Biochemical characterization of Limulus clotting factors and inhibitors which interact with bacterial endotoxins.
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Antimicrobial Cationic Peptides↗
Biomedical subjects
S Iwanaga
Publications and source records attributed to S Iwanaga.
Alterations in tight junctions of human endometrial epithelial cells during normal menstrual cycle--freeze-fracture electron microscopic study.
The structural change in the tight junction between human endometrial epithelial cells during the normal menstrual cycle was observed by means of freeze-fracture replica electron microscopy. Tight junctions between epithelial cells of the endometrial basal layer showed no alterations in structure during the menstrual cycle, while those between epithelial cells of the functional layer exhibited remarkable changes synchronized with the menstrual cycle; in the early proliferative phase, the tight junctions were composed of strands running mainly parallel to the luminal surface and the depth of the junctions from the luminal surface was 0.57 +/- 0.04 micron (Mean +/- S.E.), whereas in the late proliferative phase the junction formed a prominent network of strands 0.80 +/- 0.04 micron deep. In the early secretory phase, the junctions developed mostly as a complex network, and the depth was 0.96 +/- 0.04 micron. In the late secretory phase, the network consisting of the junctions looked disorderly and the depth of the junctions was reduced to 0.71 +/- 0.04 micron. As a conclusion, it was suggested that the tight junction in the human endometrial epithelium tended to develop or diminish according to the phase of the menstrual cycle.
Endometrium↗
Demonstration of the third kininogen in high and low molecular weight kininogens-deficient Brown Norway Katholiek rat.
A gel filtration profile of the plasma of Brown Norway Katholiek (B/N-Ka) rat was compared with those of B/N-Kitasato (B/N-Ki) and Sprague-Dawley (SD) rats. In the chromatograms of B/N-Ki and SD rat plasmas, high-molecular weight (HMW) kininogen was eluted together with pre-kallikrein. In lower molecular weight fractions, there were two kininogens, one of which released kinin by urinary kallikrein, snake venom kininogenase (SVK) and trypsin, and the other released kinin only by trypsin. In the chromatogram of B/N-Ka rat plasma, there was no fraction which released kinin by plasma kallikrein, urinary kallikrein or SVK. However, the kinin-release only by trypsin was found in the lower molecular weight fraction, which corresponds to the third peak of kininogen in the chromatograms of B/N-Ki and SD rat plasmas. These results indicate that B/N-Ka rat plasma is deficient in HMW kininogen, and also deficient in the LMW kininogen susceptible to urinary kallikrein and SVK, but it contains the third kininogen responsive only to trypsin.
Animals↗
Structure and expression of the alkA gene of Escherichia coli involved in adaptive response to alkylating agents.
Nucleotide sequence of a DNA fragment containing the alkA gene and its control region has been determined using a chemical method. Only one open reading frame responsible for 3-methyladenine DNA glycosylase II was found. The hypothetical polypeptide deduced from the DNA sequence, with a molecular weight of 31,400, has an amino-terminal sequence and total amino acid composition identical to that of purified 3-methyladenine DNA glycosylase II. We constructed hybrid plasmids carrying an alkA'-lacZ' fusion, with the proper control region for alkA expression. A hybrid polypeptide with beta-galactosidase activity was formed when lac mutant cells harboring such plasmids were incubated with low doses of N-methyl-N'-nitro-N-nitrosoguanidine or methylmethane sulfonate. Other DNA-damaging agents, such as ethylmethane sulfonate, nalidixic acid, and ultraviolet light did not induce the enzyme activity. The induction was controlled by the ada and adc, but not by the recA and lexA genes.
Alkylating Agents↗
Anti-LPS factor in the horseshoe crab, Tachypleus tridentatus. Its hemolytic activity on the red blood cell sensitized with lipopolysaccharide.
Anti-LPS factor, which inhibits the endotoxin mediated coagulation system in the horseshoe crab, Tachypleus tridentatus, was found to lyse red blood cells sensitized with gram-negative bacterial LPS, but not to lyse unsensitized cells. This hemolysis occurred even at 0 degree C and was completed within 1 min. The binding of anti-LPS factor to LPS must be essential for the hemolysis, because free LPS inhibited the hemolytic action of anti-LPS factor.
