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Biomedical subjects

L Yu

Publications and source records attributed to L Yu.

At least 685 records · Page 38Linked to original sources
PubMed1992

Sucrase-isomaltase gene expression along crypt-villus axis of human small intestine is regulated at level of mRNA abundance.

The mucosal lining of the small intestine is a complex epithelium that is continually renewed by division of a stem cell population located in intestinal crypts, migration of daughter cells along the villus, and, finally, extrusion of senescent cells into the lumen. The majority of cells in both crypt and villus cell compartments are enterocytes that acquire differentiated functions as they migrate out of the crypt. Sucrase-isomaltase (SI) is an enterocyte-specific, brush-border enzyme that has little activity in crypt cells and maximal activity in low and mid villus cells. The mechanism by which enterocytes acquire SI enzymatic activity as they move from crypt to villus is controversial. In this study we examined the distribution of SI mRNA along the crypt-villus axis of human small intestine using isolated epithelial cells and in situ hybridization. A complementary DNA to the 5' portion of the human SI mRNA was amplified and cloned using the polymerase chain reaction. Hybridization analysis of RNA extracted from human intestinal epithelial cells showed that the cloned cDNA recognized a single 6.5-kb mRNA. In situ hybridization of duodenal biopsy specimens was performed using a single-stranded RNA probe derived from this cDNA. This analysis showed that there was little SI mRNA in crypt cells and appearance of mRNA in enterocytes located at the crypt-villus junction. The mRNA levels were maximal in lower and mid villus cells with decreased levels noted in villus tip cells. These results are identical to those previously described in rat intestine and suggest that expression of the SI gene as enterocytes emerge from intestinal crypts is regulated primarily at the level of mRNA accumulation. Study of SI gene regulation may provide a useful model to investigate the mechanisms that regulate enterocyte-specific gene expression and intestinal differentiation.

Amino Acid Sequence↗
PubMed1992

Differential regulation of cytochrome P-450 genes along rat intestinal crypt-villus axis.

Mammalian small intestine contains cytochrome P-450-dependent monooxygenase enzymes that are capable of metabolizing a wide variety of xenobiotics and activating procarcinogens to mutagenic compounds. The epithelial cells lining the small intestine are separated into a proliferating undifferentiated compartment located in crypts and a nonproliferating differentiated compartment located on villi. The constitutive expression and induction by xenobiotics of genes that encode components of the cytochrome P-450-dependent mono-oxygenase system along the rat intestinal crypt-villus axis were investigated using isolated epithelial cells and in situ hybridization. For each gene examined, hybridization analysis of RNA obtained from isolated epithelial cells correlated with findings on in situ RNA hybridization. Cytochrome P-450IA1 mRNA (CYP1A1), the major aromatic hydrocarbon-inducible P-450, and cytochrome P-450IIB1 mRNA (CYP2B1), the major phenobarbital-inducible P-450, were constitutively expressed in villus cells with no detectable mRNA present in crypts. Treatment with several chemical inducers resulted in a marked increase in CYP1A1 mRNA in both crypt and villus cells. In contrast, although CYP2B1 mRNA was inducible in villus cells, CYP2B1 mRNA was not detected in crypts after treatment with chemical inducers. NADPH cytochrome P-450 reductase, a necessary component for the activity of all P-450 enzymes, was expressed constitutively at low levels only in villus cells. Treatment with dexamethasone induced reductase mRNA in both crypt and villus cells. Taken together, these results demonstrate that there is a complex gene-specific pattern of expression of the microsomal monooxygenase system along the crypt-villus axis of rat small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Acute renal failure following hemorrhagic shock: protective and aggravating factors.

