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Biomedical subjects

T Lu

Publications and source records attributed to T Lu.

At least 109 records · Page 6Linked to original sources
PubMed1996

Relationship between the concentrations of glycolytic intermediates and expression of the L-type pyruvate kinase gene in cultured hepatocytes.

Previous studies have suggested that some glycolytic intermediates are involved in the regulation of L-type pyruvate kinase gene expression by carbohydrates such as glucose and fructose. To find such intermediates, we examined the relationship between the levels of L-type pyruvate kinase mRNA and glycolytic metabolites in hepatocytes cultured under various conditions. Of the metabolites, the levels of 3-phosphoglycerate and phosphoenol-pyruvate only increased significantly under conditions under which the expression of the L-type pyruvate kinase gene was stimulated. The level of glucose 6-phosphate, which was reported to be involved in dietary stimulation of this gene expression, was not correlated with the mRNA level since marked accumulation of deoxyglucose 6-phosphate occurring on the addition of deoxyglucose, a nonmetabolizable glucose analog, was not accompanied by an increase in the L-type pyruvate kinase mRNA level. In addition, we found that fructose at a low concentration in the presence of glucose failed to increase the mRNA and metabolite levels in contrast to other reports that the promoter activity of the L-type pyruvate kinase gene is stimulated by this treatment. Thus we propose that 3-phosphoglycerate and/or phosphoenolpyruvate are involved in the carbohydrate regulation of L-type pyruvate kinase gene expression.

Animals↗
PubMed1995

Kinetics and mechanism of BAL 31 nuclease action on small substrates and single-stranded DNA.

Kinetic and mechanistic aspects of the action of two forms of the BAL 31 nuclease (EC 3.1.11) from Alteromonas espejiana on model substrates, small oligonucleotides, larger oligonucleotides and poly[d(A)] have been examined. The minimal oligonucleotide substrate is a 5'-phosphorylated dinucleotide and a phosphodiester not containing a nucleotide residue is not cleaved. Both forms act predominantly in an exonucleolytic fashion on single-stranded DNA polymers in a highly processive manner; however, the mechanism becomes distributive for small oligomers (3-4 nucleotide residues). The direction of attack is from the 5' end, in contrast to the mode of digestion of duplex DNA which involves attack at the 3' termini. An endonucleolytic mode of attack also exists, but at a level 2-3% or less of that of the terminally directed cleavage. Apparent values for the catalytic efficiency of the action on long DNA polymers are too large to fit a simple kinetic scheme involving a direct enzyme-substrate encounter and lead to an interpretation in which nuclease molecules are non-productively bound away from the 5' ends and undergo facilitated diffusion to yield productive (terminally bound) enzyme-substrate complexes.

Autoradiography↗
PubMed1995

Evidence for a (triosephosphate isomerase-like) "catalytic loop" near the active site of glyoxalase I.

The conformational mobility of glyoxalase I (Glx I) during catalysis has been probed using stable analogs of the enediol intermediate that forms along the reaction pathway: GSC(O)N(OH)R, where GS = glutathionyl and R = CH3 (1), C6H5 (2), C6H4Cl (3), or C6H4Br (4). For human erythrocyte Glx I, catalysis is unlikely to be coupled to major changes in protein secondary structure, as the circular dichroism spectrum of the enzyme (190-260 nm) is insensitive to saturating concentrations of either enediol analog or S-D-lactoylglutathione, the product of the Glx I reaction. However, a small conformational change is indicated by the fact that binding of enediol analog to the active site decreases intrinsic protein fluorescence by 11%, and protects the enzyme from proteolytic cleavage by Pronase E at the C-side of Ala-92 and Leu-93. In contrast, binding of S-D-lactoylglutathione does not affect protein fluorescence, and increases the rate of proteolytic cleavage by 1.5-fold. These observations are consistent with a model of catalysis in which a flexible peptide loop folds over and stabilizes the enediol intermediate bound to the active site. Indeed, a highly conserved sequence of amino acid residues is found near the proteolytic cleavage sites, for human Glx I (100-111) and Pseudomonas putida Glx I (93-105), that shows significant sequence homology to the "catalytic loop" of chicken muscle triosephosphate isomerase (TIM) (165-176). The active site base (Glu-165) of TIM, which catalyzes the proton transfer reaction during isomerization, corresponds in position to Glu-93 of P. putida Glx I. Consistent with a functional role for Glu-93, a mutant enzyme in which Glu-93 is replaced by Asp shows no detectable catalytic activity.