Animals↗
Scanning and transmission electron microscopic evidence of epithelial phagocytosis of spermatozoa in the terminal region of the vas deferens of the cat.
Scanning and transmission electron microscopic observations have been made in the terminal region of the vas deferens of the cat, with emphasis on the occurrence of spermiophagy. The present study has revealed that epithelial cells as well as luminal macrophages are extensively and actively involved in phagocytosis of spermatozoa. The mechanism of the spermiophagy is discussed, in relation to a possible role of the epithelial cells, as one function of the vas deferens.
Animals↗
Amino acid sequence of the coagulogen from Limulus polyphemus hemocytes.
The complete amino acid sequence of the coagulogen from hemocyte lysates of Limulus polyphemus has been determined by sequencing the peptides obtained from tryptic, chymotryptic, staphylococcal protease V8 and lysyl endopeptidase digestions. These results established the following sequence: (formula; see text) Limulus coagulogen consists of a single chain with a total of 175 amino acid residues and the molecular weight is calculated to be 19,675. It contains 16 half-cystines in disulfide linkages, with 5 half-cystines located in a cluster in the COOH-terminal 14 residues. The sequence of Limulus coagulogen is very close to that for the coagulogen of Tachypleus tridentatus (Japanese horseshoe crab), having 69% sequence homology. The 16 half-cystines of these coagulogens are in the same positions, suggesting a very similar conformation. Moreover, the COOH-terminal tripeptide regions of the A chain (from the NH2-terminal end to Arg-18) and peptide C (from Lys-19 to Arg-46), both of which seem to interact with a Limulus clotting enzyme to liberate peptide C, are completely conserved. From secondary structure predictions by the method of Chou and Fasman (Chow, P.Y., and Fasman, G. D. (1974) Biochemistry 13, 211-222), the coagulogen appears to contain an alpha-helical region in the peptide C segment, released by the clotting enzyme, suggesting a marked conformational change in the transformation of the coagulogen to the coagulin gel. beta-sheet and reverse turn regions are distributed in the B chain segment (from Gly-47 to the COOH-terminal end). It is likely that the 16 half-cystines and abundant beta-sheet structure make the coagulogen molecule compact.
Amino Acid Sequence↗
Amino acid sequence of Japanese horseshoe crab (Tachypleus tridentatus) coagulogen B chain: completion of the coagulogen sequence.
The complete amino acid sequence of the B chain derived from Tachypleus tridentatus coagulogen was determined. It consisted of a total of 129 amino acid residues with a NH2-terminal glycine and COOH-terminal phenylalanine. Sequence studies of the whole B chain and the fragments obtained from the digests with trypsin, alpha-chymotrypsin, thermolysin and Staphylococcal protease V8 showed the following sequence: (sequence; see text) These structural studies of the B chain and the previously established amino acid sequences of the A chain and peptide C derived from T. tridentatus coagulogen, now make it possible to complete the whole sequence of coagulogen consisting of 175 amino acid residues with the molecular weight of 19,723.
Amino Acid Sequence↗
The amino acid sequence of coagulogen isolated from southeast Asian horseshoe crab, Tachypleus gigas.
The amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab (Tachypleus gigas) has been determined. The NH2-terminal sequence of the first 51 residues was obtained by automated Edman degradation. The intact protein was then treated with a Tachypleus clotting enzyme, to form a gel and to remove an internal peptide C (28 residues) located near the NH2-terminal portion. The gel protein, which consisted of A chain (18 residues) and B chain (129 residues), was S-alkylated and the resulting two chains were separated by acetone precipitation. Among these segments, A chain and peptide C were assigned to the NH2-terminal portion of whole coagulogen, as judged from their amino acid compositions. On the other hand, the covalent structure of B chain was determined by sequencing the peptides obtained from its tryptic digest. The alignments of the tryptic peptides were deduced from the sequence homology in comparison with the previously established B chain sequence of Japanese horseshoe crab (T. tridentatus) coagulogen. T. gigas coagulogen had a total of 175 amino acids and a calculated molecular weight of 19,770. When the sequence was compared with those of Japanese and American horseshoe crab (Limulus polyphemus) coagulogens, extensive structural homology was found: T. tridentatus/T. gigas, 87% and L. polyphemus/T. gigas, 67%. This comparison suggests that Japanese and Southeast Asian horseshoe crabs have a crab, based on amino acid sequence data.
