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PubMed1991

Red blood cells augment transport of reactive metabolites of monocrotaline from liver to lung in isolated and tandem liver and lung preparations.

Monocrotaline (MCT) is a pyrrolizidine alkaloid that causes pulmonary hypertension in rats by mechanisms which remain largely unknown. MCT is thought to be activated in the liver to a reactive intermediate that is transported to the lung where it causes endothelial injury. Our previous pharmacokinetic work demonstrated significant sequestration of radioactivity in red blood cells (RBCs) of rats treated with [14C]MCT. To determine whether this RBC sequestration might be important in the transport of reactive MCT metabolites, we compared the effect of inclusion of RBCs in the perfusion buffer on the extent of covalent binding of [14C]MCT to rat lungs in tandem liver-lung preparations. The potential effect of RBCs in stabilizing reactive intermediates was evaluated by preperfusion of isolated liver preparations with [14C]MCT with and without RBCs, separation and washing of the RBC fraction, and subsequent (90 min later) perfusion of washed RBCs or buffer alone in isolated perfused lungs. Covalent binding to lung tissues was determined by exhaustive methanol/chloroform extractions of unbound label from homogenized lung tissue followed by scintillation counting of residual 14C. Covalent binding was expressed as picomole MCT molecular weight equivalents/mg protein. Comparison of the relative capability of these isolated organ preparations for conversion of MCT to polar metabolites was done by extraction and HPLC analysis of perfusate at the end of the experiment. Isolated livers converted 65-85% of MCT to polar metabolites compared with less than 5% conversion in the isolated lungs. Inclusion of RBCs in the buffer of tandem lung liver preparations perfused with 400 microM [14C]MCT increased the covalent binding to the lung from 97 +/- 25 (buffer alone) to 182 +/- 36 (buffer + RBC) pmol/mg protein. At the end of these perfusions, RBCs contained 1552 +/- 429 pmol/mg hemoglobin of which 333 +/- 98 pmol/mg hemoglobin resisted exhaustive solvent extraction. After 90 min at room temperature, buffer with 400 microM [14C]MCT preperfused in isolated livers resulted in covalent binding to isolated perfused lung of 0.8 +/- 0.4 pmol/mg protein while washed RBCs isolated from buffer of similar liver preperfusions preparations resulted in 53 +/- 7 pmol/mg protein bound to lung. Control groups perfused with 400 microM [14C]MCT in buffer or buffer + RBCs through isolated lungs only resulted in covalent binding of 2 +/- 1 or 1 +/- 0.6 pmol/mg protein respectively. We conclude: (1) RBCs significantly augment the transport of lung reactive MCT metabolites from the liver to the lung.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2002

Propane and butane emission sources to ambient air of Mexico City metropolitan area.

Samples of volatile organic compounds (VOCs) were collected in a smog chamber in order to determine whether automotive exhausts or LP Gas emissions play a greater role in the source of propane and butane, which affect ozone formation and other pollutants in the ambient air of the Mexico City metropolitan area (MCMA). These samples were collected in April 1995 during mornings and evenings. The testing methodology used for measuring exhaust emission were FTP or EPA-74 tests, and SHED type tests were also conducted in order to evaluate evaporative emissions. The finding from analysis of the VOCs collected in the morning demonstrate that in the atmosphere, propane concentrations are higher than that of butane but the reverse in evaporative and exhaust emissions, with the concentration of propane lower than that of butane. Our conclusion is that most of C3 and C4 in the ambient air comes from LP gas and not vehicle exhaust or evaporative emission, due to the higher levels of propane than butane in its formulation. The analysis of VOCs also indicates that although the conversion (in the smog chamber) of alkanes is low during the day, due to the high initial concentration, their contribution in the reaction mechanism to produce ozone can be appreciable.

Butanes↗
PubMed1983

Five years' experience of a gastroenterology day-case unit.

