG1-EGFP. HEK293 or TSA-201 cells (a transformed HEK293 cell line that stably expresses the SV40 T-antigen) were used for transfection to express those of two different constructs. The angiogenin-GFP protein expression was monitored by a confocal microscopy and the expression pattern of angiogenin-GFP was examined using western blot analyses. Both of angiogenin-GFP proteins containing the signal peptide or not migrated to the same position of 43 kD. The angiogenin-GFP protein containing the signal peptide in the cell lysates began to be detected at 48 hr post-transfection, whereas the angiogenin-GFP protein of the mature peptide in the cell lysates was first detected at 24 hr after transfection. On the other hand, the secreted angiogenin-GFP from the cells expressing pSPANG1-EGFP was first detected at 72 hr post-transfection, in contrast, the secreted angiogenin-GFP from the cells expressing pANG1-EGFP was first measurable at 24 hr after transfection. The angiogenin-GFP expression from the cells transfected with both type of constructs was maintained to 96 hr post-transfection. The functional activity of the expressed angiogenin-GFP proteins was tested by a wound-healing assay. More studies using pCIneo-SPANG1 and pCIneo-ANG1 are currently under investigation to verify that GFP tagging does not interfere angiogenic activity of angiogenin. Animal studies using four different types of naked DNA containing angiogenin cDNA remain to be done. Taken together, these data suggest that the naked DNA containing angiogenin cDNA may be a potential therapeutic angiogenesis for the treatment of cardiovascular diseases.

Cloning of transforming growth factor-beta 1 (TGF-beta 1) and its type II receptor from zebrafish ovary and role of TGF-beta 1 in oocyte maturation.

TGF-beta is a multifunctional factor involved in regulating a variety of cellular activities. In mammals, TGF-beta is known to regulate reproduction, including ovarian functions. The role of TGF-beta in lower vertebrates, such as fish, is poorly understood. To examine the role of TGF-beta in fish reproduction, cDNAs encoding TGF-beta 1 and the type II TGF-beta receptor (T beta RII) were cloned from the zebrafish ovary using PCR- based strategies. The mature peptide region of the zebrafish TGF-beta 1 shows 70-85% identity with TGF-beta 1 from other species. The zebrafish T beta RII cDNA sequence is the first to be reported from a fish species, and it shows a high level of conservation at the kinase domain. Using RT-PCR, we have detected mRNA expression of TGF-beta 1, T beta RII, as well as its downstream signaling molecules Smad2, 3, and 4 in ovarian follicles at different stages of development. In addition, we have examined the effect of TGF-beta 1 on oocyte maturation. TGF-beta 1 significantly inhibited both gonadotropin- and 17 alpha, 20 beta-dihydroxyprogesterone-induced oocyte maturation in a dose- and time-dependent manner. These findings demonstrate, for the first time, that TGF-beta 1 plays a role in regulating oocyte maturation in fish and suggest that a TGF-beta/Smad signaling pathway is present in the zebrafish ovary.

Molecular characterisation of sea bream (Sparus aurata) transforming growth factor β1

A transforming growth factor β1 (TGF β1) full length cDNA was characterised and sequenced from the head kidney of sea bream (Sparus aurata) previously challenged with a nodavirus. The cloned cDNA of 1778bp contains a predicted open reading frame of 379 amino acids, which includes the mature peptide region of 112 amino acids. The regulating region of the peptide possesses four potential N-linked glycosylation sites (N-X-T/S), as well as an RGD integrin binding site, an RKKR tetrabasic cut site and nine conserved cysteines all characteristic of the TGF β superfamily. Compared to other teleost TGF β1 genes, the sea bream TGF β1 is most closely related to hybrid striped bass (Moronesaxatilis ×M. chrysops) TGF β1 (80% amino acid identity).The genomic organisation of TGF β1 was determined through the generation of contiguous PCR clones. The sea bream TGF β1 gene is approximately 3·6kb in length and consists of five coding regions. Two introns are absent in comparison to the genomic organisation of rainbow trout Oncorhynchus mykiss TGF β1, whilst an additional intron not present in other sequenced TGF β genes, but present in the trout TGF β1 gene, is conserved in sea bream.A reverse transcription polymerase chain reaction (RT-PCR) assay was developed to study TGF β expression in different sea bream tissues. Constitutive TGF β1 expression was detected in the liver, brain, muscle, kidney, heart, gills and spleen of sea bream, as well as in head kidney macrophages and blood leucocytes.

