Biomedical subjects
Publications and source records attributed to T Sako.
We have cloned and sequenced some prlA mutant alleles of the Escherichia coli secY gene. From the mutation sites determined, it is strongly suggested that distinct regions in the SecY (PrlA) protein are involved in the recognition of different structural features of a signal peptide as it functions.
Bryostatin 1, a macrocyclic lactone, activated protein kinase C purified from mouse brain in a dose-dependent fashion to the same degree as phorbol 12-myristate 13-acetate (PMA). There was no significant difference in calcium and phosphatidylserine requirements for activation of protein kinase C between bryostatin 1 and PMA. We also found no significant difference in the inhibitory effect between staurosporine and H-7 known to be potent inhibitors of protein kinase C. These data suggest that bryostatin 1 and PMA activate protein kinase C in a similar way. We found, however, that negative modulation of protein kinase C with bryostatin 1 was weaker than that with PMA. The reason of this difference was unclear. It may possibly suggest that there is some difference in configuration of protein kinase C after binding between these activators.
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Bryostatin 1, a macrocyclic lactone, functions like the phorbol esters biochemically in binding to and activating protein kinase C. Biologically, however, although it induces some phorbol ester responses such as mitogenesis in Swiss 3T3 cells, it paradoxically blocks the effects of the phorbol esters on differentiation in HL-60 promyelocytic leukemia cells and Friend erythroleukemia cells. Since the phorbol esters induce proliferation and terminal differentiation in distinct subpopulations of epidermal basal cells, we have now examined the action of bryostatin 1 in that system. Bryostatin 1 decreased epidermal growth factor binding and induced ornithine decarboxylase activity, the latter a marker of proliferation. The magnitude of the maximal induction of ornithine decarboxylase was less than for phorbol 12,13-dibutyrate. Bryostatin 1 only transiently caused the morphological change typical of phorbol ester treatment and did not induce transglutaminase or cornified envelope production, markers of the differentiative pathway. Combined treatment with bryostatin 1 and phorbol 12,13-dibutyrate gave similar results to treatment with bryostatin 1 alone, i.e., slight reduction to complete inhibition of phorbol ester action, depending on the response. The mechanism may reflect time dependent block of the protein kinase C pathway by bryostatin 1 in this system; although bryostatin 1 inhibited epidermal growth factor binding at short incubation times (1-2 h), by 4 h of incubation its inhibition was markedly reduced and it correspondingly blocked inhibition of epidermal growth factor binding by phorbol 12,13-dibutyrate. Since induction of terminal differentiation is proposed to be an essential component of phorbol ester mediated tumor promotion in skin, our findings suggest that bryostatin 1 may function as an inhibitor of phorbol ester promotion.
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Staphylokinase mutants having amino acid substitutions within the amino-terminal charged segment of the signal peptide have been produced by in vitro oligonucleotide-directed mutagenesis. When the processing of the gene products was analyzed in Escherichia coli cells, the rate of processing of the mutant staphylokinase precursor decreased as the net charge became more negative. A net positive charge, but not specific amino acid residues, was required on the amino-terminal segment for efficient processing. Staphylokinase precursor having a net negative charge accumulated in the cytoplasm, tending to bind to the cytoplasmic membrane as determined by subcellular fractionation and immunoelectron microscopy. Although a mutant carrying an amino acid substitution in the hydrophobic segment and wild-type staphylokinases had an interfering effect on the processing of other normal secreted proteins, this effect was lost when they also contained charge-altering substitutions in the amino-terminal region. From these results, we concluded that a positive charge on the amino-terminal segment of the staphylokinase signal peptide is required for entrance into the protein export process.
We determined the partial primary structures of and identified the genes for new basic proteins A and B in Escherichia coli ribosomal 50S subunits, found by means of an improved two-dimensional gel electrophoresis method. The sequence up to the 17th amino acid of protein B was in agreement with that of the X gene in the spc operon. The gene for protein A was searched for in the GenBank data base using the sequence up to the 35th amino acid, and was found at a locus between infC and rplT. The base sequence indicated that protein A contained 64 amino acids and had a molecular weight of 6,984. We conclude that proteins A and B are intrinsic ribosomal proteins, and propose calling their genes, rpmI and rpmJ, respectively.
