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Biomedical subjects

C Tu

Publications and source records attributed to C Tu.

At least 73 records · Page 4Linked to original sources
PubMed1996

[Thoracoscopy in malignant pleural effusions].

To assess the value of thoracoscopy in malignant pleural effusions, the procedure and results of thoracoscopy by using a fiberoptic bronchoscope and a rigid cold-light thoracoscope in 130 cases with malignant pleural effusion are reported. The overall diagnostic rate was 91.5% (119/130). The malignant pleural mesothelioma in 24 cases and metastatic cancers in 95 cases were histopathologically confirmed. Talcum powder, tetracycline and Corynebacterium parvum were separately sprayed through thoracoscope into pleural cavity in 69, 10 and 10 patients, and the success rates of complete and lasting pleurodesis were 87.0%, 5/10 and 8/10 respectively. Postoperative complications included transient fever and chest pain, local subcutaneous emphysema in 6 cases and tumor seeding at thoracoscopy site in 4 cases. It is concluded that thoracoscopy is simple, safe, reliable and of high practical value in the diagnosis of malignant pleural effusions and in assessment before exploratory thoracotomy, and that transendoscopical administration of drugs for pleurodesis is a very effective method for controlling malignant pleural effusions. The efficacy of the talc poudrage is better than tetracycline and Corynebacterium parvum.

Adenocarcinoma↗
PubMed1996

[The role of thoracoscopy in the diagnosis and management of pleural effusion].

To assess the value of thoracoscopy in the diagnosis and management of pleural effusion, 146 patients with pleural effusion of unknown causes had this examination by using fiberoptic bronchoscope the rigid cold light thoracoscope. 127 of these cases were histopathologically diagnosed. 109 had malignant diseases and 18 benign specific diseases. The histologic diagnoses following thoracoscopic biopsy in all the patients were compared with the clinical findings at follow-up, the results showed that the sensitivity was 92.7%, specificity 100.0% and diagnostic accuracy 93.2%. 72 patients with more than moderate volume of pleural effusion were treated with intrapleural talcum powder suspensions (3% 100 ml) under thoracoscopic control, 63 of them obtained complete pleurodesis. The success rates of talc poudrage pleurodesis were 88.1% in 67 cases with malignant effusion (59/67) and 80.0% in 5 cases with benign pleural effusion (4/5). There were only minor postoperative complications: transient fever in 54 cases, local subcutaneous emphysema in 6 and thoracoscopy site tumor seeding in 2. It was shown that thoracoscopy is simple, safe, reliable and practical in the diagnosis of pleural effusion and that talc pleurodesis is a very effective method for controlling refractory pleural effusion transendoscopically.

Adult↗
PubMed1995

Proton transfer by histidine 67 in site-directed mutants of human carbonic anhydrase III.

The ability of a histidine residue at position 67 in human carbonic anhydrase III to transfer protons in the catalytic pathway for the hydration of CO2 was investigated for a series of site-specific mutants. Wild-type carbonic anhydrase III has an arginine at this position with the C alpha of residue 67 about 9.4 A from the zinc. The active-site cavity contains no other residues capable of facile proton transfer. Rate constants for proton transfer from His 67 to the zinc-bound hydroxide were determined from the rate constants for the exchange of 18O between CO2 and water measured by mass spectrometry. A range of values for the pKa of zinc-bound water was achieved by replacement of phenylalanine with leucine and aspartate at position 198 adjacent to the zinc. Application of Marcus rate theory showed that intramolecular proton transfer involving His 67 had an intrinsic energy barrier of 1.3 +/- 0.3 kcal/mol and a thermodynamic work function for a preceding unfavorable equilibrium of 10.9 +/- 0.1 kcal/mol. We previously showed that proton transfer from histidine 64 in carbonic anhydrase III could be described by Marcus rate theory [Silverman, D. N., Tu, C. K., Chen, X., Tanhauser, S. M., Kresge, A. J., & Laipis, P. J. (1993) Biochemistry 32, 10757-10762]. In comparison, proton transfer from His 67 must overcome a more unfavorable preceding equilibrium (a larger work function) that probably represents an energy requirement for proper alignment of donor and acceptor groups plus the intervening hydrogen-bonded water. Once this alignment is achieved, the intrinsic energy barrier appears the same for His 67 or His 64.

