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D Yang

Publications and source records attributed to D Yang.

At least 361 records · Page 20Linked to original sources
PubMed1995

Limitations of the mass isotopomer distribution analysis of glucose to study gluconeogenesis. Substrate cycling between glycerol and triose phosphates in liver.

Mass isotopomer distribution analysis allows studying the synthesis of polymeric biomolecules from 15N, 13C-, or 2H-labeled monomeric units in the presence of unlabeled polymer. The mass isotopomer distribution of the polymer allows calculation of (i) the enrichment of the monomer and (ii) the dilution of the newly synthesized polymer by unlabeled polymer. We tested the conditions of validity of mass isotopomer distribution analysis of glucose labeled from [U-13C3]lactate, [U-13C3]glycerol, and [2-13C]glycerol to calculate the fraction of glucose production derived from gluconeogenesis. Experiments were conducted in perfused rat livers, live rats, and live monkeys. In all cases, [13C]glycerol yielded labeling patterns of glucose that are incompatible with glucose being formed from a single pool of triose phosphates of constant enrichment. We show evidence that variations in the enrichment of triose phosphates result from (i) the large fractional decrease in physiological glycerol concentration in a single pass through the liver and (ii) the release of unlabeled glycerol by the liver, presumably via lipase activity. This zonation of glycerol metabolism in liver results in the calculation of artifactually low contributions of gluconeogenesis to glucose production when the latter is labeled from [13C]glycerol. In contrast, [U-13C3]lactate appears to be a suitable tracer for mass isotopomer distribution analysis of gluconeogenesis in vivo, but not in the perfused liver. In other perfusion experiments with [2H5]glycerol, we showed that the rat liver releases glycerol molecules containing one to four 2H atoms. This indicates the operation of a substrate cycle between extracellular glycerol and liver triose phosphates, where 2H is lost in the reversible reactions catalyzed by alpha-glycerophosphate dehydrogenase, triose-phosphate isomerase, and glycolytic enzymes. This substrate cycle presumably involves alpha-glycerophosphate hydrolysis.

Animals↗
PubMed1995

Rat serum carboxylesterase. Cloning, expression, regulation, and evidence of secretion from liver.

Multiple forms of carboxylesterase have been identified in rat liver, and five carboxylesterases (designated hydrolases A, B, C, S, and egasyn) have been cloned. Hydrolases A, B, C, and egasyn all have a C-terminal consensus sequence (HXEL) for retaining proteins in the endoplasmic reticulum, and these carboxylesterases are found in rat liver microsomes. In contrast, hydrolase S lacks this C-terminal consensus sequence and is presumed to be secreted. In order to test this hypothesis, a polyclonal antibody was raised against recombinant hydrolase S from cDNA-directed expression in Escherichia coli. In addition to hydrolases A, B, and C (57-59 kDa), this antibody recognized a 67-kDa protein in rat liver microsomes and a 71-kDa protein in rat serum. The 71-kDa protein detected in rat serum was also detected in the extracellular medium from primary cultures of rat hepatocytes. Non-denaturing gel electrophoresis with staining for esterase activity showed that a serum carboxylesterase comigrated with the 71-kDa protein. Immunoprecipitation of the 71-kDa enzyme from rat serum decreased esterase activity toward 1-naphthylacetate and para-nitrophenylacetate. The 71-kDa protein immunoprecipitated from rat serum had an N-terminal amino acid sequence identical to that predicted from the cDNA encoding hydrolase S, providing further evidence that hydrolase S is synthesized in and secreted by the liver. The levels of the 67-kDa protein in rat liver microsomes and the levels of the 71-kDa protein in rat serum were co-regulated. Deglycosylation of microsomes and serum converted the 67- and 71-kDa proteins to a 58-kDa peptide, which matches the molecular mass calculated from the cDNA for hydrolase S. These results suggest that the 67-kDa protein in liver microsomes is a precursor form of hydrolase S that undergoes further glycosylation before being secreted into serum. In rats, liver appears to be the only source of hydrolase S because no mRNA encoding hydrolase S could be detected in several extrahepatic tissues. Serum carboxylesterases have been found to play an important role in lipid metabolism and detoxication of organophosphates, therefore, the secretion of hydrolase S and the modulation of its expression by xenobiotics may have physiological as well as toxicological significance.

