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.     

Nucleosidediphosphate-kinase induction by human recombinant IL-2 in mouse NK cells

The ability of human recombinant IL-2 to induce NDP-kinase in mouse NK cells has been studied. A significant increase in the amount of NDP-kinase was observed when the cells were exposed to IL-2 (100 units/ml) for 3 h at 37 degrees C. The enzyme inducting ability of human recombinant IL-2 was similar to that of native mouse IL-2 in the cells. The enzymatic characteristics [chemical requirements for the phosphoenzyme formation and molecular size of two distinct subunits (18,000 and 20,000 daltons)] of NDP-kinase from IL-2 treated cells were similar to those of the enzymes from EAT cells. The enzyme's biological role in the initiation of cell proliferation by IL-2 has been discussed.     

Nucleosidediphosphate kinase from Ehrlich ascites tumor cells

A phosphate-incorporating protein has been highly purified from the cytosol of Ehrlich ascites tumor cells (EAT cells). The nitrocellulose membrane method was used to follow the progress of the purification by quantitation of the [32P]phosphorylated form of the protein. The purified protein was identified as an NDP-kinase since it exhibited NDP-kinase activity and had enzyme characteristics in common with other NDP-kinases from various mammalian cells. The purified NDP-kinase was found to have a molecular weight of approximately 76,000 daltons. Moreover, the enzyme appears to consist of two distinct polypeptides (18,000 and 20,000 daltons). This enzyme contained 19 amino acids, with high levels of glycine (9.8%) and lysine (9.0%). The enzyme rapidly formed a [32P]phosphoenzyme when incubated with [gamma-32P]ATP in the presence of Mg2+ (1 mM) at the optimum pH of 7.5 even at low temperature (below 4 degrees C). This phosphoenzyme is an enzyme-bound, high-energy-phosphate intermediate, because ATP was formed from it on incubation with ADP in the presence of Mg2+ (1 mM). This finding suggests that the phosphoenzyme functions as an intermediate in NDP-kinase action.