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S S Li

Publications and source records attributed to S S Li.

At least 73 records · Page 4Linked to original sources
PubMed1991

Analysis of genetic mutations in human lactate dehydrogenase-A(M) deficiency using DNA conformation polymorphism in combination with polyacrylamide gradient gel and silver staining.

Human lactate dehydrogenase (LDH)-A mutant gene was analyzed by polymerase chain reaction - DNA conformation polymorphism (DCP). We used polyacrylamide gradient gel and silver staining procedures for DCP analysis and observed abnormal migration patterns in individuals heterozygous for LDH-A deficiency. Further sequence determination of the mutant alleles consistently resulted in detection of base substitutions, a G to T transversion at codon 328 (GAG----TAG), and synonymous substitutions at codon 115, 160 and 172. Such mutations were easily detectable using the DCP technique. The DCP technique using the polyacrylamide gradient gel and silver staining method seems likely to be useful for the rapid screening of mutations and for further genotype detection.

Amino Acid Sequence↗
PubMed1991

Homodimer and heterodimer subunits of human prostate acid phosphatase.

Human prostatic acid phosphatase (PAP) isoenzymes, designated PAP-A and PAP-B, were isolated from human seminal plasma by sequential affinity chromatography on concanavalin A and L(+)-tartrate, a classic inhibitor of PAP. Both the major PAP-A and the minor PAP-B isoenzymes exhibited a similar molecular mass (100 and 105 kDa respectively), multiple pI values (5.05-5.35 and 5.05-5.12), and substrate and inhibitor specificity. Immunological characterization revealed that PAP-B possesses distinct antigenic determinants, in addition to the common sites shared with PAP-A. SDS/PAGE indicated that both isoenzymes are composed of two subunits of 50 kDa each. At high salt concentration, PAP-B dissociated completely into single subunits of 50 kDa, whereas PAP-A remained intact at 100 kDa. PAP-B was resolved by reverse-phase h.p.l.c. into three components, designated alpha, beta and gamma, each of 50 kDa, at a molar ratio of approx. 2:1:1. PAP-A contained a single component of molecular mass 50 kDa. The single component of PAP-A and the alpha component of PAP-B possessed identical amino acid compositions and N-terminal sequences, which were different from those of the beta and gamma components. These results indicate that human PAP contains three isoforms, alpha 2, alpha beta and alpha gamma. PAP-A, the major isoenzyme, is a homodimer consisting of two identical subunits (alpha 2), and PAP-B, the minor isoenzyme, is a mixture of two heterodimers, consisting of non-identical subunits (alpha beta and alpha gamma).

Acid Phosphatase↗
PubMed1991

Genotypic analysis of families with lactate dehydrogenase A (M) deficiency by selective DNA amplification.

Genomic DNA prepared from LDH-A-deficient whole blood was amplified by the polymerase chain reaction technique using two primers specific for the active human LDH-A gene. The amplified fragment was examined by direct agarose gel electrophoresis, and a deletion of 20 base pairs (bp) in exon 6 of the LDH-A gene was found. The results permitted a clear distinction between the homozygous mutant, the heterozygous mutant, and wild-type genotypes. Moreover, HinfI digestion and direct sequencing of the amplified product confirmed the results from direct agarose gel electrophoresis. Four families, including 18 individuals, were shown to contain the same mutation, that is a 20-bp deletion in exon 6. All genotypes were consistent with their biochemical phenotypes as evaluated by the ratio of LDH-B to LDH-A subunits in erythrocytes. Thus, all four known affected families in Japan have been shown to carry the same mutant gene, which may have been derived from a single mutational event.

Amino Acid Sequence↗
PubMed1991

Comparison of the antigenic peptides between human prostatic and lysosomal acid phosphatases.

The relative antigenicity (capacity to bind antibodies raised against the intact prostatic acid phosphatase) of the selected peptides from human prostatic and lysosomal acid phosphatases was evaluated in a competitive assay. Both prostatic and lysosomal acid phosphatases were shown to possess similar antigenic determinants on both terminal regions, along with more similarity on NH2-terminal peptide than COOH-terminal site. At least one additional antigenic site is present at the internal region of prostatic acid phosphatase, since the mixture of both amino- and carboxyl-terminal peptides exhibited only 70% inhibition.

Acid Phosphatase↗
PubMed1991

Modulation of splanchnic vascular sensitivity to angiotensin II.

