Cloning and characterization of genes encoding four additional human aldehyde dehydrogenase isozymes.
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Aldehyde Dehydrogenase↗
Biomedical subjects
W C Chang
Publications and source records attributed to W C Chang.
Evaluating the dose to the contralateral breast when using a dynamic wedge versus a regular wedge.
The incidence of secondary cancers in the contralateral breast after primary breast irradiation is several times higher than the incidence of first time breast cancer. Studies have shown that the scatter radiation to the contralateral breast may play a large part in the induction of secondary breast cancers. Factors that may contribute to the contralateral breast dose may include the use of blocks, the orientation of the field, and wedges. Reports have shown that the use of regular wedges, particularly for the medial tangential field, gives a significantly higher dose to the contralateral breast compared to an open field. This paper compares the peripheral dose outside the field using a regular wedge, a dynamic wedge, and an open field technique. The data collected consisted of measurements taken with patients, solid water and a Rando phantom using a Varian 2300CD linear accelerator. Ion chambers, thermoluminescent dosimeters (TLD), diodes, and films were the primary means for collecting the data. The measurements show that the peripheral dose outside the field using a dynamic wedge is close to that of open fields, and significantly lower than that of regular wedges. This information indicates that when using a medial wedge, a dynamic wedge should be used.
Breast↗
Rasch analysis for outcomes measures: some methodological considerations.
OBJECTIVE: To assess the four approaches to applying Rasch analysis to ordinal, repeated measures: (1) to perform separate Rasch analyses; (2) to perform a single analysis by treating the subjects measured on different occasions as distinct ones; (3) to perform a single analysis by treating items used on different occasions as distinct ones; and (4) to construct a 3-facet model by adding occasions as the third facet. DESIGN: Various Rasch and 3-facet models applied to retrospective, cohort data. SETTING: A stroke program at a rehabilitation hospital. PATIENTS: 99 of the 107 consecutive patients admitted between March and October, 1990, average age 69 years, 40% of whom were women, with an average stay of 54 days. INTERVENTION: Medical inpatient rehabilitation. MAIN OUTCOME MEASURE: The Functional Independence Measure (FIM), administered within 1 week of admission and discharge, and approximately 6 weeks after discharge. RESULTS: The four approaches resulted in various occasion-specific and generalized measures of item difficulty, patient ability, and occasion severity. Although these measures turned out to be congruent with each other as they should, they generated different sources of information needed for formative and summative evaluation of health care outcomes. All four approaches were found to be useful, although each had its own limitations. A four-step strategy based on these approaches was therefore designed and recommended. CONCLUSION: Rasch analysis of ordinal, repeated measures poses a special challenge to those interested in health care outcomes. A multi-method strategy incorporating various approaches is therefore needed to optimize its use in health care research.
Activities of Daily Living↗
Long-term expression of the biologically active growth hormone in genetically modified fibroblasts after implantation into a hypophysectomized rat.
We employed the hypophysectomized rats as an animal model to explore the feasibility of using genetically engineered fibroblast cells for growth hormone gene therapy. An internal ribosome entry site (IRES)-directed bicistronic retroviral vector, PSN, which contained a porcine growth hormone (pGH) cDNA at the first cistron and a Neo(r) gene at the second cistron was used to infect primary rat embryo fibroblast (REF) cells. The infected cells (5 x 10(6) cells/rat) were injected directly into the peritoneum of syngeneic hypophysectomized rats. We demonstrate that the implanted PSN-infected REF cells could secrete biologically active pGH in vivo, leading to significant growth of the tibia at day 15 and day 57 post-implantation. We also treated the PSN-infected REF cells with collagen to form a tissue-like structure. The skin-like discs were grafted underneath the skin on the back of rats and cells were retrieved at different times. Using two criteria, semiquantitative reverse transcription-polymerase chain reaction on the pGH RNA extracted from the explants and G418 resistance conferred from the explanted cells, we demonstrate that pGH was expressed in the implanted fibroblasts up to 70 days. Despite the fact that the total pGH RNA level was reduced in the explants of long-time post-implantation, which was probably due to the reduction of transduced cells retained in the explants, the specific efficiencies of pGH RNA expression from these explants were maintained as high as the primary PSN-infected REF prior implantation. These results suggest that fibroblast cells are capable of expressing the foreign genes persistently in vivo.
3T3 Cells↗
Dietary fat and fiber alter rat colonic protein kinase C isozyme expression.
