Mitochondrial DNA polymorphism among five Asian populations

Mitochondrial DNA (mtDNA) polymorphisms were detected using 13 restriction enzymes on the total DNA obtained from blood samples of five Asian populations: Japanese and Ainu of northern Japan, Korean, Negrito (Aeta) of the Philippines, and Vedda of Sri Lanka. Of a total of 28 restriction-enzyme morphs detected, eight had not been reported previously. By combining the morphs, we were able to classify mtDNAs of 243 individuals into 20 mtDNA types. Phylogenetic analyses using maximum parsimony and genetic distance methods both showed that the Japanese, Ainu, and Korean populations were closely related to each other. Aeta was found to show a relatively close relationship to these three populations, confirming the conclusion from previous studies of blood markers. In contrast, Vedda was quite different from the other four populations.     

On some freshwater harpacticois from Japan,closely related to Canthocamptus mirabilis Štěrba

Preliminary comparisons are presented, of certain diagnostic characters of four species of Canthocamptus, all the specimens examined here being collected from freshwater bodies throughout Japan. The geographical distribution of each species is also discussed.Contribution No. 69 from the Itako Hydrobiological Station, Ibaraki University.     

Molecular cloning of complementary DNA encoding one of the human pancreatic protease E isozymes

We have cloned a DNA from a human pancreatic cDNA library using a cloned rat pancreatic elastase 1 cDNA as a probe, and determined its nucleotide sequence. This cDNA contains a coding region of 810 nucleotides which encodes a 270-amino-acid protein. The deduced amino acid sequence shows less than 60% homologies with rat and porcine pancreatic elastase 1, although its substrate binding region is homologous with those of the above elastases 1. When this deduced amino acid sequence was compared with known amino acid sequences of pancreatic proteases other than elastases, it was found to contain an amino acid sequence which was highly homologous with the N-terminal amino acid sequence of porcine pancreatic protease E. We also purified human pancreatic protease E isozymes from human pancreatic juice, and determined their N-terminal amino acid sequences. One of the isozymes does not hydrolyze elastin but does hydrolyze a synthetic substrate. Endoglycosidase F digests glycoside bonds of the isozyme. These results suggest that the cDNA cloned by us corresponded to one of the human protease E isozymes.     

A single amino acid substitution converts cytochrome P450(14DM) to an inactive form, cytochrome P450SG1: complete primary structures deduced from cloned DNAS

Genes for lanosterol 14-demethylase, cytochrome P450(14DM), and a mutated inactive cytochrome P450SG1 were cloned from S. cerevisiae strains D587 and SG1, respectively. A single nucleotide change resulting in substitution of Asp for Gly-310 of cytochrome P450(14DM) was found to have occurred in cytochrome P450SG1. In this protein the 6th ligand to heme iron is a histidine residue instead of a water molecule, which may be the ligand for the active cytochrome P450(14DM). Molecular models of the active sites of the cytochrome P450(14DM) and cytochrome P450SG1 were built by computer modeling on the basis of the known structure of that of cytochrome P450CAM whose crystallographic data are available. The mechanisms which may cause a histidine residue to gain access to the heme iron are discussed.     

Possible involvement of 1 alpha,25-dihydroxyvitamin D3 in proliferation and differentiation of 3T3-L1 cells

Growth of 3T3-L1 cells was inhibited by 10(-10)-10(-7)M of 1 alpha,25-dihydroxy vitamin D3 [1 alpha,25(OH)2D3] in a dose- and time-dependent manner. The potency of 1 alpha,25(OH)2D3 in inducing differentiation was low, since 3T3-L1 cells cultured with 1 alpha,25(OH)2D3 did not become mature adipocyte-like cells but were changed to slightly rounded cells containing small droplet-like substances in the cytoplasm and glycerophosphate dehydrogenase (sn-glycerol-3-phosphate: NAD+2-oxidoreductase, EC 1.1.1.8), the marker enzyme of differentiation to adipocyte, did not increase. These results together with the natural occurrence of this vitamin indicate that 1 alpha,25(OH)2D3 may play an important role in the cell growth and differentiation besides such known action as intestinal calcium transport and bone mineral mobilization.     

Stacking and hydrogen bonding interactions between phenylalanine and guanine nucleotide: crystal structure of L-phenylalanine-7-methylguanosine-5'-monophosphate complex

The crystal structure of title complex has been analyzed by X-ray diffraction method as a model for elucidating the possible interaction between the phenylalanyl residue of proteins and the N7-protonated or methylated guanine base of nucleic acids. The guanine base is associated with the benzene ring of phenylalanine by stacking interaction, and further connected with the carboxyl group by the formation of a pair of hydrogen bonds. These two interaction modes are suggested to be responsible for the specific recognition of base sequence by protein.     