An inpatient ward has been converted, at low cost, to a gastroenterology day-case unit, which includes a purpose-built endoscopy room and a day-case area. The presence of a day-case ward permits the nursing establishment to be sufficiently flexible to cover the endoscopy room fully, and the hours are suitable for married nurses with families. Patients remain on the same trolley throughout the procedure so that portering delays are avoided, and a good flow system operated by the unit's staff enables endoscopies to be carried out at the rate of one every ten to fifteen minutes. Admissions rose from 1900 to 2700 per year (an average of 12 X 5 admissions per working day, theoretical bed occupancy = 140%). Cost per patient is about half the estimated cost without a day-case unit. The increasing numbers of routine upper gastrointestinal endoscopies are mirrored by a halving of barium-meal examinations.

Bed Conversion↗
PubMed1999

Self-curing membranes of chitosan/PAA IPNs obtained by radical polymerization: preparation, characterization and interpolymer complexation.

Chitosan/polyacrylic acid I PN's were prepared by radical polymerization of acrylic acid, AA, activated at low temperature, in an aqueous/alcoholic chitosan dispersion. AA monomer to polymer conversion and membrane compositions were determined by elemental analysis and FTIR. Evidences of interpolyelectrolite complex formation were found from the FTIR spectra as well. The gravimetric measurements and the elemental analysis after some exhaustive PAA extraction support the existence of some PAA grafting on the reactive amine group of the chitosan. Swelling degree of the membranes is highly dependent on pH and composition, showing a higher swelling in membranes richer in AA and increased pH due to the breaking of interpolyelectrolite salt bonds.

Acrylic Resins↗
PubMed1999

Carotenoid hydroxylase from Haematococcus pluvialis: cDNA sequence, regulation and functional complementation.

A cDNA homologous to beta-carotene hydroxylase from Arabidopsis thaliana was isolated from the green alga Haematococcus pluvialis. The predicted amino acid sequence for this enzyme shows homology to the three known plant beta-carotene hydroxylases from Arabidopsis thaliana and from Capsicum annuum (38% identity) and to prokaryote carotenoid hydroxylases (32-34% identities). Heterologous complementation using E. coli strains which were genetically engineered to produce carotenoids indicated that the H. pluvialis beta-carotene hydroxylase was able to catalyse not only the conversion of beta-carotene to zeaxanthin but also the conversion of canthaxanthin to astaxanthin. Furthermore, Northern blot analysis revealed increased beta-carotene hydroxylase mRNA steady state levels after induction of astaxanthin biosynthesis. In accordance with the latter results, it is proposed that the carotenoid hydroxylase characterized in the present publication is involved in the biosynthesis of astaxanthin during cyst cell formation of H. pluvialis.

Amino Acid Sequence↗
PubMed2000

High-performance liquid chromatographic determination of 17beta-estradiol and 17beta-estradiol-3-acetate solubilities and diffusion coefficents in silicone elastromeric intravaginal rings.

A rapid, sensitive reversed-phase high-performance liquid chromatographic method has been developed for the determination of in vitro release of 17beta-estradiol and its ester prodrug, 17beta-estradiol-3-acetate, from silicone intravaginal rings. Partial hydrolysis of the acetate under the aqueous conditions provided by the 1% benzalkonium chloride release medium necessitates its conversion to 17beta-estradiol prior to HPLC analysis. Both steroid peaks have been fully resolved from the benzalkonium chloride peaks by the reported chromatographic method, which employs a C18 bonded reversed-phase column, an acetonitrile-water (50:50, v/v) mobile phase and a UV detection wavelength of 281 nm. The peak area versus 17beta-estradiol concentration was found to be linear over the range of 0.0137-1347 microkg ml(-1). The HPLC method has also been used to determine the silicone solubilities and diffusion coefficients of the two related steroids. The almost 100-fold increase in 17beta-estradiol-3-acetate release from the silicone core-type intravaginal rings compared to 17beta-estradiol is shown to be due to a 60-fold increase in silicone solubility and a one and a half-fold increase in diffusitivity. The results demonstrate that an effective estrogen replacement therapy dose of 17beta-estradiol may be administered from a silicone intravaginal reservoir device containing the labile 17beta-estradiol-3-acetate prodrug.

Administration, Intravaginal↗
PubMed2004

Tissue trauma in abdominal and laparoscopic-assisted vaginal hysterectomy.