Acute renal failure following hemorrhagic shock was studied in awake rats. The animals were bled to maintain the mean arterial blood pressure between 40 and 60 mm Hg during 180 min. After this period, the blood was reinfused and the rats were studied 24 h later. Hemorrhagic shock caused a less intensive renal injury than 60-min bilateral renal artery clamping. Renal function in the latter model was worse (p less than 0.05) as shown by serum creatinine (SCr) (0.75 +/- 0.10 vs 1.2 +/- 0.2 mg/dL), blood urea nitrogen (BUN) (26.0 +/- 2.8 vs 53.0 +/- 8.5 mg/dL), fractional excretion of sodium (FENa, %) (0.3 +/- 0.1 vs 1.8 +/- 1.0) and potassium (FEK, %) (41.4 +/- 5.7 vs 76.3 +/- 14.2) and urine/plasma creatine (U/PCr (86.4 +/- 15.7 vs 38.8 +/- 15.5). The rats which received verapamil (10 micrograms/kg/min) prior and during the HS did not show increase in SCr (0.5 +/- 0.06 vs 0.75 +/- 0.1 mg/dL, p less than 0.05). This effect was also observed in the rats which received intravenous allopurinol (40 mg/kg) before HS, SCr did not increase (0.5 +/- 0.04 vs 0.75 +/- 0.1 mg/dL, p = 0.05), suggesting a protective effect of those substances in HS.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Kidney Injury↗
PubMed1992

Late evaluation of glomerular filtration rate, proteinuria, and urinary acidification after acute tubular necrosis.

Fourteen patients were studied 2 to 36 months after acute tubular necrosis. It was observed that 43% of the patients had decreased glomerular filtration rate. These patients were older and had lower urinary excretion of ammonium and titratable acidity. Proteinuria greater than 150 mg/day, without reaching a nephrotic level, was found in 92% of the patients. The presence of oliguria, the demand of dialysis, and the acute tubular necrosis etiology were not statistically different among the patients who recovered their glomerular filtration rate either totally or partially.

Adult↗
PubMed1992

[Preliminary study on RFLPs for dystrophin gene in Chinese].

We have studied the RFLPs distribution and frequency of dystrophin gene in Chinese by using 14 subclones of complete 14 kb cDNA for the dystrophin gene as hybridization probes. Allelic fragments are detected in hybridization patterns of Pvu II/1a, Taq I/2b-3, Taq I/5b-7, Xba I/10. Among them, the allelic fragments (26 kb and 3.8 kb) in Pvu II/2b-3 patterns and the allelic fragments (10 kb and 8.4 kb) in Taq I/5b-7 patterns are the new RFLPs which have never been reported. From the comparison of our data with those of Caucasian and Japanese respectively and their statistical analysis, we can obtain the results as follows: There is remarkable difference (p less than 0.01) of the allelic fragment frequency in Taq I/2 b-3 (A1 = 3.4 kb, fre. 0.04; A2 = 3.2 kb, fre. 0.96) and Xba I/10 (A1 = 7.4 kb, fre. 0.41; A2 = 6.7 kb, fre. 0.59) between Chinese and Caucasian. The frequency of the allelic fragments A2 in Taq I/8 (A1 = 6.5 kb, A2 = 5.6 kb) and EcoR V/9 (A1 = 11.8 kb, A2 = 10.7 kb) are high in Caucasian, but have not been detected in Chinese. These differences are also highly significant. But the B1B2 allelic frequencies in Taq I/5 b-7 (B1 = 3.2 kb, B2 = 1.6 kb) are the same. There is no significant difference in the frequency of the allelic fragments A1A2 and B1B2 in Pvu II/1 a between Chinese and Japanese. Preliminary results suggest that there probably are high frequencies for spontaneous neutral mutations in the evolution process of the huge dystrophin gene (about 2,300 kb). In the meantime, the neutral mutation frequencies of various sectional sequences have remarkable differences, and that of some sectional sequences of the gene between Chinese and Caucasian may also have remarkable differences.

Adult↗
PubMed1992

[Effect of 764-3 on bleomycin activation of alveolar macrophages].

The effect of pretreatment with 764-3, a huo xue hua yu medicine, on bleomycin A6 activation of alveolar macrophages of rats was observed. Results showed that bleomycin A6 could activate alveolar macrophages directly and that 764-3 could inhibit the activating effect of bleomycin A6. When alveolar macrophages are activated, they release not only PMN chemotactic factor, but also many destructive lysosomal enzymes, superoxide anions, etc, possibly resulting in serious injury to the lungs.

Animals↗
PubMed1991

Protein-ubiquinone interaction in bovine heart mitochondrial succinate-cytochrome c reductase. Synthesis and biological properties of fluorine substituted ubiquinone derivatives.

