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C L Bevins

Publications and source records attributed to C L Bevins.

54 records · Page 3Linked to original sources
PubMed1992

Localization of xenopsin and xenopsin precursor fragment immunoreactivities in the skin and gastrointestinal tract of Xenopus laevis.

Xenopsin (Xp) and xenopsin precursor fragment (XPF) are bioactive peptides derived from a single precursor molecule; both were isolated previously from extracts of Xenopus laevis skin. The present immunohistochemical study was undertaken to determine the specific cellular localization of these two peptides in the skin and also in the gastrointestinal tract of adult Xenopus. We report here that Xp-like and XPF-like immunoreactivities co-exist in the granular glands of the skin and specific granular cells in the lower esophagus and stomach. However, only Xp-like immunoreactivity, not XPF-like immunoreactivity, was detected in tall, thin cells of the duodenum and in club-shaped cells of the large intestine. The immunochemical co-localization of the two peptides in specific cells of the skin, lower esophagus and stomach suggests that the same gene is expressed in each of these cells, and that the precursor molecule undergoes similar post-translational processing. In contrast, the observation that certain cells of the duodenum and large intestine display only one peptide immunoreactivity suggests an alternative phenomenon, possibly involving selective peptide accumulation or expression of a different gene.

Amino Acid Sequence↗
PubMed1992

A novel peptide-producing cell in Xenopus: multinucleated gastric mucosal cell strikingly similar to the granular gland of the skin.

We have characterized a novel peptide-containing cell within the gastric mucosa of Xenopus laevis. The cell is a spherical, multinucleated syncytial structure containing a cytoplasmic space filled with dense rice-shaped granules, and is strikingly similar in morphology to the well-studied granular gland of the amphibian skin. Immunohistochemical and immunogold methods were used to demonstrate that several peptides previously isolated from the granular glands of the skin, including the antimicrobial peptides magainin and PGLa (a peptide with amino-terminal glycine and carboxy-terminal leucinamide), are also stored in granules present in these enteric cells. These data demonstrate that this enteric peptide-producing cell is strikingly similar both morphologically and biochemically to the granular gland, previously considered a highly specialized structure of the amphibian integument. This novel gastric mucosal cell, which we have designated a "granular multinucleated cell," is distinct in its morphology and its diversity of stored peptide products from other well-characterized peptide-containing cells in the vertebrate gastrointestinal tract.

Amino Acid Sequence↗
PubMed1991

A cross-species analysis of the cystic fibrosis transmembrane conductance regulator. Potential functional domains and regulatory sites.

To help elucidate the function of the cystic fibrosis transmembrane conductance regulator (CFTR), we have undertaken a cross-species analysis of the DNA sequence which encodes this protein. We have isolated and characterized the cDNA of the bovine homologue of CFTR. The deduced amino acid sequence shows high overall identity with the published sequences from human and mouse, although there is marked variability between the different potential functional domains. The region around human amino acid 508, which is deleted in 70% of cystic fibrosis chromosomes, is highly conserved across species; of the missense cystic fibrosis mutations reported to date, all of the amino acids in the normal human sequence are conserved in the bovine and mouse sequences. A single amino acid encoded by the human cDNA (Ser-434) is missing in the bovine sequence, and there are two amino acids encoded by the bovine sequence which are absent in the human. These all stem from in-frame 3-base omissions within the sequences. In addition to the cow, we amplified the DNA sequences encoding a portion of the R-domain from sheep, monkey, rabbit, and guinea pig. These sequences show relatively low overall sequence identity (63%), but nearly all of the potential protein kinase A and protein kinase C phosphorylation sites are conserved over all of the species examined. Our results suggest functional significance for certain highly conserved residues and putative domains within CFTR.

Amino Acid Sequence↗
PubMed1991

Antimicrobial peptides in the stomach of Xenopus laevis.

Antimicrobial peptides are widely distributed in nature and appear to play a role in the host defense of plants and animals. In this study we report the existence of antimicrobial peptides in the stomach of the vertebrate Xenopus laevis, an animal previously shown to store high concentrations of antimicrobial peptides in its skin. Antimicrobial activity was detected in extracts of X. laevis stomach tissue and nine antimicrobial peptides were then purified. A novel 24-amino acid peptide, designated PGQ, was isolated from these extracts, and has the following amino acid sequence: GVLSNVIGYLKKLGTGALNAVLKQ. PGQ is relatively basic and has the potential to form an amphipathic alpha-helix. The other peptides isolated are members of the magainin family of antimicrobial peptides, and include magainins I and II, PGLa, xenopsin precursor fragment, and four caerulein precursor fragments. None of these peptides had been previously identified in tissues other than the skin. The purification of the peptides from stomach extracts and subsequent protein sequence analysis reveals that the peptides have undergone the same processing as their dermal counterparts, and that they are stored in their processed forms. Northern blot analysis indicates that the magainin family of peptides are synthesized in the stomach, and immunohistochemical studies demonstrate that magainin is stored in a novel granular multinucleated cell in the gastric mucosa of Xenopus. This study demonstrates that the magainin family of antimicrobial peptides is found in the gastrointestinal system of X. laevis and offers an opportunity to further define the physiological role of these defense peptides.