Signal sequence conservation and mature peptide divergence within subgroups of the murine beta-defensin gene family.

Beta-defensins are two exon genes which encode broad spectrum antimicrobial cationic peptides. We have analyzed the largest murine cluster of these genes which localizes to chromosome 8. Using hidden Markov models, we identified six beta-defensin exon 2-like sequences and subsequently found full-length expressed transcripts for these novel genes. Expression was high in brain and reproductive tissues. Eleven beta-defensins could be grouped into two clear subgroups by virtue of their position and high signal sequence (exon 1 encoded) identity. In contrast, however, there was a very low level of sequence conservation in the exon 2 region encoding the mature antimicrobial peptide. Examination of the gene sequences of orthologs in other rodents also revealed an excess of nucleotide changes that altered amino acids in the mature peptide region. Evolutionary analysis revealed strong evidence that following gene duplication, exon 1 and surrounding noncoding DNA show little divergence within subgroups. The focus for rapid sequence divergence is localized in the DNA encoding the mature peptide and this is driven by accelerated positive selection. This mechanism of evolution is consistent with the role of this gene family as defense against bacterial pathogens and the sequence changes have implications for novel antibiotic design.

Characterization of complementary deoxyribonucleic acids encoding preprogalanin and its alternative splice variants in the goldfish

This paper reports the identification of five preprogalanin complementary deoxyribonucleic acids encoding preprogalanin peptides in the goldfish. Preprogalanin 1A, 1B and 1C are encoded in galanin gene 1 and 2A and 2B are encoded in gene 2. Preprogalanin 1B and 2B have a 24 amino acids insert in the mature peptide region and form 1C has a deletion of 23 amino acids in the middle of the galanin message associated peptide region. The mature peptides from the preprogalanin 1A and 1C are 29 amino acids. However, the mature peptide is 31 amino acids from preprogalanin 2A, 53 amino acids from 1B and 55 amino acids from 2B. The physiological significance of multiple forms of galanin peptide is unknown. Organization of galanin gene 1, which is similar to the mammalian galanin gene has been identified. Expression of preprogalanin messenger ribonucleic acids was widely detected in goldfish brain and several peripheral tissues.

Goldfish ghrelin: molecular characterization of the complementary deoxyribonucleic acid, partial gene structure and evidence for its stimulatory role in food intake.

Complementary deoxyribonucleic acid (cDNA) encoding goldfish preproghrelin was identified using rapid amplification of the cDNA ends (RACE) and reverse transcription (RT)-polymerase chain reaction (PCR). The 490 bp cDNA encodes a 103 amino acid preproghrelin which has a 26 amino acid signal region, 19 amino acid mature peptide and a 55 amino acid C-terminal peptide region. The mature peptide region of goldfish ghrelin has two putative cleavage sites and amidation signals (GRR); one after 12 amino acids and the other after 19 amino acids. The serine (S) in the second amino acid position in the "active core" of ghrelin is substituted with threonine (T). The goldfish ghrelin gene has four exons and three short introns and resembles the human ghrelin gene. Ghrelin messenger RNA (mRNA) expression was detected in the brain, pituitary, intestine, liver, spleen and gill by RT-PCR followed by Southern blot analysis, and in the intestine by Northern blot. Intracerebroventricular (ICV) injection of n-octanoylated goldfish ghrelin (1-19) stimulates food intake in goldfish.

Differential Expression of Insulin-like Growth Factor I and II mRNAs during Embryogenesis and Early Larval Development in Rabbitfish, Siganus guttatus