Animals↗
PubMed1995

Permeation of small molecules into the cavity of ferritin as revealed by proton nuclear magnetic resonance relaxation.

The NMR relaxation technique was used to investigate the permeation of molecules into the cavity of ferritin. Spin-lattice relaxation times in the rotating frame of various probe molecules were measured for solutions of recombinant horse L-apoferritin without iron and horse spleen apoferritin with very small amounts of ferric ions. The results show that molecules larger than the size of the ferritin channels can pass through the channels into the ferritin interior, and that the maximum size of molecules for the permeation is smaller than maltotriose.

Animals↗
PubMed1995

Cloning and expression of hydrolase C, a member of the rat carboxylesterase family.

Using polymerase chain reaction (PCR), we have isolated a cDNA that encodes a rat liver carboxylesterase. This novel enzyme, designated hydrolase C, is structurally very similar to hydrolase B, a microsomal carboxylesterase expressed in rat liver and kidney. Hydrolase B and C are 96% identical in nucleotide sequence and 93% identical in deduced amino acid sequence. Both enzymes have an 18-amino-acid signal peptide at the N-terminus. The C-terminus of hydrolase B and C contains an HXEL consensus sequence for retaining proteins in the endoplasmic reticulum. As expected, when the cDNA encoding hydrolase C was expressed in a baculovirus/Sf21 cell system, the recombinant enzyme was localized in the endoplasmic reticulum. Hydrolase B and C both have putative N-linked glycosylation sites at Asn1 and Asn61. The active site of hydrolase B and C appears to be composed of a nucleophile, Ser203, a basic residue, His448, and an acidic residue, either Asp97 or Glu228. Based on cloning experiments, restriction endonuclease mapping and Northern blotting, hydrolase B is expressed in both rat liver and kidney, whereas hydrolase C is expressed predominantly, perhaps exclusively, in liver. When expressed in Escherichia coli, hydrolase C was catalytically inactive and unstable, but when expressed in the baculovirus/Sf21 cell system hydrolase C it was stable and catalytically active toward 1-naphthylacetate and esters of para-nitrophenol. Hydrolase C is the fourth member of the rat carboxylesterase family to be cloned and sequenced. In terms of nucleotide and deduced amino acid sequence, hydrolase C is highly similar to hydrolase B, but differs from hydrolase B in terms of its catalytic activity and tissue distribution. Recombinant hydrolase C has properties similar to those described for esterase RL2, which was purified from rat liver microsomes by Hosokawa et al. (Arch. Biochem. Biophys. 277, 219-227, 1990), although additional studies will be required to establish conclusively the identity of this enzyme. The high degree of sequence identity (96%) between hydrolase B and C, particularly in the 3' untranslated region, suggests that the genes encoding these two carboxylesterases evolved by duplication and divergence of a common ancestral gene.

Amino Acid Sequence↗
PubMed1995

Rat testicular carboxylesterase: cloning, cellular localization, and relationship to liver hydrolase A.