Amino Acid Sequence↗
PubMed1991

Tracheal antimicrobial peptide, a cysteine-rich peptide from mammalian tracheal mucosa: peptide isolation and cloning of a cDNA.

Extracts of the bovine tracheal mucosa have an abundant peptide with potent antimicrobial activity. The 38-amino acid peptide, which we have named tracheal antimicrobial peptide (TAP), was isolated by a sequential use of size-exclusion, ion-exchange, and reverse-phase chromatographic fractionations using antimicrobial activity as a functional assay. The yield was approximately 2 micrograms/g of wet mucosa. The complete peptide sequence was determined by a combination of peptide and cDNA analysis. The amino acid sequence of TAP is H-Asn-Pro-Val-Ser-Cys-Val-Arg-Asn-Lys-Gly-Ile-Cys-Val-Pro-Ile-Arg-Cys-Pr o- Gly-Ser-Met-Lys-Gln-Ile-Gly-Thr-Cys-Val-Gly-Arg-Ala-Val-Lys-Cys-Cys-Arg- Lys-Lys - OH. Mass spectral analysis of the isolated peptide was consistent with this sequence and indicated the participation of six cysteine residues in the formation of intramolecular disulfide bonds. The size, basic charge, and presence of three intramolecular disulfide bonds is similar to, but clearly distinct from, the defensins, a well-characterized class of antimicrobial peptides from mammalian circulating phagocytic cells. The putative TAP precursor is predicted to be relatively small (64 amino acids), and the mature peptide resides at the extreme carboxyl terminus and is bracketed by a short putative propeptide region and an inframe stop codon. The mRNA encoding this peptide is more abundant in the respiratory mucosa than in whole lung tissue. The purified peptide had antibacterial activity in vitro against Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia, and Pseudomonas aeruginosa. In addition, the peptide was active against Candida albicans, indicating a broad spectrum of activity. This peptide appears to be, based on structure and activity, a member of a group of cysteine-rich, cationic, antimicrobial peptides found in animals, insects, and plants. The isolation of TAP from the mammalian respiratory mucosa may provide insight into our understanding of host defense of this vital tissue.

Amino Acid Sequence↗
PubMed1990

Mutagenesis of lysine 460 in the human insulin receptor. Effects upon receptor recycling and cooperative interactions among binding sites.

Mutations in the insulin receptor gene can cause insulin resistance. Previously, we have identified a mutation substituting glutamic acid for lysine at position 460 in the alpha-subunit of the insulin receptor in a patient with a genetic form of insulin resistance. In the present work, we have investigated the effect upon receptor function of amino acid substitutions at position 460. Decreasing the pH from 8.0 to 5.5 caused a progressive acceleration of the dissociation of 125I-insulin from the wild-type insulin receptor. Substitution of acidic amino acids (Glu or Asp) for Lys460 decreased the ability of acid pH to accelerate dissociation of 125I-insulin. In contrast, substitution of Arg or neutral amino acids (Val, Met, Thr, or Gln) had no effect upon the sensitivity to acid pH. Correlated with decreased sensitivity to acid pH, substitution of Glu or Asp at position 460 retarded the dissociation of 125I-insulin from intracellular receptors subsequent to receptor-mediated endocytosis. Furthermore, retardation of dissociation of 125I-insulin from the internalized receptor was associated with a decreased half-life of the receptor. In summary, the Glu460 mutation appears to cause insulin resistance by accelerating receptor degradation and, thereby, decreasing the number of insulin receptors on the cell surface. Additional studies suggested that Lys460 may provide the amino groups whereby disuccinimidyl suberate cross-links the two alpha-subunits to each other. Consistent with the hypothesis that Lys460 is located at the interface between adjacent alpha-subunits, substitutions at position 460 impair cooperative interactions among insulin binding sites. The Glu460 mutation decreases positively cooperative binding interactions; the Arg460 mutation impairs negative cooperativity. Mutations at position 460 in the alpha-subunit did not decrease the ability of insulin to stimulate receptor tyrosine kinase.

Amino Acid Sequence↗
PubMed1990

Magainin-like immunoreactivity in human submandibular and labial salivary glands.