STUDY OBJECTIVE: To compare the degree of tissue damage between abdominal and laparoscopic-assisted hysterectomy. DESIGN: Prospective, randomized, controlled study (Canadian Task Force classification I). SETTING: University hospital. PATIENTS: Forty-six women. INTERVENTION: Laparoscopic-assisted and abdominal hysterectomy. MEASUREMENTS AND MAIN RESULTS: The degree of tissue injury caused by surgical trauma was assessed by measuring plasma levels of creatine phosphokinase (CPK), C-reactive protein (CRP), lactic dehydrogenase (LDH), and CA 125 in patients undergoing laparoscopic-assisted (n = 23) and abdominal hysterectomy (n = 23). Plasma levels of CPK-MB isoenzyme were measured in order to exclude the possibility of myocardial injury. Blood samples were taken preoperatively, and on the first and second postoperative days. There was no difference in demographic characteristics between the groups. One patient in the laparotomy group and two patients in the laparoscopy group were excluded from analysis due to bladder injury, postoperative thrombophlebitis, and conversion to laparotomy because of severe adhesions. The mean operation time was longer (105.5 +/- 23.1 minutes vs 77.3 +/- 18.7 minutes, p < .001), but the duration of hospital stay was shorter (2.7 +/- 0.8 days vs 4.3 +/- 1.4 days, p < .001) and analgesic requirement was lower in the laparoscopy group compared with the abdominal group. We observed significant postoperative increases in CRP and CPK in both groups on postoperative days 1 and 2, whereas the increase in LDH was significant only in the laparotomy group on postoperative day 2. There were no significant changes in CPK-MB and CA 125. Moreover, mean plasma levels of CRP and CPK were significantly higher in the laparotomy group on postoperative days 1 and 2 than in the laparoscopy group (p < .05). CONCLUSION: Surgery is a significant cause of tissue injury that can be assessed by specific enzymes and proteins. We suggest that laparoscopic surgery causes less tissue damage as assessed by lower postoperative CRP and CPK values, and that less tissue trauma related with laparoscopic surgery may account for early mobilization and reduced analgesic requirement in these patients.

Adult↗
PubMed2002

Relative value of analog and digital teaching files.

RATIONALE AND OBJECTIVES: This study was performed to determine if it is financially reasonable for radiology residency programs that create and maintain their own teaching files to switch from analog teaching files (ATFs) to digital teaching files (DTFs). MATERIALS AND METHODS: Radiology residency program directors were surveyed electronically about the monetary value and importance of conventional ATFs and DTFs. The costs for maintaining each type of file were calculated at the authors' institution. RESULTS: Surveys were sent to the program directors of all 197 accredited radiology residencies. Responses were received from 48 (24%). DTFs were scored as more important than ATFs, but the difference was not significant (P = .22). DTFs were rated as less complete (P = .01) but more current (P << .001) than ATFs. DTFs included the American College of Radiology Learning File (85%), in-house productions (77%), and other commercially available products (63%). Thirty percent of respondents had a DTF integrated into a picture archiving and communication system, and 28% reported having a technician dedicated to the teaching file. Program directors ascribed total median dollar values of $250 and $3,000 per year to their ATFs and DTFs, respectively. The annual costs at the authors' institution were much higher than these ascribed values: $44,720 ($91 per case) for maintaining a DTF produced in house and $24,601 ($50 per case) for maintaining an ATF, excluding physician time. CONCLUSION: Program directors are more willing to pay for a DTF than an ATF. For both, the costs of maintenance are great and the relative monetary value is low.

Analog-Digital Conversion↗
PubMed1992

Structure of the gene for human coagulation factor V.

