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Biomedical subjects

G C Tu

Publications and source records attributed to G C Tu.

At least 19 recordsLinked to original sources
PubMed2002

Gene and antisense delivery in alcoholism research.

This article represents the proceedings of a symposium at the 2001 annual meeting of the Research Society on Alcoholism in Montreal, Canada. Drs. Yedy Israel and Fulton Crews were organizers and co-chairpersons. The presentations were (1) Introduction to the symposium, by Yedy Israel; (2) Gene delivery to the brain, by Fulton T. Crews; (3) Gene therapy in alcoholic liver injury, by Ronald Thurman; and (4) Antisense oligonucleotides and antisense-gene delivery, by Yedy Israel.

Alcoholism↗
PubMed2001

Detection of usual and atypical aldehyde dehydrogenase alleles by mismatch amplification mutation assay.

The genotypes of liver mitochondrial high-affinity aldehyde dehydrogenase-2 (ALDH2) are strongly associated with the drinking behavior and the alcohol liver diseases, since the individuals with atypical ALDH2(2) allele have higher levels of acetaldehyde in their plasma. The atypical ALDH2(2) allele has a nucleotide base transition (G-->A) in its exon 12. Based on this point mutation, we developed a rapid, reliable and inexpensive method, mismatch amplification mutation assay (MAMA), for the determination of human ALDH2 usual and atypical alleles. Two pairs of primers were designed for the amplification of the usual ALDH2(1) allele and the atypical ALDH2(2) allele, respectively. If the sample for the detection was heterozygous, it could be amplified by both of the primers. The product of polymerase chain reaction (PCR) of ALDH2 exon 12 could be easily screened by electrophoresis on a 2% agarose gel. The results of the MAMA method were further confirmed by sequencing. In the total of fifty samples from unrelated healthy Chinese Han people from Wuhan, China, the frequency of atypical ALDH2(2) allele was found to be 12%.

Aldehyde Dehydrogenase↗
PubMed2001

In vivo delivery of antisense oligonucleotides in pH-sensitive liposomes inhibits lipopolysaccharide-induced production of tumor necrosis factor-alpha in rats.

Kupffer cells play an important role in the pathogenesis of liver diseases. During endotoxemia and alcohol-induced liver disease, tissue injury is preceded by an excessive release of cytokines by these macrophages. Tumor necrosis factor-alpha (TNF-alpha) is one of the key cytokines associated with liver injury. Pre-exposure of animals to TNF-alpha antibodies has been shown to prevent macrophage-mediated liver injury in experimental animals. In this article, we describe a method to encapsulate in pH-sensitive liposomes and to deliver an antisense phosphorothioate oligonucleotide (TJU-2755) against TNF-alpha. We describe the efficacy of this formulation in inhibiting endotoxin-mediated production of TNF-alpha. The liposomes prepared were stable for over 4 weeks at pH 7.4, but readily released their contents when exposed to an acidic environment below pH 6, similar to the pH that exists in early endosomes. Male Sprague-Dawley rats were administered (i.v.) liposome-encapsulated TJU-2755 (1-2 mg/kg body wt.). Empty liposomes served as controls. Forty-eight hours postinjection, the animals were administered a single dose of lipopolysaccharide (50 microg/kg body wt.) and were sacrificed 90 min later. The TNF-alpha produced by excised liver incubated ex vivo and the levels of plasma TNF-alpha were determined. After a single administration of liposome-encapsulated antisense TJU-2755, a 30% reduction in TNF-alpha produced by liver slices was observed. Two daily doses of the antisense oligonucleotide inhibited TNF-alpha production by 50%. This was associated with a 65 to 70% reduction in plasma levels of TNF-alpha, compared with controls. These results indicate that oligonucleotide TJU-2755 encapsulated in pH-sensitive liposomes can be used to effectively reduce endotoxin-mediated production of TNF-alpha in macrophages in vivo and thus may be of value in attenuating or preventing macrophage-mediated liver injury.

Animals↗
PubMed2001

GTI-2501. Lorus Therapeutics.

Lorus Therapeutics (formerly GeneSense Technologies) is developing GTI-2501, directed against the R1 component of ribonucleotide reductase, for the potential treatment of cancer [348928]. In November 2000, the company filed an IND with the FDA in preparation to begin a phase I trial in the US in the first quarter of 2001 [389975], [396537], [397960]. In March 2001, the FDA approved the IND application [402350], and in June 2001, a phase I, dose-escalating trial in patients with lymphomas or solid tumors which have not responded to standard therapy commenced in the US [412225]. The company expected the phase I trial of GTI-2501 to be completed within 12 to 15 months [420582]. In November 1999, the company reported that in preclinical trials, GTI-2501 was an effective anticancer agent when tested in standard mouse models bearing a variety of different human cancer lines including tumor cells derived from lung, breast, colon, kidney, ovary pancreas and skin cancers [348928]. Lorus received a patent from the US PTO in January 2000, covering the design and use of unique anitisense anticancer drugs, including GTI-2501 and GTI-2040 [353538]. In June 2000, the USPTO allowed a patent to specifically protect GTI-2501 [370810].