In rodents, the expression of insulin-like growth factor II (IGF-II) is higher than that of insulin-like growth factor I (IGF-I) during fetal life while the reverse is true after birth. We wanted to examine whether this is also true in fish and whether IGF-I and IGF-II are differentially regulated during different stages of embryogenesis and early larval development in rabbitfish. We first cloned the cDNAs of rabbitfish IGF-I and IGF-II from the liver. Rabbitfish IGF-I has an open reading frame of 558 bp that codes for a signal peptide of 44 amino acids (aa), a mature protein of 68 aa, and a single form of E domain of 74 aa. Rabbitfish IGF-II, on the other hand, has an open reading frame of 645 bp that codes for a signal peptide of 47 aa, a mature protein of 70 aa, and an E domain of 98 aa. On the amino acid level, rabbitfish IGF-I shares 68% similarity with IGF-II. We then examined the relative expression of the two IGFs in unfertilized eggs, during different stages of embryogenesis, and in early larval stages of rabbitfish by a semiquantitative reverse transcription-polymerase chain reaction. Primers that amplify the mature peptide region of both IGFs were used and PCR for both peptides was done simultaneously, with identical PCR conditions for both. The identity of the PCR products was confirmed by direct sequencing. Contrary to published reports for seabream and rainbow trout, IGF-I mRNA was not detected in rabbitfish unfertilized eggs; it was first expressed in larvae soon after hatching. IGF-II mRNA, however, was expressed in unfertilized eggs, albeit weakly, and was already strongly expressed during the cleavage stage. mRNAs for both peptides were strongly expressed in the larvae, although IGF-II mRNA expression was higher than IGF-I expression.

Gonad-inhibiting hormone of the Norway lobster (Nephropsnorvegicus): cDNA cloning, expression, recombinant protein production, and immunolocalization

The gonad-inhibiting hormone (GIH) belongs to a neuropeptide family synthesized and released in a neurohemal complex of crustacean eyestalks. The GIH is involved in gonad maturation and plays a more complex role in the control of reproduction and molting. With a combination of reverse transcription–polymerase chain reaction (RT–PCR) and rapid amplification of cDNA ends approaches we determined the cDNA sequence of the Norway lobster Nephropsnorvegicus prepro GIH. The open reading frame of 339 bp codes for a polypeptide of 112 amino acids showing 96% identity with the other known GIH of Homarusamericanus. The precursor peptide consists of a putative signal peptide of 31 amino acids and a putative mature peptide region of 81. RT–PCR analysis shows that GIH mRNA is expressed mainly in eyestalks, both in female and male; the expression of GIH mRNA also in supraesophageal ganglia suggests the existence of additional GIH-producing neurons besides those of eyestalks. A specific polyclonal antibody was raised against a portion of the mature peptide region obtained through expression in Escherichiacoli fused to glutathione-S-transferase. Immunocytochemical studies were carried out by using this antibody in N. norvegicus and in other crustaceans, Munidarugosa and Squillamantis; these locate GIH in superficial axon terminals of the releasing organ, the sinus gland. The identification of a second GIH sequence in crustaceans allows to hypothesize the occurrence, within the neuropeptide family, of three subfamilies probably involved in different functions: crustacean hyperglycemic hormones, GIHs and molt-inhibiting hormones/mandibular organ-inhibiting hormones.

Molecular cloning and expression analysis of the complementary deoxyribonucleic acid for chicken inhibin/activin beta(B) subunit.

Inhibins and activins are dimeric peptide hormones that play an integral role in the intraovarian regulation of folliculogenesis. The domestic hen, with its well-defined follicular hierarchy, provides a unique model in which to study the role of these hormones in follicular development. In the present study, the complete coding sequence and deduced amino acid sequence for the chicken inhibin/activin beta(B) subunit has been determined from cDNA clones isolated from a chicken ovarian granulosa cell library. This beta(B)-subunit cDNA predicts a precursor protein of 392 amino acids containing the mature C-terminal 115 amino acid beta(B) subunit. When compared to the beta(B) subunit isolated from a variety of species, the chicken cDNA clone showed high nucleotide identity in the full-length coding region (>70%) and in the mature coding region (>80%). In addition, the deduced amino acid sequence of chicken beta(B) subunit showed greater than 95% identity compared to other species in the mature peptide region. Expression of the beta(B)-subunit mRNA was detected by reverse transcription-polymerase chain reaction in both gonadal and extragonadal tissues. Northern blot analysis detected expression in the gonadal tissues only, specifically in the granulosa tissue from the F3-F5 follicles, small yellow follicles (SYF), large white follicles, and immature and mature rooster testes. A major transcript of approximately 4.1 kilobases (kb) and three minor transcripts of approximately 8.4 kb, 6.5 kb, and 1.7 kb were detected in the SYF granulosa samples. To examine the expression pattern of the beta(B) subunit around the stage of follicle selection, the SYF granulosa was subdivided into two groups: 6-8 mm and 9-12 mm. Quantification of RNA expression (n = 3) showed that expression of the beta(B) subunit was maximal in the 6-8 mm SYF. Activin B, as well as other intraovarian signals, may regulate early follicle selection and/or development in the chicken.

