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T Date

Publications and source records attributed to T Date.

At least 91 records · Page 5Linked to original sources
PubMed1989

Reconstituted F1-ATPase complexes containing one impaired beta subunit are ATPase-active.

Homogeneous populations of hybrid alpha 3 beta 3 gamma complexes of the thermostable F1-ATPase containing one, two, or three copies of the mutationally impaired beta subunits were produced using the solid phase reconstitution method. Two kinds of mutated beta subunits were used for the reconstitution, one of which lacked the ability to bind any adenine nucleotides. The complexes containing one impaired beta and two wild-type beta subunits retained a significant amount of ATPase activity with cooperative kinetics, whereas those containing two or three impaired beta subunits showed very little ATPase activity. These results imply that the catalysis of steady-state ATP hydrolysis can proceed even if one of the three beta subunits in F1-ATPase is not functional.

Adenosine Diphosphate↗
PubMed1988

Isolation of human cDNA clones of myb-related genes, A-myb and B-myb.

cDNA clones of the myb-related genes A-myb and B-myb were obtained by screening human cDNA libraries. The predicted open reading frame of B-myb could encode a protein of 700 amino acid residues. Although the C-terminal end has not been cloned yet, an almost entire coding region of A-myb, which is 745 amino acid long, was determined. The A-myb and B-myb proteins are highly homologous with the myb protein in three regions. Domain I, which is 161 amino acid long, is well conserved in the myb gene family. The homology between human-myb and A-myb in domain I is 90% at the amino acid level. Domain II, which is about 85 amino acid long, is less well conserved. Although it is a short stretch, domain III is found in the C-terminal region. The mRNAs of A-myb and B-myb were 5.0 and 2.6 kb, respectively. The mRNA expression pattern of the myb gene family in various tumors is presented.

Amino Acid Sequence↗
PubMed1988

Molecular cloning of the beta-subunit of a possible non-F0F1 type ATP synthase from the acidothermophilic archaebacterium, Sulfolobus acidocaldarius.

The gene which encodes the beta subunit of the novel membrane-associated ATPase has been identified and characterized. The beta subunit, which is most likely the soluble part of the non-F0F1 type H+-ATPase, was obtained from the archaebacterium, Sulfolobus acidocaldarius. In terms of its location, it follows just after the gene for its alpha subunit. It is comprised of 1398 nucleotides, corresponding to a protein of 465 amino acids, and the consensus sequence in the nucleotide binding proteins is poorly conserved. Together with previously described results, the distant homology of the S. acidocaldarius ATPase alpha and beta subunits when compared to those of F0F1-ATPases indicates that this archaebacterial ATPase belongs to an ion-translocating ATPase family uniquely different than F0F1-ATPases even if S. acidocaldarius ATPase and F0F1-ATPases have been derived from a common ancestral ATPase.

Amino Acid Sequence↗
PubMed1988

Expression of synthetic thaumatin genes in yeast.

Thaumatin is a plant protein that contains 8 disulfides and 207 amino acids in the mature form. The protein is of potential commercial interest since microgram quantities elicit an intense sweetness sensation. Two major variants of thaumatin have been identified in our laboratory by using sequence data obtained from thaumatin tryptic peptides. These differ by one amino acid at position 46 (asparagine or lysine), and both proteins differ from previously published sequences. We have synthesized DNA-coding sequences for three of these thaumatin variants using yeast preferred codons. The genes were inserted into an expression vector that contained a yeast 3-phosphoglycerate kinase promoter and terminator, and the vectors were transformed into yeast for expression of the recombinant protein. Upon lysis of the yeast cells, all thaumatin was localized in the insoluble cell fraction. Analysis of the sodium dodecyl sulfate solubilized yeast extracts by gel electrophoresis and Western blotting showed that thaumatin represented about 20% of the insoluble yeast protein. Although expressed at high levels, none of the thaumatins was biologically active (sweet). Preliminary protein folding experiments showed that two of three thaumatin variants could be folded to the sweet conformation.

Amino Acid Sequence↗
PubMed1988

The membrane-associated ATPase from Sulfolobus acidocaldarius is distantly related to F1-ATPase as assessed from the primary structure of its alpha-subunit.

