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.     

Demonstration of neutralizing autoantibodies against IL-1 alpha in sera from patients with rheumatoid arthritis

We have recently reported the presence of IgG which has a potent inhibitory activity against IL-1 alpha in some sera from patients with rheumatoid arthritis. The mechanism of this inhibition by IgG against IL-1 alpha is now elucidated. IgG with IL-1 alpha-inhibitory activity inhibited the binding of 125I-IL-1 alpha to receptors on rheumatoid synovial cells. In addition, preincubation of synovial cells with the inhibitory IgG did not block the binding of 125I-IL-1 alpha to receptors, suggesting a direct interaction between IgG and IL-1 alpha. To examine which region of the IgG, namely Fab or Fc region, has the inhibitory activity, the IgG was digested with papain, and Fab and Fc fragments were purified. Fab fragments, but not Fc fragments, inhibited both IL-1 alpha-induced thymocyte-proliferation and the binding of 125I-IL-1 alpha to receptors. We further demonstrated that the inhibitory IgG which was bound to protein A Sepharose could bind a significant amount of 125I-IL-1 alpha, whereas only a negligible binding of the radiolabeled ligand was detected when IgG without the inhibitory activity was used as control. Moreover, the binding of 125I-IL-1 alpha to IgG with the inhibitory activity was clearly blocked by Fab fragments of IgG having the inhibitory activity. Finally, affinity-purified IgG over an IL-alpha affinity column showed approximately 100-fold more potent inhibitory activity on IL-1 alpha-induced thymocyte proliferation compared with untreated IgG. From these results, we conclude that IgG molecules with IL-1-alpha-inhibitory activity are neutralizing autoantibodies against IL-1 alpha.     

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)     

Spontaneous Release of γ-Aminobutyric Acid Formed from Putrescine and Its Enhanced Ca2+-Dependent Release by High K+ Stimulation in the Brains of Freely Moving Rats

The spontaneous release of [3H] gamma-aminobutyric acid ([3H]GABA) in various areas of rat brain injected with [3H]putrescine was examined using a push-pull perfusion technique. The release in a 25-min perfusate was highest in the caudate-putamen. The effect of high K+ stimulation on the release of [3H]GABA formed from [3H]putrescine was examined in the caudate-putamen. The release was enhanced by high K+ solution in a Ca2+-dependent manner.     

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.     

Three separate mitochondrial DNA sequences are contiguous in human genomic DNA

We isolated from a HeLa genomic library 38 plaques that hybridized to total mitochondrial (mt) DNA isolated from human placenta. One clone (HLmt-17.8) hybridized to a 740 base-pair (12 S ribosomal RNA gene and displacement loop) mtDNA probe and was characterized in more detail. Within its 17.8 x 10(3) base-pair insert a 1.6 x 10(3) base-pair mtDNA fragment was similar to three non-sequential coding genes of human mtDNA, including a part of the 12 S ribosomal RNA (684-971), the cytochrome oxidase I (6553-7302), and two NADH dehydrogenase [ND4L/ND4] (10,606-11,159). The similarity to human mtDNA sequences was 92.0%, 92.3% and 92.4%, respectively, the highest degree of similarity to human mtDNA so far reported. This is also the first report of several adjacent mtDNA-like sequences in cellular chromosomes. The mtDNA-like sequences in HLmt-17.8 was found in the DNAs of human placenta, freshly isolated human leukocytes, foreskin and several human cell lines; but it was not present in other primates or lower organisms. The HLmt-17.8 mtDNA-like region appears to be a pseudogene that transferred into the nucleus in humans more recently than nine million years ago.     

Particle size of airborne mouse crude and defined allergens

Laboratory animal allergy is a serious occupational diseases of many workers and scientists engaged in animal experimentation. Control measures depend upon characterization of allergens including airborne particles. This study measured the particle size of crude mouse urine and pelt aeroallergens generated in mouse housing rooms and compared them with mouse serum albumin, a defined major allergen. Allergens were detected by specific immunological methods. Most crude and defined allergens (74.5-86.4%) concentrated on a filter with a retention size greater than 7 microns. In distrubed air, allergen concentration increased 1.4 (albumin) to 5 (crude) fold and the proportion of small particles increased from 1.4% in calm air to 4.5% in distrubed air. This information on the generation and size distribution of aeroallergens will be important in the development of effective counter measures.