To better understand the biochemical mechanisms by which select fats and fibers modulate colonic cell proliferation, we determined the profile of protein kinase C (PKC) isozymes and cell proliferation in rat proximal and distal colonic mucosa following diet manipulation, because enhanced cell proliferation has been correlated with colon cancer incidence. Rats were assigned to one of four diets (each with 15 g fat + 6 g fiber/100 g diet) for 3 wk: fiber-free fish oil (FF), fiber-free corn oil (FC), cellulose + corn oil (CC), or pectin + corn oil (PC). Stead-state levels of colonic mucosal cytosolic and membrane PKC isozymes were determined. In vivo cell proliferation was determined by bromodeoxyuridine incorporation into DNA. In addition, viable exfoliated colonic epithelial cells were isolated from feces using Percoll-bovine serum albumin gradients. We found that 1) proximal and distal colonic mucosa possessed different steady-state levels and relative proportions of PKC isozymes; 2) PKC alpha and delta expression were significantly greater in distal membrane of the PC-fed group compared with the other dietary groups; 3) the number of exfoliated cells per 4-h fecal collection generally was proportional to the diet-induced changes in cell proliferation (PC > FC > CC > FF). These data demonstrate that dietary treatment altered colonic PKC isozyme expression, with animals fed the fiber-containing diets generally expressing higher steady-state levels of PKC alpha and delta.
Animals↗
Pharmacokinetic properties of recombinant porcine growth hormone in pigs.
The plasma pharmacokinetic profile of recombinant porcine growth hormone (rpGH) was studied in six healthy gilts after a rapid single i.v. injection of 250 micrograms/kg rpGH. The linear pharmacokinetic properties of rpGH were investigated by giving four different doses of the hormone at intervals of 2 days. The plasma rpGH concentration was determined by radioimmunoassay with a lowest detection limit of 50 pg/ml. Plasma levels of rpGH were fitted to a three-compartment open model; a triexponential decrease in plasma rpGH with a rather long mean terminal half-life of 40.22 +/- 5.623 min was obtained in these pigs. Linear pharmacokinetic properties of rpGH were confirmed in the additional pig that received doses between 25 and 250 micrograms/kg rpGH. The small volume of distribution, 0.029 +/- 0.003 l/kg, indicated that most of the injected rpGH was confined to the blood and probably lymph. The total plasma clearance of rpGH was 31.80 +/- 2.802 ml/h x kg. The results not only contribute significantly to our knowledge about rpGH pharmacokinetics, but also to future studies for the rational design of optimal dosage regimens and the development of a controlled-release device for rpGH.
Animals↗
Characterization of gamma-crystallin from a catfish: structural characterization of one major isoform with high methionine by cDNA sequencing.
gamma-Crystallin is the major and most abundant lens protein present in the eye lens of most teleostean fishes. To facilitate structural characterization of gamma-crystallins isolated from the lens of the catfishes (Clarias fuscus), a cDNA mixture was synthesized from the poly(A)+mRNA isolated from fresh eye lenses, and amplification by polymerase chain reaction (PCR) was adopted to obtain cDNAs encoding various gamma-crystallins. Plasmids of transformed E. coli strain JM109 containing amplified gamma-crystallin cDNAs were purified and prepared for nucleotide sequencing by the dideoxynucleotide chain-termination method. Sequencing more than five clones containing DNA inserts of 0.52 kb revealed the presence of one major isoform with a complete reading frame of 534 base pairs, covering a gamma-crystallin (gamma M1) with a deduced protein sequence of 177 amino acids excluding the initiating methionine. It was of interest to find that this crystallin of pI 9.1 contains a high-methionine content of 15.3% in contrast to those gamma-crystallins of low-methionine content from most mammalian lenses. Sequence comparisons of catfish gamma M1-crystallin with those published sequences of gamma-crystallins from carp, bovine and mouse lenses indicate that there is approx. an 82% sequence homology between the catfish and the carp species of piscine class whereas only 51-58% homology is found between mammals and the catfish. Moreover the differences in the hydropathy profiles for these two groups of gamma-crystallins, i.e. one with a high-methionine content from teleostean fishes and the other with a low-methionine content from mammalian species, reflect a distinct variance in the polarity distributions of surface amino acids in these crystallins.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Double pylorus: report of a longitudinal follow-up in two refractory cases with underlying diseases.