Amino Acid Sequence↗
PubMed1995

[Pharmacokinetics study of intraperitoneal carboplatin in gynecologic carcinoma].

Twenty patients with surgically and pathologically confirmed gynecologic cancer from December, 1991 to December, 1992 were treated by intraperitoneal (i.p.) chemotherapy with carboplatin in 15 and by intravenous (i.v.) chemotherapy with carboplatin in 5, patients had normal hepatic and renal function test and were nonsmoker. Using Grathite atomic absorption spectrum method, total mean maximal plasmic carboplatin concentration in the cases by intravenous infusion was 8 times greater than those by intraperitoneal infusion. The half-life of plasmic elimination in the cases treated by i.v. infusion and by i.p. infusion were respectively 10.82 +/- 4.95 hours and 54.04 +/- 10.75 hours (P < 0.05). But total carboplatin plasmic clearance in the cases were 1.88 +/- 0.53 ml/min and 1.12 +/- 0.58 ml/min, respectively (P < 0.05). Pharmacokinetic profiles suggest a possible therapeutic advantage by giving the drug intraperitoneally for the treatment of tumour nodules situated in the peritoneum. This research also suggest the method has the possibility in increasing antitumn effect and decreasing bone marrow in intoxication because of the wide distribution, low plasmic concentration and slow clearance of drugs.

Adult↗
PubMed1995

[Study of autologous bone marrow transplantation in the treatment of gynecologic carcinoma].

OBJECTIVE: To investigate the role of autologous bone marrow transplantation (ABMT) in patients with gynecologic carcinoma treated by high-dose chemotherapy. METHODS: This was a presentation of four patients with ovarian carcinoma, two patients with primary fallopian tubal carcinoma, and 5/6 of them clinically staged as III and IV, treated with combination chemotherapy of cytoxan and carboplatin by intraperitoneal and intravenous infusion, the dose being 2.5-5 times those employed conventionaly. Autologous bone marrow collection and storage were conducted under strictly sterile conditions. Chemotherapy was given pre- and post-operatively and ABMT was performed immediately after chemotherapy. Careful follow-up was observed at monthly intervals. RESULTS: All of the six patients tolerated the treatment well, with no death within a median follow-up of 26 months (range 1532). Five of them lived more than 6 months with no evidence of disease. Two patients without gross residual tumor, who could not tolerate conventional chemotherapy which had to be discontinued, successfully received high-dose chemotherapy, supported by ABMT. All four patients with gross residual tumor benefited by immediate clinical remission after treatment. Median survival for all patients was 28 months (range 1253), median survival for stage IV patients was 24 months (range 1230). CONCLUSIONS: Patients subjected to high-dose chemotherapy supported by ABMT are exempted from suffering the cyclic multiple courses of chemotherapy, repeated or long-term hospitalization and may lead a life of good quality.

Adult↗
PubMed1994

Sex-specific processing of the Drosophila exuperantia transcript is regulated in male germ cells by the tra-2 gene.

The Drosophila exuperantia (exu) gene encodes overlapping sex-specific, germline-dependent mRNAs. In this work, the structural differences between these sex-specific exu mRNAs were determined by sequence analysis of 9 ovary and 10 testis cDNAs. The transformer 2 (tra-2) gene functions in sex determination of female somatic cells through its role in regulating female-specific splicing of doublesex (dsx) RNA. We report here that tra-2 is required in male germ cells for efficient male-specific processing of exu RNA; in the absence of tra-2, X/Y males produce a new mRNA which is processed at its 3' end so that it contains sequences normally specific to the female 3' untranslated region. Although the processing event that requires tra-2 occurs in an untranslated region of the exu transcript, the isolation and characterization of a male-specific exu allele which deletes male 3' untranslated sequence indicate that this processing is biologically significant.