Binding and crosslinking of 125I-labeled recombinant human tumor necrosis factor to cell surface receptors

Highly purified recombinant human tumor necrosis factor (TNF) (molecular mass determined as 17 kilodaltons (kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as 36 kDa by Sephadex G-100 gel chromatography) was labeled with 125I to a specific activity of 5 microCi/micrograms without appreciable loss of activity. The binding of 125I-TNF to eighteen human and twelve animal cell lines was examined. The binding varied considerably among different cell lines. In most cell lines, the binding was inhibited up to greater than 90% by the addition of a 100-fold excess of unlabeled TNF. Some human and mouse cell lines showed no significant binding above background levels, suggesting that these cell lines had no receptors for TNF. Among the TNF receptor-positive cell lines, there was no direct correlation between the level of specific TNF binding and the level of sensitivity to the cytotoxic or cytostatic effect of TNF. Some cell lines were sensitive to TNF, whereas others were not affected at all by TNF. The TNF receptor-negative cell lines were also resistant to TNF. Therefore, although the existence of TNF receptor seems to be necessary, it does not alone determine cellular sensitivity to TNF. Scatchard analysis of the binding data revealed that human HeLa S3 and THP-1 had about 50,000 and 10,000 receptors/cell with a dissociation constant (KD) of 0.3-0.5 nM, respectively. Similarly, mouse L-929 and L-M cells had about 5,000 receptors/cell with KD of 3-5 nM. 125I-TNF bound to HeLa S3 cells was rapidly internalized at 37 degrees C, presumably by receptor-mediated endocytosis, and degraded to acid-soluble products. The turnover of TNF receptors on HeLA S3 cells seemed to be rapid, since the level of specific binding quickly decreased after treatment with 100 micrograms/ml of cycloheximide at 37 degrees C with a half-life of about 1.5 h. The crosslinking of the cell-bound 125I-TNF with the use of disuccinimidyl suberate yielded a complex of 105 kDa for HeLa S3 and THP-1 cells, and a complex of 100 kDa for U937 cells. The crosslinking was completely inhibited by the addition of a 100-fold excess of unlabeled TNF. Assuming that the complex was due to a one-to-one association of the dimeric form of TNF (34 kDa) with the receptor, we estimated the molecular size of the human TNF receptor to be 71 kDa for HeLa S3 and THP-1, and 66 kDa for U937.     

Purified protein kinase C phosphorylates microtubule-associated protein 2

We have investigated actions of purified protein kinase C on microtubule- and microfilament-related proteins. Among the cytoskeletal proteins examined, microtubule-associated protein 2 (MAP2) was found to serve as a good substrate. Other cytoskeletal proteins, tubulin, fodrin, cofilin, tropomyosin, and 53,000-Da protein, were very poorly phosphorylated. The amino acid residues of MAP2 that were phosphorylated by the protein kinase C were almost exclusively serine. The peptide mapping analysis indicated that protein kinase C and cAMP-dependent protein kinase phosphorylate MAP2 differently. The ability of MAP2 to interact with actin was markedly reduced by this protein kinase C-mediated phosphorylation. These data raise the possibility that phosphorylation of MAP2 by activated protein kinase C may be involved in cell-surface signal transduction.     

Technique for the long-term storage of lobster (Homarus) spermatophores

We have developed a procedure for the long-term storage of lobster (Homarus) sperm. Sperm are collected from males by electrically stimulating the area around the gonopore. They are transferred with a bamboo stick to a plastic test tube containing paraffin oil and are stored for variable periods of time at 4–7°C. Sperm stored up to 289 days appear morphologically normal (equivalent to unstored sperm) when examined by phase contrast microscopy and electron microscopy, and morphologically normal sperm are able to undergo acrosome reactions. After longer periods of storage, degenerative changes begin to develop in sperm. These include loss of the nuclear spikes, condensation of the subacrosomal material, and distortion of the acrosome. Sperm stored better in spermatophores that had thick walls than in those with thin walls. In some spermatophores, bacteria were found in the sperm mass after storage; in general, sperm in these spermatophores were morphologically abnormal. This technique provides a means for saving lobster sperm for subsequent use in experiments or for artificial insemination of female lobsters. It may be adaptable to other invertebrate species that produce spermatophores.     

Genome type analysis of adenovirus type 4 isolates, recently obtained from clinically different syndromes in some areas in Japan

DNA cleavage analyses with EcoRI, HindIII and BamHI were carried out to investigate genome types of recent Ad 4 isolates obtained from acute respiratory disease (8 strains), and ocular disease (11 strains) in Japan. DNA cleavage patterns of all 19 isolates studied were identical regardless of whether they were recovered from respiratory tract or conjunctiva, but were distinct from that of the prototype strain.