Purification and characterization of Escherichia coli heat-stable enterotoxin II.

Escherichia coli heat-stable enterotoxin II (STII) was purified to homogeneity by successive column chromatographies from the culture supernatant of a strain harboring the plasmid encoding the STII gene. The purified STII evoked a secretory response in the suckling mouse assay and ligated rat intestinal loop assay in the presence of protease inhibitor, but the response was not observed in the absence of the inhibitor. Analyses of the peptide by the Edman degradation method and fast atom bombardment mass spectrometry revealed that purified STII is composed of 48 amino acid residues and that its amino acid sequence was identical to the 48 carboxy-terminal amino acids of STII predicted from the DNA sequence (C. H. Lee, S. L. Mosely, H. W. Moon, S. C. Whipp, C. L. Gyles, and M. So, Infect. Immun. 42:264-268, 1983). STII has four cysteine residues which form two intramolecular disulfide bonds. Two disulfide bonds were determined to be formed between Cys-10-Cys-48 and Cys-21-Cys-36 by analyzing tryptic hydrolysates of STII.     

Human endometrial stromal cells and decidual cells express cluster of differentiation (CD) 13 antigen/aminopeptidase N and CD10 antigen/neutral endopeptidase.

With specific monoclonal antibodies, we found that human endometrial stromal cells and decidual cells express two function-related surface antigens. Indirect immunofluorescence staining revealed that both endometrial stromal cells and decidual cells during the first trimester of pregnancy expressed cluster of differentiation (CD) 13 antigen and CD10 antigen, which are identical to aminopeptidase N and neutral endopeptidase, respectively. By flow cytometric analysis, CD13 antigen was detected on 82-93% of the examined cells, and CD10 antigen was detected on 75-93% of the examined cells in endometrial stromal cell-enriched preparations. Furthermore, peptidase activity was detected in these cell preparations by an assay based on the hydrolysis of alanine-p-nitroanilide into p-nitroaniline and alanine.     

The primary structure of rat rig/ribosomal protein S15 gene.

We have isolated rat rig/ribosomal protein S15 gene from a DNA library derived from a rat insulinoma and determined the complete nucleotide sequence. The rat rig/S15 gene is composed of four exons and three introns spanning 2 kbp and exhibits distinctive structural features unique for a ribosomal protein gene.     

Factor XIII-dependent generation of 5th complement component(C5)-derived monocyte chemotactic factor coinciding with plasma clotting.

Blood coagulation or plasma clotting caused generation of a monocyte chemotactic factor(s) in vitro. The chemotactic factor, of which the apparent molecular mass was 75 kDa, shared antigenicity with complement C5 and possessed the affinity to monocytes, but not to polymorphonuclear leukocytes. The generation of the chemotactic factor was hindered in the presence of a thiol enzyme inhibitor, p-chloromercuriphenyl sulfonic acid, at the concentration of 1 mmol/l, although the gelation of plasma was apparently completed. Furthermore, the generation of chemotactic factor was not observed when a plasma deficient in blood coagulation factor XIII, which is a precursor of a thiol enzyme, plasma transglutaminase, was used; and the activity normally appeared when the deficient plasma was reconstituted with purified factor XIII or with a tissue transglutaminase prior to clotting. When the human sera were injected into guinea pig skin, the serum derived from normal plasma or from the reconstituted factor XIII deficient one caused mononuclear cell infiltration, however, the serum from the deficient plasma without reconstitution infiltrated to a significantly smaller extent. These results indicated that the complement system was initiated somehow during the clotting process resulting in the generation of the C5-derived monocyte chemotactic factor in cooperation with factor XIIIa (activated factor XIII).     

Alpha-elastin coacervate as a protein liquid membrane: effect of pH on transmembrane potential responses.

A protein liquid membrane composed of coacervated alpha-elastin, a chemical fragmentation product of the biological elastic fiber protein, functioned as an amphoteric liquid ion-exchange membrane. Ionic permselectivities of the alpha-elastin coacervate membrane to a series of metal chlorides were investigated for the concentration-cell systems by the ordinary electrochemical measurements. Effects of pH on the transmembrane potential responses for NaCl, CaCl2, and MgCl2 systems were examined. Only in the Ca(2+)-containing system did potential responses stay at constant levels against the pH changes, whereas in the other systems, increasing pH caused potential changes, indicating an improvement of cationic permselectivity across the alpha-elastin coacervate membrane. It was suggested that the characteristic Ca2+ transport mechanisms across the alpha-elastin coacervate membrane are related in some way to the polypeptide backbone interactions specific and selective to Ca2+ ions.     