Amino Acid Sequence↗
PubMed1995

Diterpenes from Solidago rugosa.

Investigation of the roots and aerial parts of Solidago rugosa afforded the known diterpenes kolavenol, hardwickiic acid, (-)-kaur-16-en-19-oic acid, (+)-manool, (+)-3 beta-hydroxymanool, manoyl oxide and ent-abietic acid. In addition, the new labdane diterpene (+)-18-tigloyloxymanool and four new ent-abietanes were obtained. The structures of all known and new compounds were elucidated by spectroscopic methods, especially high-field 1H and 13C NMR, and inverse 1H-13C-correlation techniques, as well as chemical transformations. Six diterpenes were tested against Mycobacterium tuberculosis and M. avium, but showed no significant activities with minimum inhibitory concentrations of > 100 micrograms ml-1.

Diterpenes↗
PubMed1995

The electrophysiologic effects of endothelin. A patch clamp study in guinea pig ventricular myocytes.

To study the electrophysiologic effects of endothelin-1 (ET-1), we used patch clamp and glass microelectrode techniques to investigate the effects of ET-1 on cardiac L-Ica, Ik and Ik2 in guinea pig ventricular myocytes. The prolongation of APD50 was induced and EADs was triggered by 50 nM ET-1 perfusion. L-Ica and Ik were enhanced by various ET-1 concentration from 1 to 50 nM with dose-dependence. Their steady-state activations of L-Ica and Ik shifted left with ET-1 concentration increments. ET-1 elicited a kind of GTP-dependent inward rectifier K+ current having a mean conductance of 82.36 +/- 1.27 pS. The open time and close time (both interburst intervals and burst durations) abbreviated with ET-1 concentration increase. The results suggested that EADs -ET evoked was ascribed to the prolongation on the plateau level, which resulted from L-Ica enhancement. The ET- evoked inward rectifier K+ current should be further studied.

Action Potentials↗
PubMed1995

[Retrosigmoid carding operation of facial nerve trunk].

Retrosigmoid carding operation of facial nerve trunk for treatment of intractable hemifacial spasm was reported in 22 patients. These cases were followed up for 1 to 4 years. The effective rate was 87.5%. Facial nerves compressed by an arterial loop was found in 54.5 per cent of all cases during the operations. The operation was safe and no death occurred. No facial palsy was seen more than 5 months after the operations. The operative indication was very wide. The operation was not confined to those having vascular compressions. Especially the operation can resolve the difficult problems when no compressing vessel can be found or vascular decompression is difficult to do.

Adult↗
PubMed1994

Combined pH gradient and anion-exchange high-performance liquid chromatographic separation of oligodeoxyribonucleotides.

A novel method of elution using a pH gradient to separate small thymidine- and guanosine-containing oligonucleotides on a Pharmacia Mono Q HR anion-exchange column is described. The method is based on the alkaline titration of ring protons of the thymine and guanine base moieties and results in excellent separations of di-, tri- and tetranucleotides that either are not resolved in salt gradients near neutral pH or require long elution times when salt gradient elution is used with strongly alkaline eluents.

Anion Exchange Resins↗
PubMed1994

The gene encoding glyoxalase I from Pseudomonas putida: cloning, overexpression, and sequence comparisons with human glyoxalase I.

The gene encoding glyoxalase I (GlxI) from Pseudomonas putida has been cloned into the high-expression plasmid pBTacI. In the presence of IPTG, JM109 cells transformed with this vector give expression levels of GlxI 4000-fold higher than wild-type Escherichia coli. Contrary to a previous report, the nucleotide sequence of the gene encodes a 173-amino-acid polypeptide. Edman analysis indicates that the predicted N-terminal methionine is lost post-translationally to yield a 19407-Da protein. Mass spectrometry of the intact protein, and of the peptides generated from treatment with CNBr, does not indicate any additional post-translational modifications of the enzyme. Contrary to previous conclusions, there are no major regions of dissimilarity between the human and bacterial enzymes.

