Cysteine proteinase inhibitor in the ascitic fluid of sarcoma 180 tumor-bearing mice is a low molecular weight kininogen. Partial NH2- and COOH-terminal sequences and susceptibility to various glandular kallikreins

It has been proposed that a cysteine proteinase inhibitor (CPI) found in the ascitic fluid of Sarcoma 180 tumor-bearing mice is a kind of kininogen (Itoh, N., Yokota, S., Takagishi, U., Hatta, A., and Okamaoto, H. (1987) Cancer Res. 47, 5560-5565). The first 40 NH2-terminal residues and 54 residues of the COOH-terminal sequence, including the bradykinin moiety of highly purified ascites CPI, were determined and compared with those of mammalian low molecular weight kininogens (LMWK). The significant identity between these amino acid sequences with those of other mammalian LMWKs suggests that ascites CPI corresponds precisely to mouse LMWK. This kininogen has a light chain composed of 43 amino acid residues, which contains a unique Met-Ala-Arg-bradykinin sequence. Hydroxyproline, which was recently identified in the bradykinin sequence of kininogen from the ascitic fluid of a cancer patient, was not found in the kinin moiety of this mouse kininogen. Among purified glandular kallikreins from human, hog, rat, and mouse, only mouse submaxillary gland kallikrein was able to release bradykinin from this kininogen. Kinetic studies using a newly synthesized fluorogenic substrate, N-t-butoxycarbonyl-Met-Ala-Arg-MCA, revealed that mouse kallikrein hydrolyzes this substrate approximately 80-fold faster than does hog kallikrein, suggesting that the unique Met-Ala-Arg-bradykinin sequence is responsible for the varied susceptibility of mouse kininogen to different kallikreins.     

Polymorphism of BF,C2, and GLO in Japanese patients with insulin-dependent diabetes mellitus: Confirmation of an increase of BF *FT

Summary The distribution of phenotypes controlled by three HLA-linked loci BF, C2, and GLO has been studied in Japanese patients with insulin-dependent diabetes mellitus (IDDM). A slight but significant higher incidence of a rare varian BF ^*FT (=^* F075) in patients was confirmed in the combined data with our previous study (Tokunaga et al. 1981 b). No significant association of C2 and GLO alleles with IDDM was found.     

Physical mapping of the genome and sequence analysis at the inverted terminal repetition of adenovirus type 4 DNA

The genome of adenovirus type 4 (Ad4), the unique member of Ad group E, has been mapped with nine restriction endonucleases. Comparison of the occurrence of restriction endonuclease cleavage sites on Ad2, Ad7, Ad12 and Ad4 indicates that there is very little homology between these serotypes. Sequence analysis at the ITR of Ad4 showed that the "CAT" box which is present in all the ITRs of Ad's so far sequenced is absent in Ad4. The length of 116 bp for the ITR of Ad4 is also different from that of other Ad subgroups.     

Photoreaction of tyrosin-iodinated bacteriorhodopsin at low temperature

To elucidate the role of tyrosine residues in the shift of max and the light-driven proton pump of bacteriorhodopsin~ the photochemical reaction of tyrosine-iodinated bacteriorhodopsin (tyr-mod-bR) was investigated by low-temperature spectrophotometry. After 4–5 of 11 tyrosine residues of bacteriorhodopsin were iodinated, the meta-intermediate of tyr-mod-bR in 75% glycerol solution became so stable that its decay could be observed even at room temperature and i t was stable in the dark for several hours at –65°C.Four batho-intermediates were formed by irradiation with green light (500 nm) at –170°C. Like native bacteriorhodopsin, these batho-intermediates were photoreversible at –170°C. Four corresponding meta-intermediates were also formed by irradiation at –60°C. Using the difference spectra between meta-intermediates and tyr-mod-bR, the absorption spectra of four kinds of tyr-mod-bRs, batho-intermediates, and meta-intermediates were estimated. Each was at shorter wavelengths than that of its corresponding type in native bacteriorhodopsin. The results indicate that two or more tyrosine residues have some role in determining color in native bacteriorhodopsin.     

Characterization of porins from the outer membrane of Salmonella typhimurium. 1. Chemical analysis.