Export through the cytoplasmic membrane and processing of the sak product in Escherichia coli cells were investigated with E. coli strains carrying pTS301, which produce large amounts of staphylokinase at 42 degrees C. High-level synthesis of the sak product caused transient accumulation not only of the staphylokinase precursor (pSAK) but also of the maltose-binding protein and outer membrane protein A precursors. Thus it was concluded that the sak product shares the export pathway with E. coli secreted proteins at least at a certain step. During high-level synthesis of the sak product, a significant amount of the newly synthesized pSAK remained unprocessed after a chase period, possibly causing the observed accumulation of pSAK. Accumulating pSAK did not mature for a long period, whereas the newly synthesized sak product was exclusively detected in the mature form. These results suggest that it is necessary for the sak product to enter the export pathway during or immediately after synthesis to be exported and processed normally.
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A recombinant plasmid which directs the overproduction in Escherichia coli of staphylokinase from Staphylococcus aureus has been constructed by placing the staphylokinase gene, sak, under the control of bacteriophage lambda PR promoter in the plasmid. When an E. coli strain having the plasmid was induced, the staphylokinase activity in the periplasmic fraction increased about 60-fold and the 15.5-kDa protein corresponding to the mature form reached about 25% of the periplasmic proteins. At the same time the 18.5-kDa protein corresponding to the precursor form was accumulated in the membrane fraction, showing that the processing and translocation of the sak gene product were restricted during high level of its synthesis. By using this strain, the mature staphylokinase has been easily purified to near homogeneity. The purification steps consisted of extraction of the periplasmic proteins by osmotic shock and CM-cellulose column chromatography. Two species of staphylokinase were identified after CM-cellulose column chromatography. Although their isoelectric points and NH2-terminal amino acid sequences were different, their specific activities were almost equal. These results strongly suggest that the NH2-terminal portion of staphylokinase is not important for its activity.
Synergistic antiproliferative effect has been proven in vitro when mouse leukemic cells were sequentially treated with MTX and ara-C. The mechanism of this combination effect not well elucidated but the intracellular uptake of ara-C was higher when cells were pre-exposed to MTX. In this experiment, the intracellular ara-CTP was measured by HPLC after MTX and ara-C were sequentially administered to BDF1 mice bearing L1210 leukemic cell, either being sensitive or resistant to MTX. When MTX at the dose of 12 mg/kg was preceded 6 h and 3 h to ara-C at the dose of 25 mg/kg, the intracellular levels of ara-CTP were found to be significantly higher as compared with those of ara-C alone as control group. At 1 h after ara-C, ara-CTP was measured about 165 and 130% of the control levels, respectively, and at 12 h, ara-CTP was over 4 times higher of control level with group of mice to which MTX was preceded 6 h prior to MTX. On the contrary, the enhancement of ara-CTP production was definitely diminished with MTX-resistant cells in the same administrative model. From our present experiment, the time sequential modulation of intracellular ara-CTP production by MTX was reconfirmed in vivo, and this modulation might depend upon the sensitivity of MTX of leukemic cells.
Ogilvie's syndrome is caused from functional obstruction of the colon without mechanical stenosis and also termed as pseudo-obstruction of the colon. This disease is seen in patient who has other causative distress. The pathogenesis of this rare entity is unknown but it is suspected that there may be an underlying autonomic disturbance which causes non-mechanical obstruction of the colon. Most of the case reports on Ogilvie's syndrome have appeared in European and North American journals but are extremely rare in Japan. Some cases which have been categorized "spastic ileus" may be included in this syndrome. We recently experienced a patient with cerebral infarction who developed non-mechanical obstruction of the colon. This patient was treated successfully with an exploratory laparotomy and sigmoidostomy. It is considered that early diagnosis is an essential part of the initial management of this syndrome. Conservative treatment is indicated for most of the patients but a definitive surgical therapy is occasionally necessary.
For use in routine clinical studies, modifications to Salmon and Hamburger's human tumor stem cell assay were made. A multiplate with 24 wells made the handling of a large number of samples feasible. The addition of anticancer drugs to the bottom layer of agar facilitated avoidance of exposure to drugs before cell plating and evaluation of the effect of long-acting drugs such as 5-fluorouracil. Storage of test plates including anticancer drugs in a freezer produced no loss of colony-forming activity. Specimens from 32 patients with advanced malignancies of the GI tract were tested for sensitivity to anticancer drugs. Forty-seven percent formed enough colonies for the performance of drug testing. Two patients showed sensitivity to drugs from both in vitro and in vivo results; the ascites in one disappeared while the other showed more than 50 percent regression of hepatic metastatic foci after treatment with suitable drugs. Nine patients showed resistance to drugs from both in vitro and in vivo results. Eight showed resistance to all tested drugs.