To investigate the protein-ubiquinone interaction in the bovine heart mitochondrial succinate-cytochrome c reductase region of the respiratory chain, three fluorine substituted ubiquinone derivatives, 2,3-dimethoxy-6-(9'-fluorodecyl)-1,4-benzoquinone (9FQ), 2-methoxy-5-trifluoromethyl-6-decyl-1,4-benzoquinone (TFQ), and 2-methoxy-5-trifluoromethyl-6-(9'-fluorodecyl)-1,4-benzoquinone (9FTFQ), were synthesized. 9FQ was synthesized by radical coupling of Q0 and bis(10-fluoroundecanoyl)peroxide. The latter was prepared by fluorination of undecylenic acid followed by thionylchloride treatment and peroxidation. TFQ was synthesized from 2,2,2-trifluoro-p-cresol by methylation, nitration, reduction, acetylation, nitration, reduction, oxidation, and radical alkylation. 9FTFQ was prepared by the radical alkylation of 2-methoxy-5-trifluoromethyl-1,4-benzoquinone with bis(10-fluoroundecanoyl)peroxide. All three fluoro-Q derivatives are active (greater than 50% the activity of 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone) when used as electron acceptors for succinate-ubiquinone reductase. However, only 9FQ is active when used as an electron donor for ubiquinol-cytochrome c reductase or as an electron mediator for succinate-cytochrome c reductase. Both TFQ and 9FTFQ are competitive inhibitors for ubiquinol-cytochrome c reductase. A 19FNMR peak-broadening effect was observed for 9FQ when it was reconstituted with ubiquinone-depleted ubiquinol-cytochrome c reductase. A drastic up-field chemical shift was observed for TFQ when it was reconstituted with ubiquinone-depleted reductase. These results indicate that the binding environments of the benzoquinone ring and the alkyl side chain of the Q molecule are different. The strong up-field chemical shift for TFQ, and lack of significant chemical shift for 9FQ, suggest that the benzoquinone ring is bound near the paramagnetic cytochrome b heme.

Animals↗
PubMed1991

Critical role of a hydrogen bond in the interaction of phospholipase A2 with transition-state and substrate analogues.

The inhibition of phospholipase A2 by an amide substrate analogue, 1-hexadecylthio-2-hexadecanoyl-amino-1,2-dideoxy-sn-glycero-3-phos phocholine, and a phosphonate transition-state analogue, 1-hexadecylthio-1-deoxy-2-hexadecylphosphono-sn-glycero-3-ph osphocholine, is dramatically influenced by pH. However, these two inhibitors show opposite pH dependencies. The amide analogue acts more potently under basic conditions, whereas the phosphonate acts more potently under acidic conditions. In both cases, ligand binding is perturbed by protonation of an enzyme functional group with an apparent pKa of 6.1, which corresponds to that of a histidine residue. Thus, His-48, which has previously been implicated in catalysis, appears to be critically involved in the hydrogen bond interactions between the enzyme and these two inhibitors. The amide analogue binds most effectively to the enzyme when His-48 is deprotonated. Upon protonation of the histidine residue, the amide cannot form a critical hydrogen bond and loses its ability to interact effectively with the enzyme. In contrast, the phosphonate analogue binds much tighter to the protonated form of the enzyme than to the deprotonated form. The phosphonate analogue needs a bridging hydrogen between the oxygen on its phosphorus atom and the N delta 1 of His-48 to form a strong hydrogen bond. At optimal pH values for inhibitor binding, both the amide and the phosphonate analogues are potent competitive inhibitors of cobra (Naja naja naja) venom phospholipase A2. The IC50 for the amide was 4.4 x 10(-4) mol fraction and for the phosphonate was 1.6 x 10(-5) mol fraction. Under the experimental conditions used, this corresponds to a bulk concentration of 2 microM and 70 nM, respectively.

Elapid Venoms↗
PubMed1991

Inhibitory effects of dicyclohexylcarbodiimide on spinach cytochrome b6-f complex.