Activated factor V (Va) serves as an essential protein cofactor for the conversion of prothrombin to thrombin by factor Xa. Analysis of the factor V cDNA indicates that the protein contains several types of internal repeats with the following domain structure: A1-A2-B-A3-C1-C2. In this report we describe the isolation and characterization of genomic DNA coding for human factor V. The factor V gene contains 25 exons which range in size from 72 to 2820 bp. The structure of the gene for factor V is similar to the previously characterized gene for factor VIII. Based on the aligned amino acid sequences of the two proteins, 21 of the 24 intron-exon boundaries in the factor V gene occur at the same location as in the factor VIII gene. In both genes, the junctions of the A1-A2 and A2-A3 domains are each encoded by a single exon. In contrast, the boundaries between domains A3-C1 and C1-C2 occur at intron-exon boundaries, which is consistent with evolution through domain duplication and exon shuffling. The connecting region or B domain of factor V is encoded by a single large exon of 2820 bp. The corresponding exon of the factor VIII gene contains 3106 bp. The 5' and 3' ends of both of these exons encode sequences homologous to the carboxyl-terminal end of domain A2 and the amino-terminal end of domain A3 in ceruloplasmin. There is otherwise no homology between the B domain exons.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1996

Thermodynamic role of the pro region of the neurophysin precursor in neurophysin folding: evidence from the effects of ligand peptides on folding.

Attention has focused recently on the role of amino-terminal precursor pro regions in protein folding, with particular emphasis on their effects on folding kinetics. We examined the kinetic and thermodynamic effects of ligand peptides on the folding of neurophysin from the reduced state; these peptides serve as analogs of the pro regions of the common precursors of the neurophysins and the hormones oxytocin and vasopressin. Folding of reduced, mononitrated bovine neurophysin-II was monitored by circular dichroism in a glutathione redox buffer. The results confirmed the ability of neurophysin to fold to a limited extent (20-25% in this system) in the absence of ligand peptides. Ligand peptides increased the efficiency of folding to 100%, the exact efficiency being dependent on peptide identity and concentration. However, the rate of folding was peptide-independent. Analysis of the folding reaction demonstrated relatively rapid conversion of the reduced state to a disulfide-scrambled state, which slowly converted (half-life of 5 h at pH 7.3) to the folded state. Native unliganded neurophysin also equilibrated with the disulfide-scrambled state in the same redox buffers. For each peptide, an equilibrium constant for the folding reaction, representing the amount of peptide bound in the folding system as a function of peptide concentration, was calculated. Comparison of this constant with the intrinsic binding constants of the native protein allowed the derivation, under conditions at or approaching thermodynamic reversibility, of the relative stability of the native and disulfide-scrambled states. The results indicate that the scrambled state, which probably represents the presence of incorrect disulfide pairs in both protein domains, is more stable than the native unliganded state by approximately 1 kcal/mol in this system. The role of ligand peptide therefore is to stabilize the folded protein after it is formed, i.e., it provides a thermodynamic sink. The results contrast with the putative behavior of exogenous peptides representative of the pro regions of subtilisin and alpha-lytic protease, which are generally considered to facilitate folding by reaction with folding intermediates. A potential alternative view of the role of propeptides in protease folding is suggested.

Animals↗
PubMedDate not supplied

alpha-Ketoglutarate assay based on fluorescence quenching by NADH.

A new assay procedure for the measurement of ketoglutarate concentrations is described which is based on substrate-induced quenching (SIQ) of a fluorophore. The method makes use of the photoreaction between a fluorophore (thionine) and NADH. The latter is consumed during an enzymatic reaction between ketoglutarate and L-glutamic dehydrogenase. The conversion yield of cofactor from its reduced form to oxidized forms represented as an overall change in the population of the excited state population of the fluorophore thionine. An empirical relation is described that correlates initial substrate concentration to the observed yield of the cofactor conversion via a fluorescence recovery constant,Kt. The analysis of data obtained over a range of 0-500 microM results in a constant of 2748 M-1. The applicability of the proposed method is demonstrated by performing the assay for alpha-ketoglutarate in human urine. The ketoglutarate SIQ assay was not affected by the background interference that is inherent to this complex matrix.

Buffers↗
PubMed2006

Chemistry surrounding monomeric copper(I) methyl, phenyl, anilido, ethoxide, and phenoxide complexes supported by N-heterocyclic carbene ligands: reactivity consistent with both early and late transition metal systems.

