Cloned cDNA sequences for human pancreatic kallikrein have been isolated and determined by molecular cloning and sequence analysis. The identity between human pancreatic and urinary kallikreins is indicated by the complete coincidence between the amino acid sequence deduced from the cloned cDNA sequence and that reported partially for urinary kallikrein. The active enzyme form of the human pancreatic kallikrein consists of 238 amino acids and is preceded by a signal peptide and a profragment of 24 amino acids. A sequence comparison of this with other mammalian kallikreins indicates that key amino acid residues required for both serine protease activity and kallikrein-like cleavage specificity are retained in the human sequence, and residues corresponding to some external loops of the kallikrein diverge from other kallikreins. Analyses by RNA blot hybridization, primer extension, and S1 nuclease mapping indicate that the pancreatic kallikrein mRNA is also expressed in the kidney and sublingual gland, suggesting the active synthesis of urinary kallikrein in these tissues. Furthermore, the tissue-specific regulation of the expression of the members of the human kallikrein gene family has been discussed.

Amino Acid Sequence↗

PubMed1985

Primary structures of the mRNAs encoding the rat precursors for bradykinin and T-kinin. Structural relationship of kininogens with major acute phase protein and alpha 1-cysteine proteinase inhibitor.

Three types of cloned cDNA sequences for rat low molecular weight prekininogens were isolated and determined by molecular cloning and sequence analysis. The deduced amino acid sequences indicated that one, termed K-prekininogen, represents the counterpart of the known low molecular weight prekininogen present in other mammals, while the other two, called T-prekininogens, contain a novel T-kinin sequence which was recently identified from rat plasma. Although T- and K-prekininogens are highly homologous with each other, both of the T-prekininogens contain methionine, instead of arginine or lysine, as an amino acid preceding T-kinin and exhibit two consecutive amino acid deletions in the preceding region of T-kinin as compared with K-prekininogen. The former finding accounts for the previous observation of strong resistance of T-kininogens to cleavage with trypsin or kallikreins, while the latter finding has been explained by the structural analysis of genomic clones in which T-kinin-coding exon is contracted at its intron junction. A partial nucleotide sequence reported recently for the rat major acute phase protein (alpha 1-MAP) mRNA was found to be extremely related to the corresponding portion of the rat T-prekininogen mRNA. Furthermore, consistent with the previous report of the structural identity of major acute phase protein and alpha 1-cysteine proteinase inhibitor, kininogen closely resembles not only the former but also the latter in the amino acid compositions. The interrelationship among the triad of these proteins has been discussed.

Acute-Phase Proteins↗

PubMed1985

Cloning and sequencing of cDNA of Sarcophaga peregrina humoral lectin induced on injury of the body wall.

A previous paper described the purification of a lectin induced in the hemolymph of larvae of Sarcophaga peregrina (flesh-fly) on injury of their body wall (Komano, H., Mizuno, D., and Natori, S. (1980) J. Biol. Chem. 255, 2919-2924). This paper describes cDNA cloning and the complete nucleotide sequence of the gene for Sarcophaga lectin. Although active lectin consists of alpha and beta subunits in a molar ratio of 2:1, the fat body of injured larvae was found to contain only mRNA for the alpha subunit, suggesting that these two subunits are derived from a common gene and that the alpha subunit is converted to the beta subunit post-translationally. The alpha subunit was found to consist of 260 amino acid residues with an additional signal sequence of 19 or 23 amino acid residues.

Amino Acid Sequence↗

PubMed1985

Differential expression of the multiple forms of rat prekininogen mRNAs after acute inflammation.

Responses of the rat liver prekininogen mRNAs after induction of acute inflammation were examined by blot-hybridization and S1 nuclease protection analyses with the aid of cDNA probes specific for rat kininogens. Marked changes in the relative levels of the low molecular weight (LMW) prekininogen mRNAs were observed after administration of Escherichia coli lipopolysaccharide, and the mRNA levels increased with a half-maximal dose of approximately 100 ng of lipopolysaccharide/100 g body weight. At maximum level of induction, the LMW prekininogen mRNAs comprised about 1% of total liver mRNA, thus representing a major component of the liver mRNA in the acutely inflamed rat. Differences in the inflammatory responses of various forms of the prekininogen mRNAs were then investigated by S1 nuclease protection analysis with the use of three different cDNA probes, each specific for either K-prekininogen or two types of T-prekininogens. Both of the T-prekininogen mRNAs increased progressively during the first 24 h after induction of inflammation, and at maximum level of induction, these two mRNAs increased about 10- and 13-fold over their normal level. In contrast, neither of the high molecular weight and LMW K-prekininogen mRNAs exhibited such an increase after induction of inflammation. Thus, the expressions of the rat T- and K-prekininogen mRNAs are differentially regulated in response to the induction of acute inflammation.