We recently purified from rat liver microsomes a carboxylesterase, designated hydrolase A, that catalyzes the hydrolysis of para-nitrophenylacetate with high affinity (Km approximately 25 microM) and is very sensitive to the inhibitory effects of phenylmethylsulfonyl fluoride (PMSF). Based on its catalytic properties, isoelectric point, and N-terminal amino acid sequence, hydrolase A corresponds to the pI 6.1 esterase cloned from a rat liver cDNA library by Robbi et al. (Biochem. J. 269, 451-458, 1990). A PMSF-sensitive esterase with high affinity toward para-nitrophenylacetate is also present in testicular microsomes at levels that slightly exceed those in liver microsomes. Antibody against purified hydrolase A recognizes a 57-kDa protein in both liver and testicular microsomes, suggesting that hydrolase A is expressed to a high degree in both tissues. To determine whether the testicular carboxylesterase is identical to hydrolase A, a rat testicular cDNA library was constructed and screened with antibody against hydrolase A. A 709-bp cDNA was isolated from immunopositive clones. Screening the same cDNA library by polymerase chain reaction (PCR) with one primer based on the sequence of the 709-bp cDNA and one primer based on the sequence of the adjoining lambda gt11 arm yielded a 1.1-kb cDNA that overlapped with the 709 bp-sequence. Together these two cDNA fragments spanned a 1792-bp sequence with an opening reading frame encoding 518 amino acids, which corresponds to approximately 95% of the C-terminal sequence of the liver pI 6.1 esterase (i.e., hydrolase A). Except for four nucleotide differences at positions 479, 855, 1335, and 1350, the sequence of the testicular cDNA was identical to the cDNA sequence of the liver pI 6.1 esterase reported by Robbi et al. None these changes results in an amino acid substitution. However, these four base substitutions were not observed when a cDNA encoding hydrolase A was isolated from a rat liver cDNA library by PCR. These results establish that the same carboxylesterase, namely, hydrolase A, is expressed in rat liver and testis. The levels of mRNA for hydrolase A in various rat tissues was estimated from Northern blots probed with the 709-bp cDNA isolated from the rat testicular cDNA library. A approximately 2-kb mRNA for hydrolase A was detected in liver, testis, lung, and prostate, which confirms the tissue distribution of hydrolase A based on catalytic activity and Western immunoblotting.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

The change of carnitine content in seminal plasma after reversible injection occlusion of vas deferens.

The change in carnitine content in seminal plasma after reversible injection occlusion of vas deferens (RIOVD) was observed. RIOVD is a safe, effective and simple method of male fertility control. Carnitine was determined by microenzymatic method. The incidence of sperm disappearance increased with the duration of RIOVD and reached 90% at the end of 12 months after operation. Before RIOVD, the mean value of carnitine in seminal plasma was 336.9 +/- 78.1 nmol/ml (X +/- SD, n = 58); after RIOVD, the mean value of seminal plasma carnitine was 112.7 +/- 50.7 nmol/ml (n = 172) in the group with sperm disappearance, and 172.5 +/- 71.7 nmol/ml (n = 51) in the group without sperm disappearance. There was a significant difference in carnitine content between pre-operation and postoperation (p < 0.01). After RIOVD, the carnitine concentration in seminal plasma of the group with sperm disappearance was lower than that of the group without sperm disappearance (p < 0.01). The results suggest that carnitine content in seminal plasma following RIOVD may be a reference index for judging the success or failure of the operation.

Carnitine↗
PubMed1995

The Ca2+ binding to deionized monomerized and to retinal removed bacteriorhodopsin.

In our continuing effort to characterize the metal cation binding in bacteriorhodopsin (bR) using Ca(2+)-specific electrodes, potentiometric titration was carried out on deionized solubilized bR (containing monomeric units) and deionized bacterioopsin (bR with its retinal removed). Scatchard plots were analyzed. The monomer was found to have plots similar to those of the trimer, suggesting that the binding sites in bR are localized within the protein monomer unit and not between the molecules within the trimer structure. This also supports the previous assumption that the curvature in the Scatchard plot of regenerated bR is not due to cooperativity of metal cation within the trimer, but rather due to multiple sites. Recent studies further support the finding that the curved Scatchard plot is not due to the cooperativity between the metal ions in the two high affinity sites, wherever they are. The results of the analysis of the Scatchard plot for deionized bacterioopsin have shown a change in the binding characteristics of the high affinity but not the low affinity sites from that observed in bR. This result supports previous conclusions that metal cations in the high affinity sites are not far from the retinal cavity.

Bacteriorhodopsins↗
PubMed1995

A novel DNA structure induced by the anticancer bisplatinum compound crosslinked to a GpC site in DNA.