Alleles↗
PubMed1994

Catalytic properties of mouse carbonic anhydrase V.

A cDNA encoding the mouse carbonic anhydrase V gene was isolated by reverse transcription and polymerase chain reaction from BALB/c mouse liver mRNA. Vectors containing the full coding sequence as well as two different NH2-terminal truncated genes expressed enzymatically active protein in Escherichia coli. The carbonic anhydrase V produced by a vector containing the full coding sequence, which includes a possible NH2-terminal mitochondrial targeting signal, was proteolytically processed by E. coli and contained several amino-terminal ends. The two NH2-terminal truncated vectors deleted, respectively, 1) the 29-amino acid putative targeting sequence and 2) 51 amino acids, yielding a protein equivalent to a carbonic anhydrase (CA) V isolated from mouse liver mitochondria; and both vectors produced homogeneous protein fractions. These latter two forms of CA V had identical steady-state constants for the hydration of CO2, with maximal values of kcat/Km at 3 x 10(7) M-1 s-1 and kcat at 3 x 10(5) s-1 with an apparent pKa for catalysis of 7.4 determined from kcat/Km. In catalytic properties, mouse CA V is closest to CA I; however, in inhibition by acetazolamide, ethoxzolamide, and cyanate, CA V is very similar to CA II. Mouse CA V has a tyrosine at position 64, where the highly active isozyme II has histidine serving as a proton shuttle in the catalytic pathway. Investigation of a site-specific mutant of CA V containing the replacement Tyr64-->His showed that the unique kinetic properties of CA V are not due to the presence of tyrosine at position 64.

Amino Acid Sequence↗
PubMed1994

Interactions of active-site residues and catalytic activity of human carbonic anhydrase III.

To elucidate the interactions between residues found in the active-site cavity of human carbonic anhydrase III, we have prepared a series of single and double mutants with Lys-64, Arg-67, and Phe-198 replaced with Ala, Asp, Glu, His, and Leu. Rates of catalysis were determined using 18O exchange between CO2 and water measured by mass spectrometry and initial velocity measured by stopped-flow spectrophotometry. Replacement of these residues resulted in increases in kcat/Km for CO2 hydration as much as 200-fold and increases in the pKa of the zinc-bound water by as much as 3.5 units. We conclude that the effect of replacements made at positions 64, 67, and 198 were in general additive for kcat/Km for CO2 hydration, indicating that there is no interaction between these sites that affects the catalytic interconversion of CO2 and HCO3-. One notable exception is the antagonism exhibited by the double mutant of human carbonic anhydrase III containing Glu-64 and Leu-198. The data also show that one source of the large enhancement of kcat/Km for the mutant containing Asp-198 in human carbonic anhydrase III is the presence of both Asp-198 and Lys-64; when Lys-64 was replaced with Ala, a reduction of catalytic activity was observed. These results provide an additional view of the independent interactions of amino acids that affect the catalytic pathway of isozyme III, the least active of the known carbonic anhydrase isozymes.

Binding Sites↗
PubMed1994

Comparison of intra- and intermolecular proton transfer in human carbonic anhydrase II.

The catalysis of the hydration of CO2 by human carbonic anhydrase II (HCA II) includes the transfer of a proton from zinc-bound water to histidine 64 utilizing a network of intervening hydrogen-bonded water molecules, then the proton is transferred to buffer in solution. We used stopped-flow spectrophotometry and 18O exchange between CO2 and water measured by mass spectrometry to compare catalytic constants dependent on proton transfer in HCA II and in the mutant H64A HCA II containing the replacement His64-->Ala. Maximal velocities and oxygen-18 exchange catalyzed by H64A HCA II showed that nearly all of the proton transfer with this mutant proceeded through the imidazole buffer. The following parameters were very similar or identical in catalysis by H64A HCA II compared with catalysis by wild-type HCA II both in the presence of large concentrations of imidazole (100 mM): the maximal rate of initial velocity and of exchange of 18O between CO2 and water, solvent hydrogen isotope effects on the maximal velocity, and the dependence of these isotope effects on the atom fraction of deuterium in solvent water. These results indicate that the proton transfer involving the zinc-bound water in catalysis is not significantly affected by the difference between the mobility of the free imidazole buffer and the side chain of His 64. Moreover, data for both the wild-type and mutant enzymes are consistent with proton transfer through intervening hydrogen-bonded water bridges in the active sites. These features of the proton transfer are discussed in terms of a model in which the first proton transfer from the zinc-bound water to an adjacent water is rate limiting.