Double pylorus is either a congenital abnormality or an acquired complication of peptic ulcer disease. We had followed two patients for 3 and 5 yr, respectively, to observe the processes of formation and the prognosis of double pylorus. Initially, duodenal ulcer was found in one patient with diabetes mellitus and chronic renal failure, and gastric ulcer was found in the other with chronic obstructive pulmonary disease. Both developed double pylorus with refractory courses. In spite of intensive medical treatment, both of them had persistent ulcers in the fistulous tract and failed to develop reepithelization. Helicobacter pylori was found in all of the specimens of gastroduodenal biopsies in both cases. Therefore, we believe that the refractory courses of double pylorus may be related to the underlying diseases and/or the presence of H. pylori. Antibacterial treatment of H. pylori or surgical intervention should be considered for patients with this condition.
Aged↗
Wegener's granulomatosis: a case report and literature review.
Wegener's granulomatosis may present with a variety of findings and be difficult to diagnose. We report a case of 27-year-old man presenting with chronic cough and hemoptysis who was diagnosed by open lung biopsy to have Wegener's granulomatosis. Based on this case, we reviewed the clinical features, pathophysiology, diagnosis, treatment, and prognosis of Wegener's granulomatosis.
Adult↗
Cloning of a cDNA encoding human ALDH7, a new member of the aldehyde dehydrogenase family.
Aldehyde dehydrogenases (ALDH; EC 1.2.1.3) are a family of isozymes which have been suggested to play a major role in the detoxification of aldehydes generated by alcohol metabolism and lipid peroxidation. Five non-allelic ALDH genes, encoding the ALDH1, 2, 3, 5 and 6 isozymes, have previously been identified and cloned in our laboratory. In this paper, we report the cloning and sequencing of a cDNA encoding a new human ALDH (ALDH7). Degenerate oligodeoxyribonucleotides derived from conserved regions of known ALDH cDNAs amplified a 408-bp product from human kidney total RNA by the reverse transcription-polymerase chain reaction (RT-PCR) procedures [Hsu et al., J. Biol. Chem. 266 (1992) 3030-3037]. This PCR product was subcloned, selected and used as a probe to screen a human kidney cDNA library. The full-length human kidney cDNA (ALDH7) is 2791 bp in length and contains an open reading frame encoding 468 amino acids (aa). The deduced sequence of ALDH7 is longer than that of the human stomach ALDH3 by 15 aa at the C terminus. The degree of identity between the two isozymes is 52% with a positional alignment of 453 aa. Northern blot analysis demonstrated that lung is another major tissue expressing ALDH7.
Aldehyde Dehydrogenase↗
Molecular cloning, genomic organization, and chromosomal localization of an additional human aldehyde dehydrogenase gene, ALDH6.
Aldehyde dehydrogenase isozymes have been suggested to play a major role in the detoxification of aldehydes generated by alcohol metabolism and lipid peroxidation. We previously cloned and characterized four human nonallelic ALDH genes encoding different isozymes. The existence of an unique ALDH isozyme in human saliva and its polymorphism has been demonstrated previously. In this paper, we describe the cloning, characterization, and chromosomal mapping of an aldehyde dehydrogenase gene (ALDH6) expressed in the human salivary gland. The cloned ALDH6 cDNA is 3457 bp in length and contains an open reading frame encoding 512 amino acid residues. The deduced amino acid sequence showed that ALDH6 is larger than the human liver ALDH1 by 11 amino acid residues at the N-terminal, and the degree of identity between the two isozymes is 70% with an alignment of 500 amino acid residues. The human ALDH6 gene spans about 37 kb and consists of 13 exons. The putative TATA and CCAAT boxes and Sp1 binding sites are found in the 5' upstream region of the gene. Northern blot analysis demonstrated that the ALDH6 gene is expressed at low levels in many tissues and at higher levels in salivary gland, stomach, and kidney. The ALDH6 gene was assigned to chromosome 15q26 using fluorescence in situ hybridization.
Aldehyde Dehydrogenase↗
Dietary modulation of rat colonic cAMP-dependent protein kinase activity.