The bifunctional platinum compound, [(trans-PtCI(NH)3)2)2(H2N(CH2)4NH2)]2+, forms a stable adduct with the self-complementary DNA oligomer CATGCATG, with the two platinum atoms coordinated at the N7 positions of the two symmetrical G4 nucleotides. The NMR-derived structure shows that the DNA octamer forms a novel hairpin structure with the platinated G4 residue adopting a syn conformation and the guanine base in the minor groove. Two such hairpins stack end-over-end and are linked together by the butanediamine tether to form a dumbbell structure. Such unusual structural distortion is different from that of the anticancer drug cisplatin-DNA adduct and may provide clues to explain the distinct biological activities of the two compounds.

Antineoplastic Agents↗
PubMed1995

A Fourier-transform infrared spectroscopic comparison of cultured human fibroblast and fibrosarcoma cells: a new method for detection of malignancies.

Infrared vibration spectroscopy appears to be a more powerful technique for tumor diagnosis than visible or UV spectroscopy. In the present work, Fourier-transform infrared (FTIR) spectroscopy was used to compare cultured normal fibroblast and fibrosarcoma cells. Significant differences were observed by comparing the spectra of the normal human cells with that of the cancer cells. The PO2 symmetric stretching mode at 1082 cm-1 is shifted to a higher frequency in the cancer cell and a broad band, whose center is located at 1064 cm-1 in the cancer cell is reduced in intensity. In addition, the decrease in intensity of the CH2 bending mode relative to that of CH3 mode is detectable only in the fibrosarcoma cell. This FTIR difference between fibroblast and fibrosarcoma cells suggests that either fatty acid chains or protein side chains of the cancer cells are partially degraded resulting in more terminal carbon (e.g., CH3). It is also possible that changes in the environment upon carcinogenesis induces a change in the relative absorption cross section for CH3 and CH2 bending vibrations. These results suggest that FTIR spectroscopy may become a promising and sensitive technique for tumor identification.

Cells, Cultured↗
PubMed1995

Type I and type II interferons upregulate functional type I interleukin-1 receptor in a human fibroblast cell line TIG-1.

The regulation of type I interleukin-1 receptor (IL-1R) expression by type I, interferon (IFN)-alpha A/D, and type II IFN, IFN-gamma, in a human fibroblast cell line TIG-1 was investigated. After 2 h stimulation with human IFN-alpha A/D or IFN-gamma, the levels of type I IL-1R mRNA increased. We previously reported that IL-1 upregulates transcription and cell surface molecules of type I IL-1R in TIG-1 cells through induction of prostaglandin (PG) E2 and cAMP accumulation. However, indomethacin was unable to inhibit the effect of IFNs, indicating that IFNs augment IL-1R expression through a pathway distinct from that of IL-1. The augmentation was also observed in other fibroblast cell lines. Nuclear run-on assays and studies of the stability of mRNA suggested that the increase in IL-1R mRNA was a result of the enhanced transcription of IL-1R gene. Binding studies using 125I-IL-1 alpha revealed that the number of cell surface IL-1R increased with no change in binding affinity by treatment with these IFNs. Pretreatment of the cells with IFNs enhanced IL-1-induced IL-6 production, indicating that IFNs upregulate functional IL-1R. IL-1 and IFNs are produced by the same cell types, as well as by the adjacent different cell types, and are concomitantly present in lesions of immune and inflammatory reactions. These results therefore suggest that IFNs exhibit synergistic effects with IL-1 through upregulation of IL-1R. Augmented production of IL-6 may also contribute to the reactions.

Cell Line↗
PubMed1995

Transfection of human melanoma cells with type I interleukin-1 (IL-1) receptor cDNA rendered them IL-1-responsive and revealed the importance of ODC activity down-regulation in IL-1-induced growth inhibition.