Isolation of novel membrane-associated ATPases, presumably soluble parts of the H+-ATPases, from archaebacteria has been recently reported, and their properties were found to be significantly different from the usual F1-ATPase. In order to assess the relationship of the archaebacterial ATPases to the F1-ATPases and other known ATPases, the amino acid sequence of the alpha subunit of the ATPase from Sulfolobus acidocaldarius, an acidothermophilic archaebacterium, was compared with the sequences of other ATPases. The gene encoding its alpha subunit was cloned from the genomic library of S. acidocaldarius, and the nucleotide sequence was determined. The 591-amino acid sequence deduced from the nucleotide sequence contains a small number of short stretches that shows sequence similarity to the alpha and beta subunits of F1-ATPase. However, the overall similarity is too weak to consider it to be a typical member of the F1-ATPase family when the highly conserved sequences of the F1-ATPase subunits among various organisms are taken into account. Moreover, most of these stretches overlap the consensus sequences that are commonly found in some nucleotide-binding proteins. There is no significant sequence similarity to the ion-translocating ATPases, which form phosphorylated intermediates, such as animal Na+,K+-ATPases. Thus, the S. acidocaldarius ATPase and probably other archaebacterial ATPases also appear to belong to a new group of ion-translocating ATPases that has only a distant relationship to F1-ATPase.

Adenosine Triphosphatases↗
PubMed1988

Expression of active rat DNA polymerase beta in Escherichia coli.

A recombinant plasmid for expression of rat DNA polymerase beta was constructed in a plasmid/phage chimeric vector, pUC118, by an oligonucleotide-directed mutagenesis technique. The insert contained a 1005 bp coding sequence for the whole rat DNA polymerase beta. The recombinant plasmid was designed to use the regulatory sequence of Escherichia coli lac operon and the initiation ATG codon for beta-galactosidase as those for DNA polymerase beta. The recombinant clone, JMp beta 5, obtained by transfection of E. coli JM109 with the plasmid, produced high levels of DNA polymerase activity and a 40-kDa polypeptide that were not detected in JM109 cell extract. Inducing this recombinant E. coli with isopropyl beta-thiogalactopyranoside (IPTG) yielded amounts of 40-kDa polypeptide as high as 19.3% of total protein. Another recombinant clone, JMp beta 2-1, which was constructed by an oligonucleotide-directed mutagenesis to use the second ATG codon for the initiation codon, thus deleting the first 17 amino acid residues from the amino terminus, produced neither high DNA polymerase activity nor the 40-kDa polypeptide. The evidence suggests that this amino-terminal structure is important for stability of this enzyme in E. coli. The DNA polymerase was purified to homogeneity from the IPTG-induced JMp beta 5 cells by fewer steps than the procedure for purification of DNA polymerase beta from animal cells. The properties of this enzyme in activity, chromatographic behavior, size, antigenicity, and also lack of associated nuclease activity were indistinguishable from those of DNA polymerase beta purified from rat cells, indicating the identity of the overproduced DNA polymerase in the JMp beta 5 and the rat DNA polymerase beta.

Amino Acid Sequence↗
PubMed1988

Mitral valve motion during diastole in patients with complete heart block: relation of pressure gradients between left atrium and left ventricle.

We analyzed the high-fidelity left atrial and left ventricular pressures and the echocardiograms of the mitral valve, left atrium, and left ventricle in patients with complete heart block. During left ventricular diastole, the mitral valve opened, and no pressure gradient was observed between the left atrium and the left ventricle before the left atrial contraction. After the left atrial contraction, the mitral valve closed with persistent higher left ventricular than left atrial pressure. These findings indicated that during the left ventricular diastole in patients with complete heart block, the mitral valve closed after the left atrial contraction, which appeared to be maintained by the pressure gradient between the left ventricle and the left atrium.

Aged↗
PubMed1988

Molecular cloning, sequence analysis, and expression in Escherichia coli of the cDNA for guanidinoacetate methyltransferase from rat liver.

Five cDNA clones encoding rat liver guanidinoacetate methyltransferase (S-adenosyl-L-methionine: guanidinoacetate N-methyltransferase, EC 2.1.1.2) were isolated from a lambda gt11 cDNA library by use of a polyclonal antibody to the purified enzyme. Sequence analysis of the longest cDNA indicated that it consisted of 711 base pairs (bp) of coding region, 51 bp of 5' noncoding region, and 162 bp of 3' noncoding region excluding the poly(A) tail. The amino acid sequence deduced from the cDNA contained the sequences of NH2-terminal and three tryptic peptides. The predicted amino acid composition and molecular weight were in excellent agreement with those obtained with the purified enzyme. Introduction of the cDNA into plasmid pUC118 having the lac promoter resulted in a production in Escherichia coli of a Mr 26,000 polypeptide in the presence of isopropyl beta-D-thiogalactopyranoside. This protein represented as much as 5% of the bacterial soluble protein and showed the guanidinoacetate methyltransferase activity. Sequence analysis and tryptic peptide mapping indicated that the enzyme obtained by the recombinant DNA procedures was structurally identical to the liver enzyme, except for the absence of the NH2-terminal blocking group. Also, the enzyme showed kinetic properties indistinguishable from those of the liver enzyme.