Amino Acid Sequence↗
Plasminogens Tochigi II and Nagoya: two additional molecular defects with Ala-600----Thr replacement found in plasmin light chain variants.
Previous studies in our laboratories (Miyata, T., et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 6132-6136) showed that the structural defect in a hereditarily abnormal plasminogen, plasminogen Tochigi, is due to replacement of Ala by Thr at position 600 from the NH2-terminal end. In the present studies, two abnormal plasminogens, plasminogens Tochigi II and Nagoya, obtained from other family members were analyzed to identify the structural impairment in these molecules. Amino acid sequence analysis of one of the tryptic peptides isolated, respectively, from plasminogens Tochigi II and Nagoya indicated that in both cases, Ala-600 (equivalent to Ala-55 of the chymotrypsin numbering system) had been replaced by Thr. No other substitutions at the active site and substrate-binding site residues, namely, His-57, Asp-102, Ser-195, and Asp-189, were found in the plasmin light chain variants, indicating that all these residues are intact. Moreover, the NH2-terminal heptapeptide sequences of the plasmin light chain variants isolated from plasminogens Tochigi II and Nagoya were identical to the sequence determined for the normal control. These results indicate that the absence of proteolytic activity of both abnormal molecules is due to the same amino acid substitution as that of previously reported plasminogen Tochigi.
Alanine↗
Amino acid sequence of NADH-cytochrome b5 reductase of human erythrocytes.
The amino acid sequence of soluble NADH-cytochrome b5 reductase purified from normal human erythrocytes was determined as one approach to understand the hereditary disease of a deficiency of this enzyme. The protein is hydrophilic as a whole, but two regions, from Phe-36 to Ile-71 and from Met-231 to Phe-275, were found to be highly hydrophobic. The sequence of the latter region is particularly unique, and rich in proline (20%). The sequence of the amino-terminal region was very similar to the partial sequences of the corresponding regions of the enzymes from pig and steer liver microsomes.
Amino Acid Sequence↗
The fine structure of the human ovarian hilus cells.
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Aged↗
[Evaluation of cancer chemotherapy in primary ovarian cancer in rats--observation of histological effect by light and electron microscopy].
In order to evaluate the effects of ifosfamide as an anticancer agent, ifosfamide was administered to rats with ovarian cancer induced by DMBA (7,12-dimethylbenz (a) anthracene). Four to five weeks after the chemotherapeutic treatment 16 rats were sacrificed and then tumor sizes were compared before and after the treatment, and in addition morphologic changes were investigated by light and transmission electron microscopy. Of 16 rats, the reductions in tumor size were recognized in six cases. A histologic effect was observed in both the tumor decreased group and the increased group. One of the latter group showed excellent histological chemotherapeutic effect. On light microscopic observation, the histologic effects seen were reduction in the amount of neoplastic tissues and proliferation of connective tissues. On transmission electron microscopic observation, destruction of tumor cells and a reduction in the amount of ribosome and chromatin were recognized as the effects of the anticancer drug. Multinuclear giant cells, presumably derived from the mesenchym, were found among the connective tissues in one case showing a remarkable histologic effect.
9,10-Dimethyl-1,2-benzanthracene↗
New 434-specific DNA binding protein copurified with the 434 tof protein from lambda imm434cI dv carrier cells of Escherichia coli.
A tof-like protein that has 434-specific DNA binding activity has been copurified with the 434 tof protein from lambda imm434cI dv carrier cells. The apparent molecular weight of the new 434-specific DNA binding protein is 9,000 to 9,500, a little higher than that of the 434 tof protein, as estimated by SDS gel electrophoresis. Amino acid analysis revealed the protein to be an arginine-rich component whereas the 434 tof protein is a lysine-rich component. The specific binding reaction of the new protein to lambda imm434dv DNA is distinct from that of the 434 tof protein in respect to the sigmoid shape of the binding curve and to the temperature dependency. This suggests that the specific binding to lambda imm434dv DNA observed with the new protein is due not to a trace of the 434 tof protein contaminating the new protein preparation but rather to the new protein itself. The NH2-terminal 11 residues of the new 434-specific DNA binding protein were sequenced by manual Edman degradation. This technique revealed that the new protein is not a fragment of the 434 tof, cII, or O protein or an NH2-terminal fragment of the cI repressor. The origin and the physiological roles of the new 434-specific DNA binding protein remain unknown.