The three species of channel-forming outer membrane proteins, porins, have been purified to homogeneity from mutant strains of Salmonella typhimurium which produce single species of porin. Purification was by stepwise solubilization with dodecylsulfate or guanidine thiocyanate, gel filtration, and preparative gel electrophoresis. Amino acid compositions and tryptic peptide maps of the three species of porins showed close resemblance, but at the same time clear differences among them. The number of amino acid residues in the porins purified from the strains SH5551, SH6377 and SH6017 were 361, 354 and 345, and their calculated molecular weights 39800, 39300 and 38000, respectively. Amino-terminal and carboxyl-terminal amino acids in all three species of porins appeared to be alanine and phenylalanine, respectively. Neither half-cystine nor hexosamine was found in these preparation of porins. The isoelectric points of porins from the strains SH5551, SH6377 and SH6017, determined by isoelectric focusing, showed slight differences from each other. These results, and the genetic experiments from another laboratory, suggest that the three species of porins in Salmonella typhimurium are distinct polypeptides, probably coded for by distinct structural genes, which might have been derived from the same ancestral gene.     

Circular dichroism of squid rhodopsin and its intermediates

Circular dichroism (CD) and absorption spectra of squid (Todarodes pacificus) rhodopsin, isorhodopsin and the intermediates were measured at low temperatures. Squid rhodopsin has positive CD bands at wavelengths corresponding the - and β-absorption bands at liquid nitrogen temperature (CD maxima: 485 nm at -band and 348 nm at β-band) as well as at room temperature (CD maxima: 474 nm at -band and 347 nm at β-band). The rotational strength of the -band has a molecular ellipticity about twice that of cattle rhodopsin. The CD spectrum of bathorhodopsin displays a negative peak at 532 nm, the rotational strength of which has an absolute value slightly larger than that of rhodopsin. The reversal in sign at -band of the CD spectrum may indicate that the isomerization of retinal chromophore from twisted 11-cis form to twisted 11-trans form has occurred in the process of conversion from rhodopsin to bathorhodopsin. Lumirhodopsin has a small negative CD band at 490 nm, the maximum of which lies at 25 nm shorter wavelengths than the absorption maximum (515 nm), and a large positive CD band near 290 nm, which is not observed in rhodopsin and the other intermediates. This band may be derived from a conformational change of the opsin. In the process of changing from lumirhodopsin to LM-rhodopsin, the CD bands at visible and near ultraviolet regions disappear. Both alkaline and acid metarhodopsins have no CD bands at visible and near ultraviolet regions.     

Formation of hypsorhodopsin in frog retina

Hypsorhodopsin was formed in frog retina by irradiation at liquid helium temperature and converted into bathorhodopsin above about 29 K.     

Modification of a novel angiogenic peptide, AG30, for the development of novel therapeutic agents

We previously identified a novel angiogenic peptide, AG30, with antibacterial effects that could serve as a foundation molecule for the design of wound-healing drugs. Toward clinical application, in this study we have developed a modified version of the AG30 peptide characterized by improved antibacterial and angiogenic action, thus establishing a lead compound for a feasibility study. Because AG30 has an α-helix structure with a number of hydrophobic and cationic amino acids, we designed a modified AG30 peptide by replacing several of the amino acids. The replacement of cationic amino acids (yielding a new molecule, AG30/5C), but not hydrophobic amino acids, increased both the angiogenic and the antimicrobial properties of the peptide. AG30/5C was also effective against methicillin-resistant Staphylococcus aureus (MRSA) and antibiotic-resistant Pseudomonas aeruginosa. In a diabetic mouse wound-healing model, the topical application of AG30/5C accelerated wound healing with increased angiogenesis and attenuated MRSA infection. To facilitate the eventual clinical investigation/application of these compounds, we developed a large-scale procedure for the synthesis of AG30/5C that employed the conventional solution method and met Good Manufacturing Practice guidelines. In the evaluation of stability of this peptide in saline solution, RP-HPLC analysis revealed that AG30/5C was fairly stable under 5°C for 12 months. Therefore, we propose the use of AG30/5C as a wound-healing drug with antibacterial and angiogenic actions.