The electron transfer activity of purified cytochrome b6-f complex of spinach chloroplast is inhibited by dicyclohexylcarbodiimide (DCCD) in a concentration and incubation time dependent manner. The maximum inhibition of 75% is observed when 300 mole of DCCD per mole of protein (based on cytochrome f) is incubated with cytochrome b6-f complex at room temperature for 40 min. The inhibition of the complex is not due to the formation of cross links between subunits but due to the modification of carboxyls. The amount of DCCD incorporation is directly proportional to the activity loss, suggesting that some carboxyl groups in the complex are directly or indirectly involved in the catalytic function. The incorporated DCCD is located mainly at cytochrome b6 protein. The partially inhibited complex shows the same H+/e-ratio as that of the intact complex when embedded in phospholipid vesicles.

Binding Sites↗
PubMed1991

Subunit IV (Mr = 14,384) of the cytochrome b-c1 complex from Rhodobacter sphaeroides. Cloning, DNA sequencing, and ubiquinone binding domain.

The Rhodobacter sphaeroides gene encoding subunit IV of the cytochrome b-c1 complex (fbcQ) was cloned and sequenced. The fbcQ cistron is 372 base pairs long and encodes 124 amino acid residues. The molecular mass of subunit IV, deduced from the nucleotide sequence, is 14,384 Da. A hydropathy plot of the predicted amino acid sequence revealed only one transmembrane helix; it is near the C-terminal end. The 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinone ([3H]azido-Q)-labeled subunit IV was isolated from the [3H]-azido-Q-treated cytochrome b-c1 complex. A ubiquinone-binding peptide was obtained by digesting the labeled subunit IV with V8 protease followed by high performance liquid chromatography separation. Amino acid analysis and partial N-terminal sequencing of this ubiquinone-binding peptide revealed that it corresponded to residues 77-124 of subunit IV. Based on the hydropathy profile and predicted tendency to form alpha-helices and beta-sheets, we propose a structural model for subunit IV. In this model the ubiquinone-binding domain is located near the surface of the membrane.

Affinity Labels↗
PubMed1991

Immunochemical study of subunit VI (Mr 13,400) of mitochondrial ubiquinol-cytochrome c reductase.

A preparation containing the Mr 13,400 protein (subunit VI), phospholipid, and ubiquinone was isolated from bovine heart mitochondrial ubiquinol-cytochrome c reductase by a procedure involving Triton X-100 and urea solubilization, calcium phosphate-cellulose column chromatography at different pHs, acetone precipitation, and decanoyl-N-methylglucamide-sodium cholate extraction. The protein in this preparation corresponds to subunit VI of ubiquinol-cytochrome c reductase resolved in the sodium dodecyl sulfate-polyacrylamidce gel electrophoresis system of Schägger et al. (1987, FEBS Lett. 21, 161-168) and has the same amino acid sequence as that of the Mr 13,400 protein reported by Wakabayashi et al. (1985, J. Biol. Chem. 260, 337-343). The phospholipid and ubiquinone present in the preparation copurify with but are not intrinsic components of, the Mr 13,400 protein. This preparation has a potency and behavior identical to that of a free phospholipid preparation in restoring activity to delipidated ubiquinol-cytochrome c reductase. Antibodies against Mr 13,400 react only with Mr 13,400 protein and complexes which contain it. They do not inhibit intact, lipid-sufficient ubiquinol-cytochrome c reductase. However, when delipidated ubiquinol-cytochrome c reductase is incubated with antibodies prior to reconstitution with phospholipid, a 55% decrease in the restoration activity is observed, indicating that the catalytic site-related epitopes of the Mr 13,400 protein are buried in the phospholipid environment. Antibodies against Mr 13,400 cause an increase of apparent Km for ubiquinol-2 in ubiquinol-cytochrome c reductase. When mitoplasts or submitochondrial particles are exposed to a horseradish peroxidase conjugate of the Fab' fragment of anti-Mr 13,400 antibodies, peroxidase activity is found mainly in the submitochondrial particles preparation; little activity is detected in mitoplasts. This suggests that the Mr 13,400 protein is extruded toward the matrix side of the membrane.

Amino Acid Sequence↗
PubMed1991

The Rhodospirillum rubrum cytochrome bc1 complex: redox properties, inhibitor sensitivity and proton pumping.