Amino Acids↗
Role of HMW kininogen in surface-mediated activation of Factor XII.
We have shown that bovine HMW kininogen remarkably accelerates the activation of Factor XII and prekallikrein in the presence of kaolin, adsorbing on kaolin through the fragment 1.2 region and forming a complex with prekallikrein through the light chain region (Sugo et al., 1980; Ikari et al., 1981). The present study was undertaken to examine the role of HMW kininogen in the activation of Factor XII and prekallikrein with other negatively-charged surfaces. The activation system used here was as follows; (1) Activation of prekallikrein by Factor XII, (2) Activation of Factor XII by plasma kallikrein and (3) Activation of prekallikrein by Factor XIIa. Among a variety of foreign surfaces, amylose sulfate and sulfatide were the most efficient in the activation reaction of Factor XII and prekallikrein. Bovene HMW kininogen accelerated all the three reactions triggered by these surfaces. However, the accelerating effect of HMW kininogen on the activation of Factor XII by plasma kallikrein was very weak, when amylose sulfate or sulfatide was used as surface. The three reactions were highly dependent on the amounts of HMW kininogen and surfaces contained in the reaction mixtures. Excess amount of them inhibited these reactions. Among the various fragments, which were prepared from HMW kininogen digests with plasma and urinary kallikreins (Sugo et al., 1980), a large fragment consisting of fragment 1.2 and light chain accelerated the reactions. Thus both fragment 1.2 and the light chain region in HMW kininogen were essential for these activation reactions.
Amylose↗
[Plasma proteases and metals].
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Amino Acid Sequence↗
[Bacterial endotoxin-mediated activation of Limulus amebocytes and its molecular mechanism].
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Amino Acid Sequence↗
Mechanism of surface-mediated activation of bovine Factor XII and prekallikrein.
The mechanism of kaolin-mediated activation of bovine Factor XII was studied in the presence of prekallikrein and HMW kininogen. The activated enzymes were assayed using fluorogenic peptides, Boc-Glu (OBzl)-Gly-Arg-4-methylcoumaryl-7-amide (MCA) for Factor XIIa and Z-Phe-Arg-MCA for plasma kallikrein. The rates of activation of the zymogens were separately measured by blocking either of the active enzymes with specific inhibitors, corn inhibitor for Factor XIIa (Ki = 6.7 nM) and Trasylol for plasma kallikrein (Ki = 3.9 nM). The result was as follows: (1) At the early stage of the activation reaction, kallikrein activity was first generated after short lag time, and then Factor XIIa activity was generated with a sigmoidal curve. In the presence of corn inhibitor, the activation of prekallikrein was observed, but in the presence of Trasylol, the activation of Factor XII was not observed. In the presence of high concentration of Ala-Phe-Arg-Ch2Cl, which inactivates immediately both of the active enzymes, the cleavage of a single chain prekallikrein into the two chain form by Factor XII was shown by SDS-PAGE, using nonlabelled and tritiated prekallikrein. (2) The incubation of Factor XII alone in a quartz cuvette or in the presence of kaolin and HMW kininogen did not result in the activation of Factor XII. The concave upward curve due to an autocatalytic activation was not observed even after the addition of Factor XIIa to Factor XII preparation. Moreover, no structural change of Factor XII during the incubation with kaolin and HMW kininogen was shown by SDS-PAGE, using 3H-Factor XII. (3) The rates of activation of prekallikrein by Factor XII and by Factor XIIa were approximately the same at higher concentration of prekallikrein. However, at lower concentration of prekallikrein the rate of activation of prekallikrein by Factor XII was shown to be a sigmoidal curve and slower than that by Factor XIIa. These results indicate that the activation of bovine Factor XII is initiated by the attack of Factor XII on prekallikrein, followed by the reciprocal activation of Factor XII by plasma kallikrein generated. The autocatalytic activation of bovine Factor XII by Factor XIIa was not demonstrated.
Amino Acid Chloromethyl Ketones↗