In a previous study, we showed that methotrexate (MTX) enhanced the intracellular production of ara-CTP. The study described in the present paper has elucidated the mechanism of this MTX-enhanced ara-CTP production, which occurs as a result of MTX reducing the intracellular dCTP pool, and the decreased dCTP pool then allowing activation of deoxycytidine Kinase. One of the mechanisms of synergistic interaction between 1-beta-D-arabinofuranosylcytosine (ara-C) and MTX might therefore be the stimulated phosphorylation of ara-C with MTX-activated deoxy-cytidine kinase. Using high-pressure liquid chromatography, the sequential changes occurring in the acid-soluble intracellular nucleotide pools of L1210 mouse leukemic cells were analysed after treatment with MTX (12mg/kg). At 3 hr after treatment with MTX a reduction of the dTTP pool to 46% of the control level was observed. The dCTP pool was also reduced to 36% of the control level after treatment with MTX. The levels of the dATP and dGTP pools were not significantly changed, at least during the observation period. Pyrimidine ribonucleotide pools were almost unchanged at 3 hr, but in the sequential changes observed in purine ribonucleotide pools after treatment with MTX, both diphosphate and triphosphate pools were seen to be on the decline, the reduction of triphosphate pools being especially marked. A decline in ATP and GPT to 24-30% of control levels was observed at 3 hr after treatment with MTX.
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We have determined the entire nucleotide sequence of a 1,4-kilobase segment containing the staphylokinase gene, sak, molecularly cloned from the bacteriophage S phi-C genome of Staphylococcus aureus. The probable coding region is 489 base pairs long and these base pairs are translated into a polypeptide of 163 amino acid residues (Mr = 18,490) with a presumed signal sequence of 27 amino acid residues at the NH2-terminal end. In regions adjacent to the sak structural gene a possible promoter sequence and three possible terminator sequences for transcription were found about 100 base pairs upstream from the initiation codon and about 300, 400, and 500 base pairs downstream from the termination codon, respectively; they are active in an in vitro transcription system using Escherichia coli RNA polymerase. The immunoactive 18,500-dalton and 15,500-dalton proteins corresponding to a precursor form before secretion and a mature form after secretion of the sak gene products, respectively, were identified by the E. coli maxicell system.
Restriction fragments of DNA from bacteriophage S phi-C of Staphylococcus aureus which carries the gene for staphylokinase, one of the plasminogen activators, were cloned onto plasmid pBR322. Recombinant plasmids carrying the 2.5 kilobase pair segment of S phi-C DNA confer on Escherichia coli cells the capacity to synthesize staphylokinase. The enzyme is synthesized in amounts comparable to that found in S. aureus, and irrespective of the orientation of cloned fragments and their insertion site on pBR322. The active enzyme produced by E. coli cells is preferentially recovered from the periplasmic space and in part excreted into the culture medium. It is indistinguishable from the enzyme produced by S. aureus in molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in antigenicity, as determined by the micro-Ouchterlony precipitation test.
Continuous illumination (LL) beginning at 22 days of age caused precocious puberty followed by persistent estrus with anovulation in female offspring originating from mother rats exposed to a 14L:10D light-dark cycle prior to and during pregnancy. However, LL had no deleterious effect on reproductive cycles of offspring reared in LL and originating from mothers exposed to LL prior to and during pregnancy. These rats had a normal onset of puberty in LL, a normal 4-day estrous cycle, a periodic rise of plasma estrogen prior to the periodic appearance of the preovulatory luteinizing hormone (LH) surge, and spontaneous ovulation in LL continued until at least 300 days of age. Also, the female offspring of these rats showed a similar resistance to the deleterious effects of LL on cyclic ovulation. These results support the following interpretation: 1) offspring from mother rats exposed to LL prior to and during pregnancy become insensitive to the deleterious effects of LL on cyclic ovulation, 2) neural elements controlling cyclic release of LH are not totally photoperiod (14L:10D)-dependent, and 3) in the absence of daily 14L:10D signals, an endogenous clock, possibly timed by daily laboratory signals (temperature, noise, taking of vaginal smears), may provide time cues for cyclic LH release.