A human type I IL-1 receptor expression plasmid has been constructed and used to transfect human melanoma cells (A375-5), which have been shown to be unresponsive to the antiproliferative effect of IL-1. Five stable transfectant cell lines have been established, of which three are sensitive and the other two resistant to the anti-proliferative effect of IL-1. All the transfectant cell lines, but not progenitor A375-5 cells, expressed functional type I IL-1 receptors and could produce IL-6 in response to IL-1, suggesting that the unresponsiveness of A375-5 melanoma cells is exactly due to an IL-1 receptor deficiency. The three IL-1-sensitive stable transfectant cell lines expressed much more type I IL-1 receptor than the IL-1-sensitive A375-6 cells, thus they are expected to be useful for investigating the signal transduction pathway of IL-1-induced growth inhibition in melanoma cells. The two resistant cell lines produced comparable amounts of IL-6 in response to IL-1, as sensitive cell lines did, indicating that IL-6 induction does not play a major role in IL-1-induced growth inhibition in these melanoma cells. The possibility of an IL-6 receptor and/or IL-6 receptor signaling deficiency was ruled out as the IL-1-sensitive and -resistant transfectants responded similarly to a high dose of exogenous human recombinant IL-6. Examination of the ornithine decarboxylase (ODC) activity of recombinant human IL-1 alpha treated cells showed that all the sensitive but none of the resistant cell lines could down-regulate their ODC activity. These results suggest that IL-1-induced ODC activity down-regulation is an important step in the signal transduction pathway of IL-1-induced growth inhibition of melanoma cells.

Cell Division↗
PubMed1995

Blood pressure lowering by pioglitazone. Evidence for a direct vascular effect.

To examine potential mechanisms for the blood pressure-lowering action of the thiazolidinedione compound, pioglitazone (PIO), we studied the effects of the drug on blood pressure and insulin action in vivo and on vascular tissue in vitro. In vivo, PIO lowered blood pressure in fructose-fed and chow-fed rats to an extent that could not be explained by alterations in fasting plasma insulin or free magnesium concentrations or by alterations in whole-body insulin sensitivity. In vitro, PIO caused significant blunting of the contractile responses of aortic rings to NE, arginine vasopressin (AVP), and potassium chloride; the blunting of responses to NE was maintained after removal of the endothelium. To assess the potential importance of extracellular calcium to the vasodepressor effect of PIO, we measured contractile responses to NE in the absence of calcium, and then after acute restoration of calcium in the presence of NE. PIO had no effect on the contractile response in the absence of calcium. By contrast, PIO blunted by 42% the contractile response that occurred when the extracellular calcium supply was acutely restored in the presence of NE, suggesting that the blunting was mediated by blockade of calcium uptake by vascular smooth muscle. Such an effect was confirmed in cultured a7r5 vascular smooth muscle cells, which exhibited a brisk increase in intracellular calcium in response to AVP that was blocked by PIO in a dose-dependent fashion. Our data indicate that PIO has a direct vascular effect that appears to be mediated at least in part by inhibition of agonist-mediated calcium uptake by vascular smooth muscle. The direct vascular effect may contribute to the blood pressure-lowering actions of PIO in vivo, because that effect could not be explained by alterations in whole-body insulin sensitivity.

Animals↗
PubMed1995

Activation and selective inhibition of a cyclic AMP-specific phosphodiesterase, PDE-4D3.

Prostaglandin E2 produces a transient increase in the intracellular concentration of cAMP in a human promonocytic cell line (U937). The temporal pattern consists of a rapid increase followed by a gradual decline to a new steady state. The decline phase coincides with an increase in the activity of a high affinity form of cAMP phosphodiesterase (PDE). Immunoprecipitation with specific antibodies revealed that the activated enzyme is a variant of PDE-4D. To confirm this observation, three isoforms of human PDE-4 (A, B, and D) were cloned and expressed in Sf9 cells with recombinant baculovirus infection. The activity of only one of the isoforms (PDE-4D3) increased after incubation with the catalytic subunit of protein kinase A and Mg-ATP. Hydrolytic activity of human PDE-4D3 was dependent on Mg2+. Before phosphorylation, the concentration-response curve for Mg2+ was biphasic and ranged from 0.1 to 100 mM. Phosphorylation of PDE-4D3 by protein kinase A produced a monophasic Mg2+ response curve (0.5 Vmax = 0.2 mM). Phosphorylation of PDE-4D3 increased the sensitivity of the enzyme to inhibition by RS-25344 (approximately 100-fold) and RS-33793 (approximately 330-fold). Thus, phosphorylation of PDE-4D3 induces an apparent conformation change that increases maximum velocity and sensitivity to inhibition by some analogues of nitraquazone. These observations provide the basis for a novel pharmacological strategy that targets an activated form of PDE in human leukocytes. Selective PDE-4D3 inhibitors may have useful anti-inflammatory properties with fewer adverse side effects than other PDE-4 inhibitors.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

[Correlation study of allelic gene deletion of nm23-H1 and human colorectal carcinoma metastasis].