We studied whether the diminished splanchnic vascular response to angiotensin II infusion in portal hypertension could be related to elevated levels of prostacyclin (PGI2). The changes in superior mesenteric artery resistance (RSMA) and systemic vascular resistance (RSYS) during angiotensin II infusion were measured by Doppler flow probe in normal rabbits (NL) and portal hypertensive rabbits (PHT), and in NL and PHT after cyclooxygenase blockade (CO) and after CO and during continuous PGI2 infusion at 300 ng/kg/min. Angiotensin II infusion in NL caused a disproportionately greater increase in RSMA than in RSYS (p less than 0.01). In PHT, angiotensin II response of RSMA was reduced from NL (p less than 0.05). CO dramatically improved the splanchnic response to angiotensin II in PHT animals, but did not significantly alter the RSMA response in NL. PGI2 in NL, NL + CO, and PHT + CO quantitatively established the splanchnic vascular hyporesponsiveness to angiotensin II seen in PHT. We conclude that PGI2 will directly diminish splanchnic response to angiotensin II in NL, and CO will ablate differences in splanchnic response between NL and PHT to angiotensin II. This strongly implies that much of the observed decrease in angiotensin II response in PHT is mediated by PGI2 and that the differences between NL and PHT vascular response is in part the result of circulating vasodilatory substances.

Angiotensin II↗
PubMed1991

Evidence for role of prostacyclin as a systemic hormone in portal hypertension.

The possibility that prostacyclin could be a systemic hormone and could mediate the splanchnic hyperemia of chronic portal hypertension was evaluated in rabbits in a normotensive state and in rabbits with chronic partial ligation of the portal vein. In rabbits with portal hypertension (PHT), 6-keto-prostaglandin F1 alpha (PGF1 alpha, a prostacyclin degradation product) was elevated twofold in all vascular beds (systemic arterial, systemic venous, and portal venous) when compared with levels in control animals. In PHT rabbits, exogenous prostacyclin infusion after cyclooxygenase blockade through the systemic arterial, systemic venous, or portal venous route resulted in an equal elevation of 6-keto-PGF1 alpha in the reciprocal vascular beds and restored the original precyclooxygenase blockade hemodynamics. These hemodynamic changes were of equal magnitude irrespective of site of infusion in PHT. In controls there was no significant change in 6-keto-PGF1 alpha or hemodynamics with intraportal infusion. We conclude that prostacyclin achieves systemic levels by escaping hepatic degradation resulting from portosystemic shunting in the animal with chronic portal hypertension.

6-Ketoprostaglandin F1 alpha↗
PubMed1990

Expression of the copy DNA for human A4 and B4 L-lactate dehydrogenases in Escherichia coli.

The human LDH-A and LDH-B cDNAs, containing the coding regions for the L-lactate dehydrogenase A4 (M) and B4 (H) polypeptides respectively have been cloned into Escherichia coli to place the cDNAs under the control of hybrid E. coli/Bacillus stearothermophilus transcriptional and translational signals. Human A4- and B4-isoenzymes are produced in E. coli cells harbouring the expression plasmids pHLDHA22 and pHLDHB10 at levels of 6.5 and 1.5% of the soluble protein of the cell, respectively. The tac promoter of these vectors was not induced by isopropyl beta-D-thiogalactopyranoside. The A4 and B4 human isoenzymes synthesized in E. coli were purified to homogeneity and show the same properties as isoenzymes isolated from human tissue. The amino acid sequences of 12 N-terminal residues of the human isoenzymes synthesized in E. coli were determined to be identical to those deduced from the DNA sequence of the cloned cDNAs except that the N-terminal methionine was absent from both. However, in contrast to LDH made in human cells, acetylation of the N-terminal alanine does not take place in E. coli cells.

Amino Acid Sequence↗
PubMed1990

Human lactate dehydrogenase-B processed pseudogene: nucleotide sequence analysis and assignment to the X-chromosome.

Two human genomic clones containing the lactate dehydrogenase-B processed pseudogene were isolated from two patients deficient in lactate dehydrogenase-B isozyme. The sequences of 3,287 nucleotides, including the pseudogenes and its flanking regions, from both clones were found to be identical except for three differences in the pseudogenes. The sequences of 1,286 nucleotides from these two pseudogenes exhibited 93% homology with the cDNA sequence of the lactate dehydrogenase-B functional gene, and the pseudogene contained 75/76 base substitutions, 11/12 single-base deletions, and 5 single-base insertions. This pseudogene was mapped to the x-chromosome by dot-blot analysis using a probe for the pseudogene or its 5' flanking sequence.

Base Sequence↗
PubMed1990

A missense mutation found in human lactate dehydrogenase-B (H) variant gene.