Monomeric copper(I) alkyl complexes that possess the N-heterocyclic carbene (NHC) ligands IPr, SIPr, and IMes [IPr = 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene, SIPr = 1,3-bis(2,6-diisopropylphenyl)imidazolin-2-ylidene, IMes = 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene] react with amines or alcohols to release alkane and form the corresponding monomeric copper(I) amido, alkoxide, or aryloxide complexes. Thermal decomposition reactions of (NHC)Cu(I) methyl complexes at temperatures between 100 and 130 degrees C produce methane, ethane, and ethylene. The reactions of (NHC)Cu(NHPh) complexes with bromoethane reveal increasing nucleophilic reactivity at the anilido ligand in the order (SIPr)Cu(NHPh) < (IPr)Cu(NHPh) < (IMes)Cu(NHPh) < (dtbpe)Cu(NHPh) [dtbpe = 1,2-bis(di-tert-butylphosphino)ethane]. DFT calculations suggest that the HOMO for the series of Cu anilido complexes is localized primarily on the amido nitrogen with some ppi(anilido)-dpi(Cu) pi-character. [(IPr)Cu(mu-H)]2 and (IPr)Cu(Ph) react with aniline to quantitatively produce (IPr)Cu(NHPh)/dihydrogen and (IPr)Cu(NHPh)/benzene, respectively. Analysis of the DFT calculations reveals that the conversion of [(IPr)Cu(mu-H)]2 and aniline to (IPr)Cu(NHPh) and dihydrogen is favorable with DeltaH approximately -7 kcal/mol and DeltaG approximately -9 kcal/mol.

Journal Article↗
PubMed2003

Effect of temperature on the enantioselectivity in the oxazaborolidine-catalyzed asymmetric reduction of ketones. Noncatalytic borane reduction, a nonneglectable factor in the reduction system.

The effect of temperature on the enantioselectivity of the oxazaborolidine-catalyzed asymmetric borane reduction of ketones has been investigated carefully using alkyl aryl ketones with a variety of functional groups and a B-methoxyoxazaborolidine derived from trimethyl borate and (S)-alpha,alpha-diphenylprolinol as a catalyst. The reductions were carried out over a range of temperatures in THF and toluene with or without the catalyst. The reductive rates increase along with increasing reaction temperature with or without the catalyst by determining the conversion of the ketones to alcohols by GC analysis. However, the rates of the catalytic reductions increase faster than those without the catalyst. The results indicate that the noncatalytic borane reduction is an important factor to the enantioselectivity in the reduction. The highest enantioselectivities were usually obtained between 20 and 30 degrees C in the asymmetric reduction.

Journal Article↗
PubMed2006

Highly selective encaging of carbon dioxide molecules in the mixed carbon dioxide and nitrogen hydrate at low temperatures.

The structural identification and guest compositions of the mixed CO(2) and N(2) hydrates at low temperature conditions were investigated by both theoretical predictions and experimental measurements. From the model calculations, at very low temperatures, the highly CO(2)-concentrated hydrates over 95 mol % CO(2) on the basis of water-free concentration could coexist with the gas mixtures of low CO(2) concentrations in equilibrium. X-ray diffraction measurements of the hydrates formed with the gas mixture of 3.16 mol % CO(2) and balanced N(2) indicate that the formed hydrates at all conditions considered in this study were identified as structure I, whereas the model predicts a structural transition to structure II around 220 K. However, it was also found that the formed hydrate samples contain a considerable amount of hexagonal ice resulting from incomplete conversion of ice to the hydrates. The compositional analysis suggests that a favorable encaging of CO(2) in the mixed hydrate can be obtained by the hydrate formation at low temperatures and relative amount of CO(2) molecules in the mixed hydrates increases with a decrease of temperature.

Journal Article↗
PubMed2002

Cytotoxic sphingosine 4-sulfates from the sponge Spirastrella abata.

Two new sphingosine 4-sulfates (1 and 2) have been isolated from the sponge Spirastrella abata as cytotoxic constituents. The gross structures of 1 and 2 were determined by spectroscopic analysis, and their stereochemistry was established by chemical conversion. The compounds exhibited significant cytotoxicity against a small panel of five human tumor cell lines.