Amino Acid Sequence↗
PubMed1988

Cloning and nucleotide sequence of the brnQ gene, the structural gene for a membrane-associated component of the LIV-II transport system for branched-chain amino acids in Salmonella typhimurium.

The genetically defined gleR-brnQ region responsible for the branched-chain amino acid transport in Salmonella typhimurium was mapped in the 3.3-kilobase SalI-PstI segment of plasmid pOH56 by complementation analysis. By subcloning and genetic recombination analysis, the gleR and brnQ3 mutational sites were localized within the 0.85-kilobase SalI-EcoRV segment, and brnQ4 within the 0.8-kilobase EcoRV-HindIII segment. The nucleotide sequence of the brnQ gene and its flanking regions was determined. The brnQ gene is encoded by the sequence starting 24 base pairs upstream from the EcoRV site. Transcription of the brnQ gene starts at three sites separated by 171, 173 and 174 nucleotides, respectively, from the initiation codon. The promoter sequences can be seen in the immediate upstream region of the transcription initiation sites. There is a long silent region between the transcription initiation sites and a potential Shine-Dalgarno nucleotide sequence. The coding sequence of the brnQ gene, which is 1317 base pairs long, specifies a very hydrophobic protein of 439 amino acid residues.

Amino Acid Sequence↗
PubMed1987

In vitro mutated beta subunits from the F1-ATPase of the thermophilic bacterium, PS3, containing glutamine in place of glutamic acid in positions 190 or 201 assembles with the alpha and gamma subunits to produce inactive complexes.

Using site-directed mutagenesis, Glu-190 or Glu-201 of the beta subunit of the F1-ATPase from the thermophilic bacterium PS3 were replaced with glutamine. It was possible to reconstitute complexes of the mutated beta subunits with alpha and gamma subunits, but the complexes did not have ATPase activity. It is concluded that carboxylic acid side chains of Glu-190 and Glu-201 of the beta subunit are essential for catalytic activity of F1-ATPase.

Bacteria↗
PubMed1986

Studies on a new antiallergic pyridinecarboxamide TA-5707F and its sodium salt (TA-5707). I. Inhibition of IgE-induced passive cutaneous anaphylaxis (PCA) in rats.

Effects of TA-5707F [6-methyl-N-(1H-tetrazol-5-yl)-2-pyridinecarboxamide] and its sodium salt (TA-5707) on IgE-induced homologous PCA in rats were investigated. 1. Both TA-5707 and TA-5707F were found to be orally effective inhibitors of rat PCA. Maximum activity by oral administration was obtained when they were administered 5 min before the challenge. Their ID50's under these conditions were both approximately 1 mg/kg. Administration 5 min after the challenge was no longer effective. 2. TA-5707 was also effective by intravenous administration, and its ID50, ca. 0.1 mg/kg, was less than that of disodium cromoglycate (DSCG). 3. The PCA-inhibitory activity of TA-5707 was not affected by adrenalectomy and adrenomedullectomy. 4. Daily administration of TA-5707 or TA-5707F for 8 days did not induce drug tolerance. 5. Tachyphylaxis and cross-tachyphylaxis were observed when the PCA-inhibitory activities of TA-5707 and DSCG were tested after intravenous pretreatment with a large dose (ca. 30 times the ID50) of either drug, but not after oral pretreatment with a therapeutic dose of TA-5707 or TA-5707F.

Adrenal Medulla↗
PubMed1986

The influence of exercise in the healing of the rabbit Achilles tendon.

Effect of exercise on tendon healing was studied in partially tenotomized rabbit achilles tendons by post-operatively applying a plaster splint to the right leg from the knee to the end of the toe, but no external fixation to the left leg. The animals were serially sacrificed, and new tendon tissues generated at the tenotomy site were examined. Collagen type analysis of the new tendon tissue showed a nearly constant ratio of type III to type I collagen of about 10% in the fixed side, but in the non-fixed side it showed an increase to 19% 2 weeks after the operation followed thereafter by a gradual decrease. Ultrastructural examination revealed an earlier increase in the diameter of collagen fibers suggesting their faster maturation in the non-fixed side.

Achilles Tendon↗
PubMed1984

Primer-directed mutagenesis of linearized plasmids.

Two novel mutagenesis techniques to specifically alter the sequence of plasmid DNA have been developed. In contrast to other primer-directed mutagenesis methods which require a single-stranded, closed-circular template, a linearized single strand was used as the mutagenesis template. The template is prepared by restriction enzyme digestion of covalently-closed-circular plasmid DNA. These methods are simple, require small amounts of plasmid DNA, and can result in a relatively high frequency of mutagenesis.

Base Sequence↗