A human lactate dehydrogenase-B mutant gene was isolated from a genomic DNA library constructed from a patient with unstable LDH-B (heart) subunit. The nucleotide sequences of seven protein-coding exons were determined and a single nucleotide substitution of G by A at Arg codon CGC in exon 4 was found. This mutation results in an amino-acid replacement of a conserved arginine by histidine at the residue "173," which is involved in an anion-binding site at the P-axis of LDH subunits.

Adult↗
PubMed1990

Molecular characterization of genetic mutation in human lactate dehydrogenase-A (M) deficiency.

Human lactate dehydrogenase-A mutant gene was isolated from the genomic DNA library of a patient deficient in LDH-A (Muscle) subunit. The nucleotide sequences of seven protein-coding exons were determined and a deletion of 20 base-pairs in exon 6 was found. This mutation results in a frame-shift translation and premature termination. The predicted incomplete LDH-A (M) subunit containing only 259 instead of 331 amino acids appears to be degraded rapidly, since no protein was detected immunologically (Maekawa et al., Am J Hum Genet 39:232-238, 1986). In addition, three synonymous (silent) substitutions, A to C, T to C, and G to A, were observed at codons 115, 160 and 172, respectively, in this LDH-A mutant gene.

Amino Acid Sequence↗
PubMed1990

The cDNA and protein sequences of mouse lactate dehydrogenase B. Molecular evolution of vertebrate lactate dehydrogenase genes A (muscle), B (heart) and C (testis).

Mouse lactate dehydrogenase-B cDNAs were isolated from cDNA libraries of macrophage (ICR strain) and thymus (F1 hybrid of C57BL/6 and CBA strains), and their nucleotide sequences determined. The lactate dehydrogenase-B cDNA insert of thymus clone mB188 consists of the protein-coding sequence (1002 nucleotides), the 5' (46 nucleotides) and 3' (190 nucleotides) non-coding regions, and poly(A) tail (19 nucleotides), while macrophage clone mB168 contains a partial lactate dehydrogenase cDNA insert from codon no. 55 to the poly(A) tail. Seven silent nucleotide substitutions at codon no. 142, 143, 186, 187, 241, 285 and 292, as well as a single nucleotide change in the 3' non-coding region, were found between these different strains of mice. The predicted sequence of 333 amino acids, excluding initiation methionine, was confirmed by sequencing and/or compositional analyses of a total of 103 (31%) amino acids from tryptic peptides of mouse lactate dehydrogenase-B protein. The nucleotide sequence of the mouse coding region for lactate dehydrogenase B shows 86% identity with that of the human isoenzyme, and only eight of the 139 nucleotide differences resulted in amino acid substitutions at residues 10, 13, 14, 17, 52, 132, 236 and 317. The rates of nucleotide substitutions at synonymous and nonsynonymous sites in the mammalian lactate dehydrogenase genes are calculated. The rates of synonymous substitutions for lactate dehydrogenase genes A (muscle) and B (heart) are considerably higher than the average rate computed from human and rodent genes. The rates of nonsynonymous substitutions for lactate dehydrogenase genes A (muscle) and B (heart), particularly the latter, are highly conservative. The rates of synonymous and nonsynonymous substitutions for the lactate dehydrogenase-C gene are about the same as the average rates for mammalian genes. A phylogenetic tree of vertebrate lactate dehydrogenase protein sequences is constructed. In agreement with the previous results, this analysis further indicates that lactate dehydrogenase-C gene branched off earlier than did lactate dehydrogenase-A and lactate dehydrogenase-B genes.

Amino Acid Sequence↗
PubMed1990

Comparison of prostate acid phosphatase with acid phosphatase isoenzymes from the lung and spleen.

Acid phosphatase was purified to electrophoretic homogeneity from human normal lung and spleen and was characterized biochemically and immunologically in comparison with prostate acid phosphatase (PAP). The apparent MW of lung acid phosphatase (LAP) and spleen acid phosphatase (SAP) was 110,000 and 100,000, respectively, similar to that of PAP (100,000). All three enzymes exhibited similar electrophoretic mobility, optimal pH, substrate, and inhibitor specificity, except that PAP dephosphorylated profoundly the phosphate group from tyrosine phosphate in phosphoangiotensin (19,700 fmol/mg/min), whereas only marginal activities were detected for LAP and SAP (19 and 73 fmol/mg/min, respectively). Amino acid analysis revealed more similarity between SAP and LAP than PAP and LAP or PAP and SAP. An immunological cross-reactivity among these three acid phosphatases was detected by polyclonal and monoclonal antibodies raised against purified PAP, although unique epitopes were detected on the PAP molecule. This study provides data explaining why conventional biochemical methods are not specific for PAP measurement and why immunologic methods still detect other acid phosphatases, as observed in clinical laboratory assays. The data also suggest the possibility of using a new substrate or antibody reagent for a more specific assay for PAP.

