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.     

A possible clinical application of multicytokine-producing cytotoxic mononuclear cell (MCCM) therapy

When peripheral blood mononuclear cells (PBMC) were incubated with a streptococcal preparation, OK-432, for 24 h, PBMC acquired cytolytic activity against cultured and fresh human tumor cells. Such PBMC were called OK-432-activated mononuclear cells (OK-MC). OK-MC produce several kinds of cytokines such as interferon (IFN), IFN, and tumor growth inhibitory factor (TGIF) bothin vitro andin vivo. OK-MC-produced cytokines also inhibited the growth of cultured and fresh human tumor cells. The growth inhibition was examined by human tumor clonogenic assay using a double-layer agar technique. The results indicate that two pathways of anti-tumor activity are induced in OK-MC, i.e., cell-mediated and cytokine-mediated.     

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.     

Isolation and characterization of gastric microsomal glycoproteins. Evidence for a glycosylated beta-subunit of the H+/K(+)-ATPase.

Detergent-solubilization of hog gastric microsomal membrane proteins followed by affinity chromatography using wheat germ agglutinin or Ricinus communis I agglutinin resulted in the isolation of five glycoproteins with the apparent molecular masses on sodium dodecyl sulfate polyacrylamide gels of (in kDa): 60-80 (two glycoproteins sharing this molecular mass); 125-150; and 190-210. In the nonionic detergent Nonidet P-40 (NP-40), the 94 kDa H+/K(+)-ATPase was recovered exclusively in the lectin-binding fraction; however, in the cationic detergent dodecyltrimethylammonium bromide, most of the ATPase was recovered in the nonbinding fraction. Detection of glycoproteins either by periodic acid-dansyl hydrazine staining of carbohydrate in polyacrylamide gels or by Western blots probed with lectins indicated that the majority of the ATPase molecules are not glycosylated. In addition, in the absence of microsomal glycoproteins, the NP-40-solubilized ATPase does not bind to a lectin column. Taken together, these results suggest that the recovery of NP-40-solubilized ATPase in the lectin-binding fraction is due to its noncovalent interaction with a gastric microsomal glycoprotein. Immunoprecipitation of the ATPase from NP-40-solubilized microsomal membrane proteins resulted in the co-precipitation of a single 60-80 kDa glycoprotein. Characterization of the 60-80 kDa glycoprotein associated with the ATPase revealed that: it is a transmembrane protein; it has an apparent core molecular mass of 32 kDa; and, it has five asparagine-linked oligosaccharide chains. Given its similarity to the glycosylated beta-subunit of the Na+/K(+)-ATPase, this 60-80 kDa gastric microsomal glycoprotein is suggested to be a beta-subunit of the H+/K(+)-ATPase.     

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.     

Seasonal and areal features of the lagoonal environment in Lake Nakanoumi,a shallow coastal lagoon in Japan

Lake Nakanoumi is a shallow coastal lagoon connected with the Japan Sea by a narrow channel. Over the past decade, land reclamation resulted in a 33% reduction of the lagoon's surface area. The remaining water basin of Lake Nakanoumi is scheduled to be artificially freshened to supply irrigation water for the newly reclaimed lands. This paper deals with the seasonal and areal features of the lagoonal environment prior to the beginning of the artificial desalinization.     

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 10. IN VIVO AND IN VITRO SYNTHESIS OF alpha-AMYLASE IN RICE SEED SCUTELLUM

Scutellar tissues were dissected from germinating rice seeds and the incorporation of [³⁵S]methionine into the α-amylase molecule was examined by in vivo and in vitro assay systems. Immunoprecipitation with monospecific anti-α-amylase immunoglobulin G raised against the purified enzyme preparation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography were used to identify α-amylase and its possible precursor molecule. Using freshly prepared scutellar tissues, it was demonstrated that α-amylase is synthesized de novo in the scutellar epithelium and secreted into endosperm. The synthesis of α-amylase directed by the polyadenylic acid-containing ribonucleic acid isolated from the scutellar tissues was also established using the translation system of either wheat germ extract or reticulocyte lysate. The immunoprecipitable product obtained in the in vitro translation system was smaller in molecular weight than that synthesized in vivo on the basis of mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results are discussed in relation to the processing of the nascent polypeptide precursor of the enzyme molecule and the introduction of the oligosaccharide chain to the cleaved polypeptide to make up the mature form of α-amylase.     

Biosynthesis and excretion of hydrolases in germinating cereal seeds

The initial formation site of hydrolases in germinating cerealseeds and their subsequent release were examined using the substrate-filmtechnique. Early in the germination of cereal seeds, e.g., barley,wheat, rye, oat and maize, -amylase invariably appeared in theregion of the epithelial cells of the scutellum, whereas itlater gradually diffused into the entire region of the endospermtissues. The initial formation site of proteinase and RNA-asein germinating barley seeds was also confirmed to be in theepithelium. We conclude that the epithelium has a more importantrole in the enzymic breakdown of reserve substances stored inthe endosperm tissues than the aleurone layer. (Received October 19, 1979; )     

Lysis of growing fissin-yeast cells induced by aculeacin A,a new antifungal antibiotic

Cells of Schizosaccharomyces pombe grown in the presence of aculeacin A, a peptide antibiotic, were lysed resulting the death of cells. Under high osmolarity, the cellular lysis induced by aculeacin A was considerably reduced. The use of synchronous-culture systems distinguished cell elongation from cell division revealed that the sites of aculeacin A-induced lysis on the fission yeast were the end(s) and the cell plate region, corresponded to the regions of the cell wall synthesis. Aculeacin A-resistant survivors exhibited morphological alterations which were swollen at one or both ends of the cell and appeared drumstick or dumbbel like; the wall of the bulge region was observed to be stained with a fluorescent brightener, as well as that of the cell plate region. These effects of aculeacin A are discussed as compared with effects of 2-deoxy-D-glucose.