Animals↗
PubMed2000

Penicillopepsin-JT2, a recombinant enzyme from Penicillium janthinellum and the contribution of a hydrogen bond in subsite S3 to k(cat).

The nucleotide sequence of the gene (pepA) of a zymogen of an aspartic proteinase from Penicillium janthinellum with a 71% identity in the deduced amino acid sequence to penicillopepsin (which we propose to call penicillopepsin-JT1) has been determined. The gene consists of 60 codons for a putative leader sequence of 20 amino acid residues, a sequence of about 150 nucleotides that probably codes for an activation peptide and a sequence with two introns that codes for the active aspartic proteinase. This gene, inserted into the expression vector pGPT-pyrG1, was expressed in an aspartic proteinase-free strain of Aspergillus niger var. awamori in high yield as a glycosylated form of the active enzyme that we call penicillopepsin-JT2. After removal of the carbohydrate component with endoglycosidase H, its relative molecular mass is between 33,700 and 34,000. Its kinetic properties, especially the rate-enhancing effects of the presence of alanine residues in positions P3 and P2' of substrates, are similar to those of penicillopepsin-JT1, endothiapepsin, rhizopuspepsin, and pig pepsin. Earlier findings suggested that this rate-enhancing effect was due to a hydrogen bond between the -NH- of P3 and the hydrogen bond accepting oxygen of the side chain of the fourth amino acid residue C-terminal to Asp215. Thr219 of penicillopepsin-JT2 was mutated to Ser, Val, Gly, and Ala. Thr219Ser showed an increase in k(cat) when a P3 residue was present in the substrate, which was similar to that of the wild-type, whereas the mutants Thr219Val, Thr219Gly, and Thr219Ala showed no significant increase when a P3 residue was added. The results show that the putative hydrogen bond alone is responsible for the increase. We propose that by locking the -NH- of P3 to the enzyme, the scissile peptide bond between P1 and P1' becomes distorted toward a tetrahedral conformation and becomes more susceptible to nucleophilic attack by the catalytic apparatus without the need of a conformational change in the enzyme.

Amino Acid Sequence↗
PubMed1998

Tetranucleotide GGGA motif in primary RNA transcripts. Novel target site for antisense design.

Selecting effective antisense target sites on a given mRNA molecule constitutes a major problem in antisense therapeutics. By trial-and-error, only 1 in 18 (6%) of antisense oligonucleotides designed to target the primary RNA transcript of tumor necrosis factor-alpha (TNF-alpha) strongly inhibited TNF-alpha synthesis. Subsequent studies showed that the area in RNA targeted by antisense oligonucleotides could be moved effectively 10-15 bases in either direction from the original area. We observed that only molecules that incorporated a tetranucleotide motif TCCC (complementary to GGGA on RNA) yielded potent antisense oligonucleotides against TNF-alpha. A comprehensive literature survey showed that this motif is unwittingly present in 48% of the most potent antisense oligonucleotides reported in the literature. This finding was prospectively used to predict the sequences of additional antisense oligonucleotides for the rat TNF-alpha primary RNA transcript. Over 50% of antisense constructs (13 of 22) containing the TCCC motif were found to effectively inhibit TNF-alpha synthesis. Marked reductions in mRNA were also observed. This motif was found to be most effective when targeting introns in the primary RNA transcript, suggesting a nuclear localization for the antisense action. Predicting target sites based on the presence of this motif in primary RNA transcripts should be of value in the development on new antisense pharmacotherapy.

Animals↗
PubMed1995

Inhibition of gene expression by triple helix formation in hepatoma cells.

The aim of this study was to selectively inhibit human mitochondrial aldehyde dehydrogenase (ALDH2) gene expression by triple helix assembly. Eight 21-mer oligodeoxyribonucleotides were designed to bind to two purine-rich sequences in the 5'-flanking region of the human ALDH2 gene. Gel mobility shift assays showed that triplex formation is sequence-specific for the target duplex and the third strand oligonucleotide. In the presence of Mg2+, but absence of K+, triplex-forming oligonucleotides bind to their target sites with apparent dissociation constants (Kd) in the 10(-7) to 10(-9) M range. Potassium cation virtually suppressed the triplex formation of G-C-rich duplex DNA with natural oligonucleotides, but did not prevent triplex formation with phosphorothioate-modified oligonucleotides. Phosphorothioate-modified oligonucleotides were delivered into human hepatoma Hep G2 cells by cationic liposomes. The reduction in ALDH2 mRNA levels in the cells was determined by the competitive reverse transcription-polymerase chain reaction. One of the phosphorothioate-modified oligonucleotides designed to forma an antiparallel triplex with a target in the 5'-flanking region of human ALDH2 gene (-105 to -125 from the translation initiation codon ATG) reduced by 80-90% the ALDH2 mRNA levels without affecting albumin mRNA levels. Data suggest that triple-helix formation may provide a means to selectively inhibit hepatic ALDH2 gene expression for therapeutic use.

