Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Age-related differences in muscle activity, stride frequency and heart rate response during walking in water.

This study aimed to examine whether walking in water produces age-related differences in muscle activity, stride frequency (SF), and heart rate (HR) response. Surface electromyography (EMG) was used to evaluate muscle activities in six older and six young subjects while they walked in water immersed to the level of the xiphoid process. The trials in water utilized the Flowmill which consists of a treadmill at the base of a water flume. The measurement of maximal voluntary contraction (MVC) of each muscle was made prior to the gait analysis. The %MVCs, which refer to the surface EMG measures, from the gastrocnemius of the older subjects were significantly lower than those of the young subjects, in every experimental condition (P<0.05). In contrast, the %MVCs from the rectus femoris (P<0.05) and the biceps femoris (P<0.001) of older subjects were significantly greater than those of young subjects in every experimental condition. Moreover, the SFs of older subjects were also significantly greater than those of young subjects (P<0.05), while the HR responses of older and young subjects were similar. In conclusion, the older subjects had increased hip musculature activity and decreased ankle plantar flexor activity while walking in water, compared with the young subjects.     

Alphoid satellite DNA is tightly associated with centromere antigens in human chromosomes throughout the cell cycle

In this study, we have examined a DNA element specific to the centromere domain of human chromosomes. Purified HeLa chromosomes were digested with the restriction enzyme Sau3AI and fractionated by sedimentation through a sucrose gradient. Fractions showing antigenecity to anticentromere (kinetochore) serum obtained from a scleroderma CREST patient were used to construct a DNA library. From this library we found one clone which has specifically hybridized to the centromere domain of metaphase chromosomes using a biotinylated probe DNA and FITC-conjugated avidin. The clone contained a stretch of alphoid DNA dimer. To determine precisely the relative location of the alphoid DNA stretch and the centromere antigen, a method was developed to carry out in situ hybridization of DNA and indirect immunofluorescent staining of antigen on the same cell preparation. Using this method, we have found perfect overlapping of the alphoid DNA sites with the centromere antigen sites in both metaphase chromosomes and nuclei at various stages in the cell cycle. We have also observed this exact correlation at the attachment sites of artificially extended sister chromatids. These results suggest the possibility that alphoid DNA repeats are a key component of kinetochore structure.     

Selective production of glutamate by an immobilized marine blue-green alga,Synechococcus sp.

Summary Among 200 strains of marine bluegreen algae isolated from the coastal areas of Japan, the marine blue-green alga Synechococcus sp. NKBG 040607 excreted glutamate at the highest rate, 82.6% of total amino acids production being glutamate. Synechococcus sp. NKBG 40607 was immobilized in calcium alginate gel. Glutamate production by immobilized cells was double that of native cells. Maximal glutamate production (25 g/cm³ gel per day) of the immobilized cells was observed under a light intensity of 144 Einstein/m² per second at a cell concentration of 7.5 mg dry cells/cm³ gel. Immobilized cells of Synechococcus sp. can use nitrate as a nitrogen source. Immobilized marine Synechococcus sp. produced 0265 mg/cm³ gel of glutamate for 7 days in the presence of chloramphenicol.     

Complete nucleotide sequence of the mitochondrial DNA from a liverwort,Marchantia polymorpha

Libraries of cosmid and plasmid clones covering the entire region of mtDNA from the liverwortMarchantia polymorpha were constructed. These clones were used for the determination of the complete nucleotide sequence of the liverwort mtDNA totally 186,608 bp (GenBank no. M68929) and including genes for 3 species of ribosomal RNAs, 29 genes for 27 species of transfer RNAs, and 30 genes for functionally known proteins (16 ribosomal proteins, 3 subunits of cytochromec oxidase, apocytochromeb protein, 3 subunits of H⁺-ATPase, and 7 subunits of NADH ubiquinone oxidoreductase). The genome also contains 32 unidentified open reading frames. Thus the complete nucleotide sequences from both chloroplast and mitochondrial genomes have been determined in the same organism. Plasmid clones are available upon the request. Gene names are represented according to Lonsdale and Leaver (1988) with modifications recommended by Lonsdale (personal communication).     

Characterization and Intracellular Distribution of Pea Phytochrome I Polypeptides Expressed in E. coli

The pea phytochrome I (PI) cDNA clone, pPP1001, was expressedin E. coli. The plasmid pPP1001 contains pea PI cDNA which coversthe entire coding region with the Shine-Dalgarno consensus sequencejoined upstream of the cDNA in an expression vector pNUT6. ThepPP1001 transformants formed typical inclusion bodies when culturedat 32?C. However, when cultured at 37?C or in the presence ofisopropyl-ß-D-thiogalactopyranoside (IPTG) at 32?C,the bacteria lysed before inclusion body formation. Immuno-stainingwith anti-PI monoclonal antibody, mAP5, of transformants fixedby cold methanol showed that stainable materials were distributedin whole cytoplasmic region. When the inclusion bodies wereobserved clearly, the regions corresponding to the inclusionbodies became difficult to stain. Western blot analysis, however,showed that a ca. 100 kDa PI polypeptide was detected in thefraction from inclusion bodies and a ca. 90 kDa PI polypeptidefrom the soluble fraction. The amino acid sequence analysisof purified 100 kDa PI sample indicated that its amino terminusis blocked. However, minor signals in one experiment yieldeda sequence corresponding to the expected amino terminus of peaPI except for the initiation methionine. One of the anti-peaPI monoclonal antibodies, mAP9, that recognizes the near N-terminusof pea phytochrome was reactive to the 100 kDa polypeptide. (Received June 22, 1990; Accepted November 18, 1990)     