CDNA cloning, overproduction and characterization of rat adrenodoxin reductase

We isolated a full-length cDNA clone for rat adrenodoxin reductase (AdR). The precursor of rat AdR was predicted to consist of 34 amino-terminal residues of extrapeptide for transport into mitochondria and the following 460 residues of the mature peptide region. The deduced amino acid sequence was 70.8 and 61.8% homologous to those of bovine and human AdRs in the extrapeptide region, respectively, and 88.5% homologous to both the sequences of bovine and human AdRs in the mature peptide region. The predicted mature form of rat AdR was directly expressed in Escherichia coli, using cDNA, and was purified with a yield of 32 mg/l of culture. The purified recombinant rat AdR showed an absorption spectrum characteristic of a flavoprotein with peaks at 270, 378 and 450 nm and shoulders at 280, 425 and 474 nm. The extinction coefficient was estimated to be 10.9 mM −1 cm −1 at 450 nm. The absorbance ratio at 270 nm/450 nm was 7.1. From the θ 208 value in the circular dichroism spectrum, the α-helix content in the rat AdR was calculated to be 30%. In NADPH-cytochrome c reductase activity reconstituted with adrenodoxin (Ad), the apparent K m value of rat AdR for NADPH was 0.32 μM, a value significantly lower than that of bovine AdR (1.4 μM). The rat AdR showed a higher affinity to the heterologous redox partner (bovine Ad, K m=9.3 nM) than to the native partner (rat Ad, K m=16.7 nM), whereas the affinity of bovine AdR was slightly higher to the native partner (bovine Ad, K m=37.1 nM) than to the heterologous partner (rat Ad, K m=46.8 nM). The K m values showed a reverse correlation to the difference of pI values between the redox partners. These results indicate that AdR binds to Ad mainly by ionic interaction.

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).

Molecular cloning and sequencing of equine inhibin α cDNA

Abstract A cDNA probe for recombinant human inhibin α subunit was used to screen an equine testis cDNA library. Approximately 220,000 plaques were screened and 10 positive clones were identified. The largest of these clones, approximately 1 kb, was sequenced. The sequence (993 nucleotides [nt]) contained the ARG‐ARG proteolytic processing site that precedes the N‐terminus of the a subunit mature peptide. The sequence that followed this processing site was 402 nt in length and encoded for a protein of 134 amino acids. Nucleotide homology was 88%, 87%, 88%, 82% and 86% with the a subunit cDNA of cattle, pigs, sheep, mice and humans, respectively. Amino acid homology within the mature peptide region was 99%, 88%, 92%, 87% and 86% with the a subunit of cattle, pigs, sheep, mice and humans, respectively. The equine a subunit peptide contained 6 cysteine residues and one potential N‐glycosylation site (Asn‐Ile‐Ser). The estimated molecular weight of the amino acid sequence for the mature protein was 14.7 kDa. T...

Guinea-pig neutrophil cationic peptides are encoded by at least three kinds of mRNA transcripts

1. 1. cDNA clones encoding antimicrobial guinea-pig neutrophil cationic peptides, GNCP-1 and GNCP-2 were isolated from a bone marrow cell cDNA library. 2. 2. Three kinds of cDNAs (GNCP-1A, GNCP-1B and GNCP-2 cDNAs) were obtained, and the analysis of these cDNAs indicated that GNCPs were produced as precursor proteins comprising 93 amino acid residues, which were composed of signal sequences (N-terminal 19 residues), pro-peptide sequences (43 residues) and mature GNCP sequences (31 residues). 3. 3. The deduced amino acid sequences showed that GNCP-1A and GNCP-1B differed by only one amino acid substitution in the pro-peptide region, and GNCP-1B and GNCP-2 differed by only one amino acid substitution in the mature peptide region. The nucleotide sequences of these cDNAs were highly homologous (99%). 4. 4. Together these observations indicate that GNCPs are encoded by at least three kinds of mRNA transcripts which are likely derived from the similar genes.