Malignant transformation of cells is associated with enhanced proliferation and alterations in cAMP-dependent protein kinase (PKA) activity. To investigate the role of PKA in normal colonic cell proliferation, PKA was characterized in rat colonic mucosa. In addition, rats were fed diets containing different fats (corn oil, fish oil) and fibers (pectin, cellulose, fiber free) to elicit varying levels of colonic cell proliferation in order to study this signaling system under normal physiologic conditions. Overall, PKA activities were higher in cytosolic compared to membrane fractions. PKA type II (PKA II) isozyme contributed 89 +/- 1% and 96 +/- 1% of total PKA activity in cytosolic and membrane fractions, respectively. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of mRNA for both the alpha and beta isoforms of the regulatory subunits of PKA II. PKA activities were 21-33% higher in distal membrane and total distal fractions in rats fed a cellulose/corn oil diet compared to animals consuming the other fiber/fat diets. These effects were seen only in the distal colon, where the number of cells per crypt column was elevated only in animals fed the cellulose/corn oil diet relative to other diets. Diet-induced mitogenic responses did not involve significant changes in the relative activity of PKA I and II isozymes. These data demonstrate that dietary effects on PKA activity in the distal colon may be related to changes in cell differentiation as indicated by the number of cells per crypt column.
Animals↗
Diacylglycerol formation induced by human plasmin in cultured endothelial cells.
Treatment of cultured bovine carotid artery endothelial cells with 10(-7) M plasmin increased the cellular diacylglycerol which was determined by the formation of [3H]palmitate-labeled diacylglycerol and diacylglycerol mass. Upon the stimulation with plasmin, a gradual increase in diacylglycerol formation was observed within 20 min then slightly declined. The maximal effect during the 1-h time course study was 45 and 55% increases in [3H]palmitate-labeled diacylglycerol and diacylglycerol mass, respectively, at 20 min after plasmin treatment. Formation of phosphatidylethanol was also studied in [3H]palmitate-prelabeled cells in the presence of ethanol. Treatment with plasmin for 20 min induced a significant 45% increase in phosphatidylethanol formation. The present results indicate that the plasmin-induced diacylglycerol formation in endothelial cells was at least in part mediated through the phospholipase D activation.
Animals↗
Characterization of gamma-crystallins from a hybrid teleostean fish: multiplicity of isoforms as revealed by cDNA sequence analysis.
gamma-Crystallins were isolated and characterized from the eye lenses of a hybrid species belonging to the teleostean fish. Isoelectric focusing of gamma-crystallin fraction obtained from gel-permeation chromatography revealed that it consists of multiple charge isomers of a protein species with a molecular mass of about 20 kDa. To facilitate the cloning of gamma-crystallin gene, cDNA was constructed from the poly(A)+mRNA isolated from fresh lenses, and amplification by polymerase chain reaction (PCR) was carried out to obtain cDNA encoding multiple gamma-crystallins. Sequencing five of more than 10 positive clones revealed that a multiplicity of isoforms exists in the gamma-crystallin class of teleostean lenses. Comparison of protein sequences encoded by these multiple cDNAs with those published sequences of gamma-crystallins from bovine, mouse an carp lenses indicated that there is about 70-80% sequence homology between different species of piscine species whereas only 50-60% is found between mammals and fishes. Structural analysis of these gamma-crystallins with high methionine contents (11-16%) suggests that there are two major subclasses of piscine gamma-crystallins, i.e. gamma M 1 and gamma M 2, existed long before the appearance of mammalian gamma-crystallin with low methionines.
Amino Acid Sequence↗
Sequence analysis and expression of phospholipase A2 from Taiwan cobra.
Polymerase chain reaction (PCR) was employed to amplify cDNAs constructed from the poly(A)+RNA of venom glands in Taiwan cobras to facilitate the cloning and sequencing of phospholipase A2 (PLA2) gene. The PCR product was then subcloned into pUC18 vector and transformed in E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by dideoxynucleotide chain-termination method. Sequencing several clones containing about 0.5 kb DNA inserts constructed a complete and unambiguous full-length reading frame of 468 base pairs covering a precursor for phospholipase A2 with a deduced mature protein sequence of 119 amino acids and a 27 amino-acid segment of signal peptide. The sequenced major PLA2 with pI 4.991 shows a high degree of sequence homology to those PLA2 of the same or closely-related genus. The deduced protein sequence allows us to correct and resolve some discrepancy between the sequences determined by conventional protein sequencing (Toxicon, 19, 141(1981)) and X-ray crystallography (Science, 250, 1560(1990)). Expression of PLA2 in E. coli vector generated a polypeptide which can cross-react with the antiserum against the native and purified PLA2 from the same cobra venom albeit with a much lower activity.