Animals↗
PubMed1998

Modulating effect of lonidamine on response to doxorubicin in metastatic breast cancer patients: results from a multicenter prospective randomized trial.

Previous results from our preclinical studies have shown that lonidamine (LND) can positively modulate the antiproliferative activity of doxorubicin (DOX) on breast cancer cell lines. To evaluate the effect of LND in a clinical setting, a multicenter randomized trial was carried out on patients with advanced breast cancer. From September 1991 to July 1993, 181 patients were enrolled in the trial and received an initial treatment of DOX at 75 mg/m2 for 3 cycles. The 137 patients who reached complete remission, partial remission, or stable disease were randomized to receive either DOX alone (75 mg/m2 day 1) (arm A) or DOX plus LND (600 mg orally/day) (arm B). The patients enrolled in the two arms were fairly homogeneous in terms of major clinical characteristics. Toxicity was similar in both arms except for myalgia: WHO grade > or=2 was observed in 57% of arm B patients. Overall response rate to DOX + LND was 50% and to DOX alone 38% in evaluable patients, and 48% vs 37% in all registered patients, as determined by an intention-to-treat analysis. The differences did not reach statistical significance. Conversely, in agreement with previous findings, we observed a significant difference in response rate in the subgroup of patients with liver metastases, regardless of the extent of hepatic involvement (DOX + LND 68% vs DOX 33%, p=0.03). This observation makes LND an important tool in association with anthracyclines in the treatment of this subgroup of patients.

Adult↗
PubMed1972

Specificity of the break produced by restricting endonuclease R 1 in Simian virus 40 DNA, as revealed by partial denaturation mapping.

Superhelical circular (form I) SV40 DNA was converted to linear molecules by the action of a partially purified restriction enzyme of Resistance Transfer Factor-R(1) of Escherichia coli. The resulting linear DNA molecules are full length, as judged by their sedimentation through alkaline sucrose gradient and by direct observation in an electron microscope. Nicked circular (form II) DNA was found as an intermediate in the conversion of form I DNA to linear DNA. Analysis of partial denaturation maps obtained by alkaline denaturation of the unitlength linear molecules showed that the break in SV40 DNA occurred at a specific site on the DNA.

Centrifugation, Density Gradient↗
PubMed1997

Altered Golgi localization of core 2 beta-1,6-N-acetylglucosaminyltransferase leads to decreased synthesis of branched O-glycans.

Mucin type O-glycans with core 2 branches are distinct from nonbranched O-glycans, and the amount of core 2 branched O-glycans changes dramatically during T cell differentiation. This oligosaccharide is synthesized only when core 2 beta-1, 6-N-acetylglucosaminyltransferase (C2GnT) is present, and the expression of this glycosyltransferase is highly regulated. To understand how O-glycan synthesis is regulated by the orderly appearance of glycosyltransferases that form core 2 branched O-glycans, the subcellular localization of C2GnT was determined by using antibodies generated that are specific to C2GnT. The studies using confocal light microscopy demonstrated that C2GnT was localized mainly in cis to medial-cisternae of the Golgi. We then converted C2GnT to a trans-Golgi enzyme by replacing its Golgi retention signal with that of alpha-2,6-sialyltransferase, which resides in trans-Golgi. Chinese hamster ovary cells expressing wild type C2GnT and the chimeric C2GnT were then subjected to oligosaccharide analysis. The results obtained clearly indicate that the conversion of C2GnT into a trans-Golgi enzyme resulted in a substantial decrease of core 2 branched oligosaccharides. These results, taken together, strongly suggest that the predominance of core 2 branched oligosaccharides in those cells expressing C2GnT is due to the fact that C2GnT is located earlier in the Golgi than alpha-2,3-sialyltransferase that competes with C2GnT for the common substrate. Furthermore, alteration of Golgi localization renders the chimeric C2GnT much less efficient in synthesizing core 2 branched oligosaccharides, indicating the critical role of orderly subcellular localization of glycosyltransferases.

Animals↗