Acute Disease↗

PubMed1985

Cloning and expression in Escherichia coli of cDNA for serine: pyruvate aminotransferase of rat liver.

Cloned cDNAs for rat liver serine: pyruvate aminotransferase were obtained by screening of a cDNA expression bank of rat liver with an antibody against the enzyme. Nineteen clones were isolated from 33 000 transformants and most of them had common fragments of cDNA on analysis by digestion with some restriction enzymes. These clones were identified as those containing cDNA for serine:pyruvate aminotransferase by the following criteria. (a) At the nucleic acid level, a 500-base-pair fragment of cDNA prepared by digestion of cDNAs with EcoRI and PstI hybridized with the mRNA coding for serine:pyruvate aminotransferase as judged by hybrid-selected and hybrid-arrested translations. (b) Specific proteins were detected in nine bacterial clones, a 40-kDa protein in one clone and a 39-kDa protein in eight clones. Among them only the 40-kDa protein was found to be solubilized from the cell by sonication, and this protein was immunoprecipitated with an antibody against serine:pyruvate aminotransferase of rat liver. (c) High activity of serine:pyruvate aminotransferase was expressed both in whole cell suspension and sonicated extract prepared from the transformant producing the 40-kDa protein, and 99% of the activity was immunoreactive with the antibody. Two types of mRNA for serine:pyruvate aminotransferase were detected on the RNA blot analysis by using cloned cDNA fragment as a probe. The larger mRNA (approximately 1600 nucleotides) was glucagon-inducible while the smaller one (approximately 1500 nucleotides) was not affected by the hormone.

Animals↗

PubMed1985

Cloning and sequence analysis of cDNAs for human high molecular weight and low molecular weight prekininogens. Primary structures of two human prekininogens.

cDNA sequences for both human high molecular weight (HMW) and low molecular weight (LMW) prekininogens have been isolated by molecular cloning and determined by sequence analysis. The sequence determination together with the S1 nuclease mapping and RNA blot-hybridization analyses indicate that human HMW and LMW prekininogen mRNAs share an identical sequence throughout the 5'-untranslated region and the protein-coding region up to the sequence encoding the 12 amino acids distal to the bradykinin sequence, and the two mRNAs then completely diverge from each other. The signal peptide, the heavy chain (H chain), and the bradykinin moiety, which are common between the two prekininogens, consist of 18, 362, and 9 amino acids, respectively, while the light chains (L chains) of the HMW and LMW prekininogens are composed of 255 and 38 amino acids, respectively. All 17 cysteine residues present in the human and bovine H chains are located at exactly equivalent positions, indicating that the human H chain, like the bovine counterpart, can form 8 loop structures, each connected by two adjacent cysteine residues. The L chains of human and bovine kininogens differ in the protein lengths as well as in some amino acids crucial for the processing of the kininogens by kallikrein. Based upon this finding, we have discussed the molecular basis for the different modes of processing of human and bovine HMW kininogens and for the different kinetics of contact activation reactions exhibited by the two HMW kininogens.

Amino Acid Sequence↗

PubMed1985

Structural organization of the human kininogen gene and a model for its evolution.

The entire human kininogen gene has been isolated as a set of overlapping genomic DNA fragments, and the 11 exons encompassing approximately 27 kilobase pairs have been mapped by restriction enzyme analysis and nucleotide sequence determination. The nine 5'-terminal exons encode the 5'-untranslated region and the protein-coding region for the signal peptide and the heavy chain, which are common for high molecular weight (HMW) and low molecular weight (LMW) prekininogen mRNAs. Exon 10 consists of the common sequence for bradykinin and the immediately following unique sequence for HMW prekininogen mRNA. Exon 11 is then located following a 90-nucleotide sequence downstream from exon 10 and precisely specifies the sequence unique to LMW prekininogen mRNA. This, together with the hybridization analysis of total human cellular DNA, leads us to conclude that human HMW and LMW prekininogen mRNAs are produced from a single gene as a consequence of alternative RNA processing events. The structural analysis of the kininogen gene also shows that each of the nine 5'-terminal exons discretely specifies the nine protein domains observed in the amino-terminal portion of the kininogens. Furthermore, these nine genetic domains can be characterized by a thrice repeated pattern of three genetic segments, and two sets of these three domains, encompassing exons 3-5 and exons 6-8, are most closely related to each other. Therefore, we have proposed two successive duplication mechanisms as a model for the generation of the structure of the kininogen gene.