Magainins, antimicrobial peptides secreted by granular glands of frog skin, may be related to the high resistance to infections of this epithelial surface. The oral mucosa of healthy individuals is another tissue in which infection is not frequent, probably owing to the activity of potent salivary and mucosal defense mechanisms. To investigate if magainin-like factors are a component of these oral defense mechanisms, human and animal minor (mucosal) and major salivary glands were examined by immunohistochemistry, using a polyclonal rabbit anti-magainin antibody. Cryostat sections of (para) formaldehyde-fixed tissues were incubated with the antibody and then stained with fluorescein-complexed anti-rabbit IgG. Specific staining was observed in the apical portion of the cytoplasm of ductal epithelial cells of human submandibular and labial salivary glands. Diffuse staining was present in submandibular acinar cells. Bovine, rat, hamster, and mouse tissues were unreactive. The presence of magainin-like substances in human salivary gland duct cells is consistent with reports of the occurrence of other biologically active substances in salivary gland ducts.

Adult↗
PubMed1989

Insulin gene expression in chicken ontogeny: pancreatic, extrapancreatic, and prepancreatic.

Insulin has metabolic, growth, and differentiation effects in chicken embryos in vivo and it is required for normal development. Whether the pancreas is the sole source of insulin in embryogenesis is controversial. In the present study we investigated (1) the developmental pattern of expression of the chicken insulin gene in the pancreas; (2) the expression of the insulin gene in three nonpancreatic tissues, liver, brain, and lower limb, during chicken development; and (3) the expression of the insulin gene at prepancreatic stages and during chicken embryo organogenesis. Hybridization of synthetic species-specific insulin oligonucleotides to pancreatic frozen section in situ and to Northern blots revealed a major increase in insulin messenger RNA (mRNA) levels during the third week of embryonic development. The hybridization histochemistry showed both an increase in the levels of insulin mRNA per pancreatic islet and, in addition, an increase in the number of insulin mRNA containing islets with development. By Northern analysis there was a major polyadenylated transcript of 0.6 kb, which increased in abundance approximately 30-fold during this interval. Under the same stringency conditions used for pancreatic RNA an insulin transcript was detected in liver RNA blots. The abundance of this hepatic insulin mRNA was about 100-fold less than the pancreatic insulin mRNA and, in contrast to the latter, did not increase in late development. Primer extension experiments demonstrated that the insulin transcripts of pancreas and liver had similar 5' ends. No insulin mRNA was detected by Northern analysis or primer extension either in whole brain or lower limb total RNA from several developmental stages. A very low abundance insulin mRNA was detected in whole embryo at Day 8 and body regions at Day 4 and Day 5 when organogenesis of the pancreas takes place. Interestingly, a polyadenylated insulin transcript was detected, as well, in whole Day 2 and Day 3 embryos (stages 10 to 20, with 20 to 40 somites) before differentiation of beta cells occurs. Thus, there is differential developmental regulation of the insulin gene in several chicken embryo tissues and the expression of insulin precedes pancreatic maturation. These findings support the proposed role of insulin in differentiation and development in vivo and suggest a paracrine type of action of the hormone in early embryos before blood circulation begins.

Animals↗
PubMed1988

Two mutant alleles of the insulin receptor gene in a patient with extreme insulin resistance.

Insulin receptor complementary DNA has been cloned from an insulin-resistant patient with leprechaunism whose receptors exhibited multiple abnormalities in insulin binding. The patient is a compound heterozygote, having inherited two different mutant alleles of the insulin receptor gene. One allele contains a missense mutation encoding the substitution of glutamic acid for lysine at position 460 in the alpha subunit of the receptor. The second allele has a nonsense mutation causing premature chain termination after amino acid 671 in the alpha subunit, thereby deleting both the transmembrane and tyrosine kinase domains of the receptor. Interestingly, the father is heterozygous for this nonsense mutation and exhibits a moderate degree of insulin resistance. This raises the possibility that mutations in the insulin receptor gene may account for the insulin resistance in some patients with non-insulin-dependent diabetes mellitus.

Alleles↗
PubMed1986

Detection of a transient enzyme-steroid complex during active-site-directed irreversible inhibition of 3-oxo-delta 5-steroid isomerase.