A detergent-solubilized, three-subunit-containing cytochrome bc1 complex, isolated from the photosynthetic bacterium R. rubrum, has been shown to be highly sensitive to stigmatellin, myxothiazol, antimycin A and UHDBT, four specific inhibitors of these complexes. Oxidation-reduction titrations have allowed the determination of Em values for all the electron-carrying prosthetic groups in the complex. Antimycin A has been shown to produce a red shift in the alpha-band absorbance maximum of one of the cytochrome b hemes in the complex and stigmatellin has been shown to alter both the Em and EPR g-values of the Rieske iron-sulfur protein in the complex. Western blots have revealed antigenic similarities between the cytochrome subunits of the R. rubrum complex and those of the related photosynthetic bacteria, Rb. capsulatus and Rb. sphaeroides. The R. rubrum complex has been incorporated into liposomes. These liposomes exhibit respiratory control and are able to couple electron transfer from quinol to cytochrome c to proton translocation across the liposome membrane in a manner consistent with a Q-cycle mechanism. It can thus be concluded that neither electron transport nor coupled proton translocation by the cytochrome bc1 complex requires more than three subunits in R. rubrum.

Antimycin A↗
PubMed1991

Essentiality of the molecular weight 15,000 protein (subunit IV) in the cytochrome b-c1 complex of Rhodobacter sphaeroides.

The cytochrome b-c1 complex from Rhodobacter sphaeroides was resolved into four protein subunits by a phenyl-Sepharose CL-4B column eluted with different detergents. Individual subunits were purified to homogeneity. Antibodies against subunit IV (Mr = 15,000) were raised and purified. These antibodies had a high titer with isolated subunit IV and with the b-c1 complex from R. sphaeroides. They inhibited 95% of the ubiquinol-cytochrome c reductase activity of the cytochrome b-c1 complex, indicating that subunit IV is essential for the catalytic function of this complex. When detergent-solubilized chromatopores were passed through an anti-subunit IV coupled Affi-Gel 10 column, no no ubiquinol-cytochrome c reductase activity was detected in the effluent, and four proteins, corresponding to the four subunits in the isolated complex, were adsorbed to the column. This indicated that subunit IV in an integral part of the cytochrome b-c1 complex. No change in the apparent Kms for Q2H2 and for cytochrome c was observed with anti-subunit IV treated complex. Antibodies against subunit IV had little effect on the stability of the ubisemiquinone radical in this complex, suggesting that they do not bind to the subunit near its ubiquinone-binding site.

Antibodies↗
PubMed1991

The catalytic role of subunit IV of the cytochrome b6-f complex from spinach chloroplast.

The catalytic role of subunit IV, the Mr 17,000 protein, in the chloroplast cytochrome b6-f complex was established through trypsinolysis of the complex under controlled conditions. When purified chloroplast cytochrome b6-f complex, 1 mg/ml, in 50 mM Tris-succinate buffer (pH 7.0) containing 1% sodium cholate and 10% glycerol is treated with 80 micrograms of trypsin at room temperature for various lengths of time, the activity of the cytochrome b6-f complex decreases as the incubation time increases. A maximal inactivation of 80% is reached at 7 min of incubation. The trypsin inactivation is accompanied by the destruction of the proton translocation activity of the complex. No alteration of absorption and EPR spectral properties was observed in the trypsin-inactivated complex. Subunit IV is the only subunit in the cytochrome b6-f complex that is digested by trypsin, and the degree of digestion correlates with the decrease of electron transfer activity. The binding of azido-Q to subunit IV of the complex decreases as the extent of inactivation of the cytochrome b6-f complex by trypsin increases. The residue molecular mass of trypsin cleaved subunit IV is about 14 kDa, suggesting that the cleavage site is at lysine 119 or arginine 125 or 126. When the thylakoid membrane was assayed for cytochrome b6-f complex activity, very little activity was observed; and the activity was not sensitive to trypsinolysis. Upon sonication, activity and sensitivity to trypsinolysis was greatly increased, suggesting that subunit IV protrudes from the lumen side of the membrane.

Affinity Labels↗