Release of newly synthesized platelet-activating factor (PAF) from human polymorphonuclear leukocytes under in vivo conditions. Contribution of PAF-releasing factor in serum.

The behavior of platelet-activating factor (PAF) produced in stimulated human polymorphonuclear leukocytes (PMN) was investigated in the presence of serum under conditions close to those existing in vivo. When the cells were stimulated in the presence of the serum obtained from a PAF acetylhydrolase (PAF-AH)-deficient Japanese subject, over 60% of synthesized PAF was detected in the extracellular medium by bioassay, scintillation proximity RIA and selected ion monitoring/gas chromatography/mass spectrography analysis. The release of PAF from PMN after stimulation with FMLP and A23187 was also observed in the presence of normal serum treated with acid to inactivate PAF-AH. The heterogeneity of the molecular species of extracellular PAF was similar to that of intracellular PAF produced in stimulated PMN in the presence of PAF-AH-deficient serum, ruling out the possibility that a specific molecular species of PAF was preferentially released from the cells in the presence of the serum. As these data suggested the occurrence of PAF-releasing factor(s) in the serum, an attempt was made to partially purify this factor from PAF-AH-deficient serum and acid-treated normal serum by ammonium sulfate fractionation and column chromatography with DEAE-Cellulofine and Sepharose CL-6B. The molecular mass of PAF-releasing factor revealed on a TSK gel G3000 SW HPLC column was 240 kDa, which was different from that of albumin. The binding assay, newly developed for this study, revealed that the PAF-binding activity of PAF-releasing factor is stronger than that of albumin, and that the PAF-releasing factor forms a complex with PAF at low concentration (10(-9) M). PAF bound to this factor was difficult to be hydrolyzed by serum PAF-AH. On the other hand, the PAF/PAF-releasing factor complex had aggregatory activity toward washed rabbit platelets. These observations suggest that certain protein(s) releases and carries the PAF newly synthesized by PMN in blood plasma/serum. Thus it appears that PAF functions as an autacoid in vivo, along with other mediators.     

Search for MHC-associated genes in human: five new genes centromeric to HLA-DP with yet unknown functions

Taking advantage of five mouse genomic or cDNA probes [KE5(probe 14), KE4 (probe11), KE3 (probe7), KE2 (probe5), and SET] mapped on the H-2K region in mouse, we have identified and localized homologues of these five genes in the human major histocompatibility complex region (HKE5, HKE4, HKE3, HKE2, and HSET, respectively). Cosmid cloning and pulsed field gel electrophoresis analyses indicated that a human homologous gene, HKE5, is located 10 kilobases (kb) centromeric of the 2(XI) collagen (COL11A2) gene followed by HKE4. HKE3, closely linked to HKE2, is located 170 kb centromeric of HKE4. Furthermore, HSET is located 50 kb centromeric of HKE2. This gene organization outside the DP subregion is completely identical to that of the mouse H-2K region centromeric of I-Pb 3, a mouse homologue of the DPB gene, except the lack of genes corresponding to the H-2K and -K2 genes in human.     

Preparation of a fluorescent derivative of benzoylecgonine, and preliminary studies of its application to the analysis of urine

A sensitive high-performance liquid chromatographic method using 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone (Br-DMEQ) as a fluorescent labeling reagent is described for the determination of benzoylecgonine (BE) and ecgonine (EC). The Br-DMEQ derivatives of BE and EC were separated on a C₁₈ column and detected at 455 nm with excitation at 370 nm. The detection limits of the proposed method were 18.7 fmol for BE and 12.5 pmol for EC at a signal-to-noise ratio of 3. Relative standard deviations of five replicate measurements were 1.94% (10 pmol) and 2.98% (50 pmol) for BE and 6.3% (250 pmol) and 5.62% (1.25 pmol) for EC. This method was applied to the determination of BE in human urine. BE was extracted from urine by solvent extraction with chloroform—isopropyl alcohol (9:1, v/v) solution. Levels of 2.5 · 10⁻⁸ M BE in urine (25 pmol/ml) could be determined.     

Isolation and functional expression of pituitary peptidylglycine alpha-amidating enzyme mRNA. A variant lacking the transmembrane domain

We demonstrate that, in rat pituitary, peptidylglycine alpha-amidating enzyme was encoded by at least 5 distinct mRNAs. Southern blot and ribonuclease protection analyses revealed that the mRNAs arose through alternative splicing. A variant lacking the transmembrane domain-coding sequence was a major mRNA species for the enzyme in the pituitary. When the cDNAs were expressed in COS-7 cells, the variant was the most efficient in producing a secretory form (37 kDA) of the enzyme.