Acid Phosphatase↗
PubMed1990

Decreased mesenteric vascular response to angiotensin II in portal hypertension.

We studied the mesenteric and systemic vascular response to angiotensin II in normotensive and portal hypertensive (PHT) rabbits, because of the documented poor tolerance of hemorrhagic shock in PHT. Normally, the hemodynamic response to angiotensin II (AII) is characterized by selective and disproportionate splanchnic vasoconstriction. We postulated that the response to AII could be diminished in PHT. Chronic PHT was induced by partial portal vein ligation 3 weeks prior to graded angiotensin II infusion. Baseline hemodynamic measurements showed markedly elevated portal pressure (PPV) and superior mesenteric artery blood flow (QSMA) compared with those of normotensive animals (P less than 0.01). Superior mesenteric artery resistance (RSMA) was markedly reduced in PHT compared to that in controls (P less than 0.05). Angiotensin II infusion in normals resulted in a marked selective rise in RSMA compared with the rise in systemic resistance (RSYS) (P less than 0.01). AII infusion in PHT resulted in a rise in RSMA and RSYS, but the disproportionate in RSMA was attenuated. Furthermore, in both normal and PHT, AII caused a significant rise in PPV (P less than 0.01). We conclude that the findings indicate that the splanchnic vasoconstrictive response to AII is substantially impaired in PHT, and that AII will paradoxically cause a rise in PPV, possibly aggravating the tendency to hemorrhage in PHT.

Angiotensin II↗
PubMed1990

Developmental regulation of expression of the lactate dehydrogenase (LDH) multigene family during mouse spermatogenesis.

Expression of the Lactate Dehydrogenase (LDH) genes during various stages of spermatogenesis was studied by using a combination of Northern blot analyses and in situ hybridization techniques. These studies have indicated that developmentally programmed expression of all three functional LDH genes occurs during differentiation of germ cells. The LDH/C (ldh-3) gene was expressed exclusively during meiosis and spermiogenesis, beginning in leptotene/zygotene spermatocytes and continuing through to the elongated spermatids. LDH/C (ldh-3) gene expression was accompanied by transient expression of the LDH/A (ldh-1) gene in pachytene spermatocytes and round spermatids. The LDH/B (ldh-2) gene was expressed mainly in Sertoli and spermatogonial cells. By using somatic cell hybrids, the LDH/C (ldh-3) gene has been mapped to mouse chromosome 7, establishing that it is syntenic with the LDH/A (ldh-1) gene locus. Experimental observations made in this study provide new insight into the order and sequence of events involved in the regulation of gene expression of the LDH gene family during spermatogenesis.

Animals↗
PubMed1989

Human prostatic acid phosphatase: cDNA cloning, gene mapping and protein sequence homology with lysosomal acid phosphatase.

The cDNAs encoding human prostatic acid phosphatase were cloned and characterized. The mRNAs contain 3' noncoding regions of heterogeneous sizes 646, 1887 or 1913 nucleotides. A dimer and a monomer of the conserved Alu-repeats are present in the longer 3' noncoding sequences. The complete sequence of 354 amino acids for the mature enzyme was determined by sequencing both cDNA and protein. Human prostatic and lysosomal acid phosphatases exhibit 50% sequence homology, including five Cys residues and two putative N-linked glycosylation sites. The Acp-3 gene coding for human prostatic acid phosphatase was mapped onto chromosome 3 in this investigation. The Acp-2 gene coding for lysosomal acid phosphatase has previously been located on chromosome 11, while the Acp-1 gene coding for red blood cell acid phosphatase is on chromosome 2.

Acid Phosphatase↗
PubMed1989

Human testicular lactate dehydrogenase-C gene is interrupted by six introns at positions homologous to those of LDH-A (muscle) and LDH-B (heart) genes.

Human genomic clones containing parts of testis-specific lactate dehydrogenase-C gene of approximately 40 kilobases in length were isolated and characterized. The protein-coding sequence of human LDH-C gene is interrupted by six introns at positions homologous to those of mammalian LDH-A (muscle) and LDH-B (heart) genes, and exhibits 21%, 24% and 34% nucleotide differences with those of mouse LDH-C, human LDH-A and LDH-B genes, respectively.

Amino Acid Sequence↗