The reaction of the active-site-directed irreversible inhibitor (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2'-oxiran]-3-ol (5 beta) with 3-oxo-delta 5-steroid isomerase has been monitored by repetitive scanning ultraviolet spectroscopy of a solution of 5 beta plus isomerase against a blank containing only 5 beta. Upon initial mixing of 5 beta with the isomerase an absorbance maximum at ca. 250 nm appears. With time, this peak decreases and is replaced with a new peak near 280 nm. These results directly demonstrate the existence of a transient enzyme-steroid intermediate in the inactivation reaction. The ultraviolet spectrum suggests that the steroid in the transient complex resembles the ionized phenol, while the phenolic group in the irreversibly bound complex is un-ionized. These spectral studies support our previous proposal that there are two enzyme-steroid complexes that are related by a 180 degree rotation about an axis perpendicular to the plane of the steroid nucleus. This hypothesis offers an explanation for the reaction of 17 beta-oxiranes with the same residue (Asp-38) that is thought to be involved in the catalytic mechanism. Two new oxiranes, (17S)-spiro[estra-1,3,5(10)-triene-17,2'-oxiran]-3 beta-ol (6 beta) and (17S)-spiro[5 alpha-androstane-17,2'-oxiran]-3-one (8 beta), were also found to be potent active-site-directed irreversible inhibitors of the isomerase (k3/KI = 31 M-1 s-1 and 340 M-1 s-1, respectively). The relationship of these results to the nature of the active site of the isomerase is discussed.

Binding Sites↗
PubMed1985

Mechanism of inactivation of 3-oxosteroid delta 5-isomerase by 17 beta-oxiranes.

The affinity label (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2'-oxiran]-3-ol (5 beta) inactivates 3-oxosteroid delta 5-isomerase from Pseudomonas testosteroni by formation of a covalent bond between Asp-38 of the enzyme and the steroid. High-performance liquid chromatography (HPLC) analysis of tryptic digests of inactivated enzyme shows that two isomeric steroid-containing peptides are formed in a ratio of 9:1 at pH 7 (TPS1 and TPS2). Hydrolysis of each of these peptides produces a different steroid: TPS1 releases 17 alpha-(hydroxymethyl)estra-1,3,5(10),6,8-pentaene-3,17 beta-diol (S1) whereas TPS2 yields 17 beta-(hydroxymethyl)estra-1,3,5(10),6,8-pentaene-3,17 alpha-diol (S2). Inactivation of the enzyme by (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2'-oxiran-18O]-3-ol, followed by mass spectral analysis of the diacetate of the steroid released upon hydrolysis of the enzyme-inhibitor bond, reveals that TPS1 is formed by attack of Asp-38 at the methylene carbon of the oxirane. In contrast, TPS2 is produced by Asp-38 attack at the tertiary carbon. These results imply that inactivation occurs through concurrent SN1 and SN2 reactions of Asp-38 with the protonated inhibitor and that Asp-38 is located on the alpha face of the steroid when it is bound to the active site in the correct manner to react for both the SN1 and SN2 processes.

Epoxy Compounds↗
PubMed1983

Affinity alkylation of bacterial delta 5-3-ketosteroid isomerase. Identification of the amino acid modified by steroidal 17 beta-oxiranes.

The two steroidal 17 beta-oxiranes, spiro-17 beta-oxiranyl-delta 4-androsten-3-one (4 beta) and spiro-17 beta-oxiranylestra-1,3,5(10),6,8-pentaene-3-ol (5 beta) are active site-directed irreversible inhibitors of bacterial delta 5-3-ketosteroid isomerase. For each inhibitor, a stoichiometry of one molecule of steroid to one enzyme subunit was found. The inhibited enzyme was denatured and subjected to digestion by trypsin. The tryptic maps show two distinct steroid-bound peptides for both 4 beta- and 5 beta-inhibited isomerase. In each case, the two modified peptides are derived from residues 14 to 45 of the isomerase. Each of the steroid-bound peptides of the 4 beta-inhibited enzyme was subjected to further proteolytic digestion and the site of steroid attachment was found to be Asp-38 in each of the inactivation products. These results are interpreted to indicate that "backwards binding" is an important feature of the binding of steroids to delta 5-3-ketosteroid isomerase.

Amino Acids↗
PubMed1980

Stabilization of 8S progesterone receptor from human prostate in the presence of molybdate ion.

In the presence of 10 mM molybdate ion, we are able to detect the appearance of a [6,7-3H]-17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione-([3H]R5020) binding moiety in human prostatic cytosol which sedimented at approximately 8S in a glycerol density gradient. The specifically bound [3H]-R5020 was displaced by progesterone, tiramcinolone acetonide, and R5020 but not by cortisol, dihydrotestosterone, 17 beta-estradiol, or diethylstilbestrol. Specific binding of [3H]R5020 in the presence of molybdate ion was shown to saturate at 7 x 10(-10) M (n = 64 fmol/mg of protein). The optimum concentration of molybdate ion for enhancing specific binding of [3H]-R5020 was determined to be between 7 and 20 mM. Molybdate ion was also effective in stabilizing the 8S R5020-binding moiety in a preincubation for 16 hr at 0 degrees in the absence of added steroid.

Binding, Competitive↗