Carbonic Anhydrases↗
PubMed1993

Rate-equilibria relationships in intramolecular proton transfer in human carbonic anhydrase III.

Maximal turnover rates for the dehydration of HCO3- catalyzed by the zinc metalloenzyme carbonic anhydrase III are limited by a proton transfer to zinc-bound hydroxide in the active site. We have used site-directed mutagenesis to place a proton donor, histidine, at position 64 and used 18O exchange between CO2 and water measured by mass spectrometry to determine the rates of intramolecular proton transfer to the zinc-bound hydroxide. In a series of site-specific mutants, the values of pKa of the zinc-bound water ranged from approximately 5 to 9. The rate constants for proton transfer obeyed a Brønsted correlation and showed sharp curvature characteristic of facile proton transfers. Application of Marcus rate theory shows that this proton transfer has the small intrinsic energy barrier (near 1.5 kcal/mol) characteristic of rapid proton transfer between nitrogen and oxygen acids and bases, but has an observed overall energy barrier (near 10 kcal/mol), indicating the involvement of accompanying, energy requiring processes such as solvent reorganization or conformational change.

Amino Acid Sequence↗
PubMed1993

Interaction and influence of phenylalanine-198 and threonine-199 on catalysis by human carbonic anhydrase III.

Site-directed mutants of human carbonic anhydrase III were used to examine the role of Thr-199 and its interaction with Phe-198 in the catalyzed hydration of CO2. Threonine-199 is a hydrogen bond acceptor for the zinc-bound water, and Phe-198 forms part of the hydrophobic side of the active-site cavity of carbonic anhydrase III. Catalytic activity for a total of five single and double mutants at residues 198 and 199 was determined by stopped-flow spectrophotometry and 18O exchange between CO2 and water measured by mass spectrometry. The replacement Thr-199-->Ala resulted in a 4-fold decrease in the kcat/Km for hydration of CO2. We tested the hypothesis that the 25-fold increase in the kcat/Km for hydration of CO2 accompanying the replacement Phe-198-->Leu in isozyme III is caused by changes in the interaction of Thr-199 with the zinc-bound water or the transition state for catalysis. Comparison of hydration of CO2 by the single and double mutants of isozyme III containing the replacements Thr-199-->Ala and Phe-198-->Leu was consistent with an interaction between these two sites.

Amino Acid Sequence↗
PubMed1993

Influence of amino acid replacement at position 198 on catalytic properties of zinc-bound water in human carbonic anhydrase III.

Carbonic anhydrase III, found predominantly in skeletal muscle, is the least efficient of the mammalian carbonic anhydrases in catalyzing the hydration of CO2. Phenylalanine-198 is located on the hydrophobic side of the active-site cavity with its phenyl ring in the proximity of the catalytically active zinc-bound water. We replaced phenylalanine-198 in human carbonic anhydrase III with seven other amino acids (Ala, Asn, Asp, His, Leu, Tyr, Val) using site-directed mutagenesis. The catalytic properties of these enzymes were determined by stopped-flow spectrophotometry, and the exchange of 18O between CO2 and water was measured by mass spectrometry. All of the mutants had maximal values of kcat/Km for the hydration of CO2 enhanced, and five of the mutants had the pKa of the zinc-bound water increased compared with the wild-type enzyme. The largest effects were observed with the replacement Phe-198-->Asp which increased the maximal kcat/Km 140-fold and increased the pKa of the zinc-bound water from near 5 to 9.2. A Brønsted correlation was observed between log(kcat/Km) for hydration of CO2 and the pKa of the zinc-bound water (correlation coefficient r = 0.92); in addition, this pKa was inversely correlated with hydrophobicity of the residue at 198 (correlation coefficient r = -0.83). A direct correlation between the logarithm of the maximal kcat/Km for hydration and the logarithm of the pH-independent value of Ki for inhibition by cyanate (r = 0.95) indicated that the effect of the mutations at residue 198 occurred in large part by enhancement of the rate of dissociation of the enzyme-bicarbonate complex.