Aldehyde Dehydrogenase↗
PubMed1995

Alcohol consumption by orientals in North America is predicted largely by a single gene.

Orientals consume significantly less alcohol, and show a lower prevalence of alcohol abuse and dependence, than Caucasians. Sociological theories propose that this difference is due mainly to cultural factors. Physiological theories have suggested that the flushing reaction experienced by some Orientals serves as a deterrent to ethanol consumption. The flushing reaction is observed mainly in individuals who possess a mutation in the high-affinity aldehyde dehydrogenase (ALDH2) which renders the enzyme inactive. However, the tendency to flush correlates poorly with alcohol consumption, thus casting doubt on the physiological interpretations. The present study investigates the influence of the ALDH2 allele and of acculturation in North America on alcohol consumption by Orientals born in Canada or the United States. Oriental males carrying the inactive ALDH2(-) allele drink two-thirds less alcohol (6.1 +/- 1.5 vs. 18.2 +/- 2.8 drinks/4 weeks; p < 0.001), show one-third the prevalence of binge drinking (15.2 vs. 42.2%; p < 0.01), and are three times more likely to be abstainers (39.4 vs. 13.3%; p < 0.01) than Oriental ALDH2(+) males carrying the gene for the active enzyme. There were no significant differences in binge drinking or abstinence rates between ALDH2(+) Orientals and Caucasian males. Acculturation in North American society accounted for only 7-11% of the variance in overall consumption (p < 0.02). It is concluded that a single mutation in the high-affinity aldehyde dehydrogenase (ALDH2) gene predicts two-thirds of the alcohol consumption and excessive alcohol use by Oriental males born in North America.

Acculturation↗
PubMed1993

A new approach for the rapid detection of common and atypical aldehyde dehydrogenase alleles.

A strong protection against the development of alcoholism is exerted by a point mutation in the gene coding for low Km aldehyde dehydrogenase (ALDH), i.e. ALDH2. We report a non-radioactive method for determining the common and atypical human mitochondrial aldehyde dehydrogenase (ALDH2) genotypes. This method is based on the fact that the base change (G-->A) in Exon 12 of the ALDH2 gene abolishes an Eco57 I restriction site (CTGAAG-->CTAAAG). A GC-clamp attached oligonucleotide was designed to yield a 176 base pair product by the polymerase chain reaction. After amplification, the resulting fragment containing the normal nucleotide sequence is cut by Eco57 I into two segments (131 base pairs + 45 base pairs) while the fragment containing the mutated sequence remains intact (176 base pairs). These are visualized by staining with ethidium bromide on agarose gels without blotting, hybridization or autoradiography.

Alcoholism↗
PubMed1992

Characteristics of a new urine, serum, and saliva alcohol reagent strip.

We have tested an ethanol reagent strip developed at the Addiction Research Foundation of Ontario. Alcohol dehydrogenase and nicotinamide adenine dinucleotide, in the presence of pyrazole, react with ethanol to yield acetaldehyde plus reduced nicotinamide adenine dinucleotide. The latter reduces iodonitrotetrazolium chloride in the presence of diaphorase, generating an intense red color. The rate of color development is proportional to the concentration of ethanol. Color is compared at a specific time against a calibrated color scale ranging from green (negative) to red, representing alcohol concentrations of 0, 25, 50, 100, 200, and 400 mg/dl (0-0.4%; 0-87 mmol/liter). We were able to interpolate the color observed between the calibrated blocks. When tested on urine, serum/plasma, and saliva, ethanol concentration determined by the reagent strip correlates well with ethanol concentration as determined by gas chromatography or by automated enzymatic analysis (r = 0.92-0.98, p less than 0.001; slope 0.83-1.16). The reagent strip was shown to be used appropriately by nonexperienced individuals following a 1-min explanation (reagent strip values, r = 0.92; p less than 0.001, slope = 0.97, versus gas chromatography). The reagent strip does not react with methanol (wood alcohol), isopropanol (rubbing alcohol), and ethylene glycol (antifreeze) often found in accidental poisonings. In 379 clinical samples obtained without exclusion criteria from 12 hospital emergency rooms and a liver clinic, the sensitivity of the reagent strip in detecting ethanol was 98%. Specificity was 99%. The reagent strip was found to have virtually unlimited stability under refrigeration (4 degrees C) and to be stable for 3 to 4 months at room temperature (22-23 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Drinking↗
PubMed1990

A ligand-induced, temperature-dependent conformational Change in penicillopepsin. Evidence from nonlinear Arrhenius plots and from circular dichroism studies.