Amino Acid Sequence↗
Public attitudes toward the right to die.
OBJECTIVE: To determine public attitudes toward the right to die, euthanasia and related end-of-life decisions. DESIGN: Mail survey based on telephone numbers randomly selected by computer. SETTING: Edmonton. PARTICIPANTS: Of 1347 computer-generated, randomly selected telephone numbers called between February and June 1992, 902 individuals were reached, and 500 eligible contacts (55%) agreed to fill out the mailed questionnaire based on 12 vignettes involving end-of-life decisions. A total of 356 usable questionnaires (71%) were subsequently returned. MAIN OUTCOME MEASURES: Attitudes toward end-of-life decisions including withdrawal of life support, euthanasia, chronic suffering and the right to die, living wills and family involvement in decision making for incompetent individuals. Comments and demographic data were also solicited. RESULTS: Of the respondents 84% supported a family's right to withdraw life support from a patient in a persistent coma, and 90% supported a mentally competent patient's right to request that life support be withdrawn. Active euthanasia was supported by 65% for only patients experiencing severe pain and terminal illness. There was marked opposition to euthanasia for patients in other circumstances, such as an elderly disabled person who feels he or she is a burden on relatives (opposed by 65%), a patient with chronic depression resistant to treatment (by 75%) or an elderly person no longer satisfied with life and who has various minor physical ailments (by 83%). In all, 63% of the respondents felt that legalization of euthanasia for terminal illnesses would lead to euthanasia for many other, unsupported reasons, and 34% supported legislation to prohibit euthanasia in all situations. CONCLUSIONS: Public support for the right to die varies depending on the circumstances of the patient. The single most significant factor determining attitudes was the level of religious activity. The family's wishes were an important factor in end-of-life decisions for patients unable to communicate their desires. Both the general public and physicians should be primary participants in determining legislation regarding the right to die.
Adolescent↗
Regional differences in prostaglandin production rates among porcine intrathoracic vessels.
To investigate the regional variability in intrathoracic vascular prostaglandin (PG) synthesis, we obtained vessel segments from porcine coronary artery (COA), thoracic aorta (AT), common carotid artery (CRA), pulmonary artery (PA), pulmonary vein (PV), and inferior vena cava (IVC). Vascular production rates of 6-keto-PGF1 alpha (an indicator for PGI2), PGF2 alpha, and PGE2 were measured both in unstimulated state and in arachidonic acid-stimulated state using immunosorbent assays. Our results indicated that PGI2 production rate in all vessel segments decayed with time after vessel dissection. In all vessel segments tested under unstimulated conditions, PGI2 production rates were about one order of magnitude higher than PGF2 alpha and PGE2 production rates of the same specimens. Results from unstimulated, 1.5 hr pre-incubated specimens indicated that i) PGI2 production rates in COA, AT, and PV were greater than those in CRA, PA, and IVC; ii) PGF2 alpha production rates from the same specimens were higher in PV than in AT, CRA, and IVC, while these in PA were higher than in IVC; and iii) PGE2 production rates from the same specimens were not significantly different from one another. Arachidonic acid added at about 1.5 hr after vessel harvest stimulated the PGI2 and PGF2 alpha synthesis rates by 3 to 15 folds. However, this arachidonic acid treatment caused 70 to 300-fold increases in PGE2 production rates, reaching levels comparable to PGI2. All three prostanoid production rates under stimulated conditions were also variable among different intrathoracic vessels. Although either physiological gas concentrations or local hemodynamic conditions alone can partially explain our results, which physiological parameter(s) actually causes these regional differences remains to be verified.
Animals↗
Stress and adenosine: I. Effect of methylxanthine and amphetamine stimulants on learned helplessness in rats.
In 3 experiments, the authors examined the effect of methylxanthine and amphetamine stimulants on deficits in shuttle-escape responding produced by earlier exposure to inescapable electric shock in rats. Caffeine completely reversed escape deficits in inescapably shocked rats when injected just before shuttle-escape testing but failed to prevent a test deficit when injected before shock pretreatment. Dose-response curves indicated that, whereas caffeine and theophylline were equally effective at reversing escape deficits, amphetamine not only failed to improve performance in preshocked rats but retarded escape in restrained (no-shock) controls. This amphetamine-induced deficit was reversed by cotreatment with caffeine. These data are discussed in terms of the role of adenosine receptor activation in helplessness and conservation-withdrawal.
Amphetamine↗