In recent years, a tumor suppressor gene nm23 has been found to be associated with decreased tumor metastatic potential. Allelic deletion, mutation and low expression of this gene has been correlated with tumor metastatic potential in a number of tumors. There are two known isotypes of human nm23 gene, named nm23-H1 and nm23-H2. We examined DNA from 23 cases of colorectal carcinomas and their corresponding normal mucosa using Southern blot hybridization with nm23-H1 cDNA probe. Five cases with allelic deletion of nm23-H1 gene were found, with allelic deletion rate of 57. 1% (4/7) in cases with metastasis to lymph node, liver or other organs, and 6.2% (1/16) in cases without metastases (P < 0.005). There is no correlation between allelic deletion and tumor size, location or differentiation. This result indicates that nm23-H1 gene plays an important role in the metastasis of colorectal carcinoma.

Alleles↗
PubMed1995

[p53 gene deletion and abnormal expression in gastric carcinoma].

We studied p53 gene at the DNA and protein level in human gastric cancer tissues and corresponding normal gastric mucosae from 20 cases of patients undergone radical surgery. By Southern blotting, the p53 gene was found to be partially deleted in 30% (6/20) of gastric cancer tissues. ABC immunohistochemical study of p53 expression was carried out on cryostat sections using monoclonal antibodies (PAb 1801) to p53. High level expression of mutated p53 protein was detected in 55% (11/20) gastric cancer tissues. The staining pattern was intranuclear and/or intracytoplamic. There was no detectable staining of any of the normal gastric tissues with 1801 antibody. The positive rate of p53 overexpression was higher in poorly-differenciated glandular carcinomas than well-differenciated cancer (P = 0.0116). Highly significant association exists between high level p53 expression and allele deletion (r = 0.59). The date indicate that inactivation of p53 gene is important in human gastric carcinogenesis and tumor progression. The assay of p53 gene structure and products may provide new biologically relevant tumor marks for predicting the behavior of gastric carcinomas, identifying more aggressive tumors, determining prognosis of the patients and guiding treatment.

Adenocarcinoma↗
PubMed1995

[Antifungal activity in vitro of Scutellaria baicalensis Georgi upon cutaneous and ungual pathogenic fungi].

The root of Scutellaria baicalensis Georgi (Lamiaceae) is one of the traditional drugs commonly used in the Far East. The extracts obtained by the successive exhaustion in chloroform and in ethyl acetat present clear fungistatic activities in vitro against to some cutaneous and ungual pathogenic fungi, and particularly upon strains of Candida albicans, Cryptococcus neoformans and Pityrosporum ovale. For each of these, its minimum inhibitory concentration is determined. The ethyl acetat extract appears the most interesting because of its efficiency and of its stability. An antifungal substance is found out as the baicalein.

Antifungal Agents↗
PubMed1995

Lymphocytic and erythrocytic k+, Na+, Ca++, Mg++ content in patients with congestive heart failure.

The content of lymphocytic and erythrocytic K+, Na+, Ca++, Mg++ changed in patients with congestive heart failure, related to clinical treatment and prognosis. The longer the course of disease, the more significant the changes, with no relation to the etiology of heart disease. The changes were partially improved after treatment of the heart failure. The content of electrolytes is not significantly different in patients in NYHA Class III, compared to those in NYHA Class IV. The metabolic changes of electrolytes occur on the myocardial cells, skeletal muscle cells and peripheral erythrocytes in patients with congestive heart failure. However, serum electrolyte content does not reflect precisely overall electrolyte metabolism in the body. Whether similar changes of electrolyte content occur in the peripheral lymphocytes is rarely reported. We studied the lymphocytic and erythrocytic electrolyte content in patients with congestive heart failure, to provide some reference in the clinical treatment and prognosis of congestive heart failure.

Adult↗