Antitumor activities and tumor necrosis factor producibility of traditional Chinese medicines and crude drugs

Summary The antitumor activities and capacity for tumor necrosis factor (TNF) production of traditional Chinese herbal preparations (Zhu-ling-tang, Xiao-chai-hu-tang), crude drugs (Polyporus, Hoelen, Bupleuri radix, Angelica radix, Cnidii rhizoma, Cinnamomum cortex), and Krestin (PSK) were investigated. These drugs were given to DDY mice in the drinking water before and after transplantation of Ehrlich tumors, and the development of the intradermally transplanted Ehrlich tumors and survival rate were observed. A good survival rate and sometimes a complete cure were found in the groups administered Bupleuri radix, Xiao-chai-hu-tang, Angelica radix, or Cinnamomum cortex, while the group given Hoelen showed poor results. To examine the capacity for TNF production these drugs were given to DDY mice PO as initial stimulating agents, to stimulate the reticuloendothelial system (RES) prior to lipopolysaccharide injection. The TNF activity was tested from the cytotoxicity against L cells. Significant differences in capacity for TNF production were observed among the drugs. Relatively high levels of TNF activity were noted in the groups given Angelica radix, Bupleuri radix, Cnidii rhizoma, or Cinnamomum cortex, very low activities in the groups given Xiao-chai-hu-tang, Zhu-ling-tang, or Krestin, and no TNF activities in the groups given Polyporus or Hoelen. The TNF capacity for production broadly paralleled the survival rate of the mice transplanted to Ehrlich tumors. Our findings suggest that one mechanism underlying the antitumor activities of these drugs is based on stimulation of the RES and is closely related of TNF production.This work was supported in part by a grant-in-aid from the Ministry of Education, Japan     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.     

Regulation of intracellular Mg2+ by superoxide in amnion cells.

Changes of intracellular free Mg2+ concentration ([Mg2+]i) in human amnion cells induced by superoxide anion were determined using a highly Mg(2+)-sensitive fluorescent dye Mg(2+)-fura2 or Mg(2+)-indol. Superoxide anion, produced by addition of xanthine oxidase to hypoxanthine, induced decrease of [Mg2+]i. The decrease was significantly inhibited by an anion channel blocker, 4,4'diisothiocyano-2,2' disulfonic acid stilbene (DIDS). Superoxide dismutase (SOD), injected into cells by cell fusion, also inhibited the change of [Mg2+]i, but catalase did not. Superoxide anion induced prompt increase of intracellular pH (pHi) as well as decrease of [Mg2+]i and subsequently activated the increase of intracellular free Ca2+ ([Ca2+]i) and the release of arachidonate. In contrast to superoxide anion, NH4Cl which induces increase of pHi in amnion cells increased [Mg2+]i. The elevation of basal level of [Mg2+]i by Mg(2+)-ionophore inhibited the change of [Ca2+]i and the release of arachidonate induced by superoxide anion. These results suggest that superoxide anion, transported through anion channels into cells, decreases [Mg2+]i directly, not due to a pH-effect and that the decrease of [Mg2+]i may regulate biological functions of the cells via increase of [Ca2+]i.     

A Novel Assay System for Anti-Human Immunodeficiency Virus Type 1 (HIV-1) Activity Using a Subclone of a Monocytic Cell Line,U937

A subclonal cl.1–14 cell was established from a monocytic cell line U937 by a limiting dilution method. The anti-HIV-1 activity of some antiviral compounds was evaluated in HIV-1-infected cl.1–14 cells. The results demonstrated that although AZT was a potent inhibitor of HIV-1 replication in cl.1–14 cells, its 50% effective concentration (EC₅₀) values was 80 times higher than that in HIV-1 infected MT-4 cells; the EC₅₀ of AZT was 0.16 μM and 0.002 μM in cl.1–14 and MT-4 cells, respectively. In contrast, the anti-HIV-1 activity of ddA, ddI and ddC in cl.1–14 cells was comparable to that in MT-4 cells. The antiviral activity of nevirapine, dextran sulfate, curdlan sulfate and T22 did not differ significantly between the cl. 1–14 and MT-4 cells. The antiviral activity of several compounds in the HIV-1-infected cl.1–14 cells was similar to that in the HIV-1jr -fl -infected human peripheral macrophages. Our results suggest that cl.1–14 cell cultures are very useful for estimating antiviral activity and more advantageous than the use of peripheral blood macrophages.