Base Sequence↗

PubMed1985

The relative frequency and topographical distribution of somatostatin-, GRP-, APP-, glucagon-, 5-HT-, and neurotensin-immunoreactive cells in the proventriculus of seven species of birds.

The relative frequency and topographical distribution of proventricular endocrine cells were examined immunohistochemically in seven species of birds: common finch, pigeon, quail, chicken, duck, gull and kite. Gastrin releasing peptide (GRP), somatostatin-, avian pancreatic polypeptide (APP)-, glucagon-, 5-hydroxytryptamine (5-HT)- and neurotensin-immunoreactive cells were observed in this study. GRP- and somatostatin-immunoreactive cells were found in all species examined. All six kinds of immunoreactive cells were found with varying frequency in the pigeon, quail and gull, but not all immunoreactives were found in the other species examined. Species differences with regard to the relative frequency and topographical distribution of proventricular endocrine cells were observed.

Animals↗

PubMed1985

Histological and immunohistochemical studies of the Segi's cap, a large aggregation of endocrine cells on the intestinal villi of porcine fetuses and neonates.

Segi's cap, a large aggregation of endocrine cells on the top of intestinal villi, was studied in porcine fetuses and neonates by histological and immunohistochemical methods. The following observations were made: 1) Segi's caps were found in the proximal small intestine in all fetuses larger than 17 cm (beyond 10 weeks of gestation), in neonates before suckling and in 1-4 day-old piglets (suckling neonates); they were not found in a 1 week-old animal. 2) Segi's caps were seen more frequently in the distal duodenum and proximal jejunum than in the proximal and middle duodenum. 3) The Segi's cap consisted mainly of numerous argyrophil cells as demonstrated by Grimelius' method and a few argentaffin cells as identified by a modified Masson-Hamperl's method. 4) Immunohistochemically, ten kinds of immunoreactive cells were dispersed in the mucosal epithelium, outside of Segi's caps, in the proximal small intestine of fetuses: 5-hydroxytryptamine (5-HT)-, gastrin-, bovine pancreatic polypeptide (BPP)-, secretin-, somatostatin-, cholecystokinin-, gastric inhibitory polypeptide (GIP)-, motilin-, leucine-enkephalin- and neurotensin-immunoreactive cells. Except for neurotensin-immunoreactive cells, all of these cells were detected also in the caps. 5) Regional differences were noted in the distribution of cells in the caps; gastrin-, BPP- and secretin-immunoreactive cells were dominant in the caps in the proximal duodenum, while 5-HT-immunoreactive cells were most numerous in those in the proximal jejunum.

Animals↗

PubMed1985

Histologic and immunocytochemical study of endocrine cells in the gastrointestinal tract of the cow and calf.

The regional distribution and relative frequency of argyrophil cells, and of cells immunoreactive for 5-hydroxytryptamine (5-HT), substance P (SP), somatostatin, glicentin, glucagon, bovine pancreatic polypeptide (BPP), gastrin, leucine-enkephalin, gastric inhibitory polypeptide (GIP), cholecystokinin, secretin, motilin, and neurotensin were studied in 9 segments from the gastrointestinal tract of cows (greater than 1 year old) and calves (less than 3 months old). Argyrophil cells, 5-HT-immunoreactive cells, and somatostatin-immunoreactive cells were distributed throughout the gastrointestinal tract, whereas the other immunoreactive cells were more restricted in distribution. Most endocrine cells were more numerous in the calf than in the cow. This feature was most conspicuous in the abomasum. In the abomasum, argyrophil cells in the cow and calf and 5-HT-immunoreactive cells in the calf were found predominantly in the fundic region, whereas somatostatin-immunoreactive cells and gastrin-immunoreactive cells in the cow and calf and 5-HT-immunoreactive cells in the cow were most numerous in the pyloric region. Substance P-, glucagon-, BPP-, and leucine-enkephalin-immunoreactive cells were rarely detected. In the small intestine, argyrophil cells, 5-HT-, SP-, somatostatin-, gastrin-, GIP-, cholecystokinin-, secretin-, and motilin-immunoreactive cells were most numerous in the duodenum. Neurotensin-, glicentin-, glucagon-, and BPP-immunoreactive cells were detected with the highest frequency in the ileum. In the large intestine, argyrophil cells and 5-HT-, glicentin-, BPP-, somatostatin-, glucagon-, and SP-immunoreactive cells occurred with the highest frequency in the rectum.

Abomasum↗

PubMed1985

[Alternative RNA processing events in kininogen gene expression].

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Animals↗