Amino Acid Sequence↗
PubMed1994

Role of the leucine zipper in the kinetics of DNA binding by transcription factor USF.

USF is a transcription factor characterized by a helix-loop-helix (HLH) DNA-binding domain that has been highly conserved through evolution. Vertebrate USFs contain an additional C-terminal leucine zipper (LZ) immediately adjacent to the HLH domain. This LZ is essential for efficient DNA binding by human USF. However, sea urchin has a USF family member that lacks a LZ and yet binds DNA efficiently. To clarify the role of the LZ in DNA binding by USF, we compared the properties of human and sea urchin USFs and found that the two proteins interacted with their specific sites on the DNA with identical affinities but very different kinetics. Association and dissociation rate constants of sea urchin USF were about 10-fold those of human USF. Domain-swapping experiments revealed that the LZ was responsible for the slower kinetics of human USF. USF heterodimers containing a single LZ displayed rates intermediate between those of dimers containing either two or no LZ, indicating that zipper-zipper interactions within USF dimers were not important for DNA binding. Temperature effects on DNA-binding parameters revealed a very high energy barrier for binding of human USF to DNA. Presence of a LZ increased the activation energy of the reaction.

Animals↗
PubMed1994

Toxicity of myristic acid analogs toward African trypanosomes.

New drugs are needed for treatment of diseases caused by African trypanosomes. One possible target for chemotherapy is the biosynthesis of the glycosyl phosphatidyl-inositol (GPI) of this parasite's variant surface glycoprotein (VSG). Unlike mammalian GPIs, the diacylglycerol moiety of the VSG anchor contains only myristate (tetradecanoate), added in unique remodeling reactions. We previously found that 11-oxatetradecanoic acid [i.e., 10-(propoxy)decanoic acid] is selectively toxic to trypanosomes. We have now assayed 244 different fatty acid analogs, most with chain lengths comparable to that of myristate, for trypanocidal effects. In these assays we surveyed the effects on toxicity of systematic alterations in the analogs' steric, conformational, and hydrophobic properties. We also used three 3H-labeled oxatetradecanoic acids to explore the mechanism of analog action. Their incorporation into VSG correlated roughly with toxicity, although they also were incorporated into phospholipids and other proteins. Myristate analogs are useful for studying the mechanism of GPI myristolyation, and they are candidates for antitrypanosomal chemotherapy.

Animals↗
PubMed1994

The substrate specificity of Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase. Polar probes of the enzyme's myristoyl-CoA recognition site.

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is a monomeric enzyme that is essential for vegetative growth. Nmt1p catalyzes the co-translational transfer of myristate from CoA to the amino-terminal Gly of cellular proteins in an ordered Bi Bi reaction mechanism that initially involves binding of myristoyl-CoA to the apoenzyme. Forty one fatty acid analogs were synthesized to define features in the acyl chain of myristoyl-CoA which are important determinants of its recognition by Nmt1p's acyl-CoA binding site as well as to help us deduce the structure of the binding site itself. These analogs included dicarboxylic acids, omega-nitrocarboxylic acids, analogs equivalent in length to C13:0-C15:0 which contain electronegative halogens at their omega-termini, hydroxytetradecanoic acids with hydrogen replaced by OH from C3 to C13, and azidophenyl-containing fatty acids with the linear azide unit attached either meta or para to phenyl and with variations in the length of their methylene chains. These compounds were converted to their CoA derivatives using Pseudomonas acyl-CoA synthetase and then surveyed as substrates for purified Nmt1p in an in vitro assay system that included an octapeptide derived from residues 1-8 of the human immunodeficiency virus Pr55gag polyprotein precursor. The results suggest that the myristoyl-CoA binding site contains a conical-shaped "receptor" that interacts with the omega-terminus of the bound acyl chain of acyl-CoAs. The acuteness of this cone determines the enzyme's capacity to accommodate steric bulk at the omega-terminus as well as Nmt1p's sensitivity to the distance between the eclipsed C5-C6 bond of a bound acyl chain and its omega-terminus. The activity profile of the various analog-CoAs also indicates that the enzyme's myristoyl-CoA binding site can accommodate fatty acid analogs with marked increases in polarity at their omega-terminus (compared to C14:0) as long as their chain length is equivalent to that of myristate.

Acyl Coenzyme A↗