Bicarbonates↗
PubMed1993

Kinetic analysis of a mutant (His107-->Tyr) responsible for human carbonic anhydrase II deficiency syndrome.

The replacement His107-->Tyr is a cause of carbonic anhydrase II deficiency syndrome in humans (Venta, P. J., Welty, R. J., Johnson, T. M., Sly, W. S., and Tashian, R. E. (1991) Am. J. Hum. Genet. 49, 1082-1090). We have prepared this mutant of human carbonic anhydrase II by site-directed mutagenesis and expressed it in Escherichia coli. The mutant was too unstable to purify; however, we were able to stabilize and store it at 4 degrees C in cell lysates containing 1-4 mg/ml bovine serum albumin. The concentration of this mutant in the lysate was determined by titration with the tight-binding inhibitor ethoxzolamide. The stability in this preparation was sufficient to determine that this mutant of carbonic anhydrase II has kcat/Km and apparent pKa for the hydration of CO2 equivalent to that of wild-type HCA II. The maximum velocity of CO2 hydration, which is dependent on the rate of proton transfer between enzyme and solution, was 3-fold smaller than for HCA II suggesting that the proton transfer pathway in the mutant is slightly less efficient than in wild type. Preliminary conformational energy calculations show that the replacement of His107 with the larger residue Tyr results in considerable distortion of the cavity surrounding site 107 and in the loss of at least two hydrogen bonds.

Animals↗
PubMed1993

A physiological role for cyanate-induced carbonic anhydrase in Escherichia coli.

Cyanate induces expression of the cyn operon in Escherichia coli. The cyn operon includes the gene cynS, encoding cyanase, which catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. A carbonic anhydrase activity was recently found to be encoded by the cynT gene, the first gene of the cyn operon; it was proposed that carbonic anhydrase prevents depletion of bicarbonate during cyanate decomposition due to loss of CO2 by diffusion out of the cell (M. B. Guilloton, J. J. Korte, A. F. Lamblin, J. A. Fuchs, and P. M. Anderson, J. Biol. Chem. 267:3731-3734, 1992). The function of the product of the third gene of this operon, cynX, is unknown. In the study reported here, the physiological roles of cynT and cynX were investigated by construction of chromosomal mutants in which each of the three genes was rendered inactive. The delta cynT chromosomal mutant expressed an active cyanase but no active carbonic anhydrase. In contrast to the wild-type strain, the growth of the delta cynT strain was inhibited by cyanate, and the mutant strain was unable to degrade cyanate and therefore could not use cyanate as the sole nitrogen source when grown at a partial CO2 pressures (pCO2) of 0.03% (air). At a high pCO2 (3%), however, the delta cynT strain behaved like the wild-type strain; it was significantly less sensitive to the toxic effects of cyanate and could degrade cyanate and use cyanate as the sole nitrogen source for growth. These results are consistent with the proposed function for carbonic anhydrase. The chromosomal mutant carrying cynS::kan expressed induced carbonic anhydrase activity but no active cyanase. The cynS::kan mutant was found to be much less sensitive to cyanate than the delta cynT mutant at a low pCO2, indicating that bicarbonate depletion due to the reaction of bicarbonate with cyanate catalyzed by cyanase is more deleterious to growth than direct inhibition by cyanate. Mutants carrying a nonfunctional cynX gene (cynX::kan and delta cynT cynX::kan) did not differ from the parental strains with respect to cyanate sensitivity, presence of carbonic anhydrase and cyanase, or degradation of cyanate by whole cells; the physiological role of the cynX product remains unknown.

Bicarbonates↗