The effect of temperature on the rate constants of hydrolysis of various substrates by penicillopepsin is dependent on the length of the substrate. For the series Ac-(Ala)m-Lys-Nph-(Ala)n-amide (where Ac- is acetyl- and Nph- is p-nitrophenylalanyl-), where m and n = 0-2, substrates lacking both P'2 and P3 residues give linear Arrhenius plots with an energy of activation of about 55 kJ.mol-1. The Arrhenius plots of substrates in which an alanine residue occupies P'2 show a sharp break at an average transition temperature of 10.5 degrees C. The activation energies are approximately 90 kJ.mol-1 below and approximately 54 kJ.mol-1 above the transition temperature, respectively. For substrates in which P3 is occupied, the average transition temperature is 14.2 degrees C. In this case, the activation energies are 66 kJ.mol-1 below and from 26 to 39 kJ.mol-1 above the transition point. The most probable explanation of these phenomena is that substrate interaction at subsites S3 and/or S'2 of the enzyme induces a temperature-dependent conformational change. Physical evidence for this comes from the observation that the temperature dependence of a CD absorption band at 242 nm of a penicillopepsin-pepstatin complex shows a sharp break that corresponds to those observed in the Arrhenius plots of substrates with alanine at P'2 and P3, whereas the same CD band in the free enzyme is linearly dependent on temperature.

Amino Acid Sequence↗
PubMed1989

Presence of cytosolic aldehyde dehydrogenase isozymes in adult and fetal rat liver.

A colony of Wistar-strain rats bred at Purdue University was composed of animals with two different isozyme patterns of liver cytosolic aldehyde dehydrogenase (EC 1.2.1.3, ALDH) as determined by isoelectric focusing. One cytosolic isozyme pattern had a major activity band with a pI = 5.8 and a minor activity band at pI = 6.2. The other pattern contained three major isozymes with pI values of 5.3, 5.4 and 5.6 along with the pI 6.2 isozyme and a trace of the 5.8 one. The 5.8 and 6.2 isozymes were recognized by antibodies produced against horse and beef liver cytosolic ALDH, whereas the set of three (5.3-5.6) were not. The cytosolic isozymes were inhibited by low levels of disulfiram and had Km values for acetaldehyde in the 100 microM range, properties typical for cytosolic ALDHs. All animals contained the same isozymes of liver mitochondrial ALDH. These were a major activity with a pI = 5.2 and minor activities associated with isozymes of pI = 6.4 and 6.6. These isozymes were recognized by antibodies produced against pure horse and beef liver mitochondrial ALDHs. Both cytosolic and mitochondrial ALDHs were found in fetal liver as early as day 15 of gestation. The total activity for mitochondrial ALDH increased between day 15 and day 21 whereas that for cytosolic ALDHs remained relatively constant during development. It appeared that both cytosolic and mitochondrial ALDH were present by at least the third trimester and could afford the fetus some protection against the toxic action of endogenous or exogenous aldehydes.

Acetaldehyde↗
PubMed1988

Identification of the cysteine residue in the active site of horse liver mitochondrial aldehyde dehydrogenase.

Aldehyde dehydrogenase catalyzes the oxidation of aldehydes to acids through the formation of a covalent intermediate. It has been postulated that a cysteine residue could be acting as the active site nucleophilic group. Although N-ethylmaleimide was found to react with many cysteines it was possible by doing the reaction in the presence of chloral hydrate, a substrate analog which functions as a competitive inhibitor, to label cysteine at position 49 in the horse liver mitochondrial enzyme. The dehydrogenase activity was lost as the residue was modified, consistent with the possibility that the residue was an integral component of the active site of the enzyme. Cysteines at positions 162 and 369 also could be modified. It is suggested that cysteine 162 may function as part of a site capable of hydrolyzing nitrophenyl acetate. Details of the second site will appear in the accompanying paper (Tu, G. C., and Weiner, H. (1988) J. Biol. Chem. 263, 1218-1222). It appeared that the substrate-binding domain was in the N-terminal portion of the enzyme while the coenzyme binding domain was in the C-terminal portion. During this investigation 133 of the 500 residues of the horse liver enzyme were sequenced. These showed about 95% sequence identity with those of the human enzyme. Inasmuch as both beef and rat liver enzymes also share 95% identity with the human enzyme it can be expected that the results found with the horse liver enzyme can be applicable to all mammalian aldehyde dehydrogenase.

Aldehyde Dehydrogenase↗
PubMed1988

Evidence for two distinct active sites on aldehyde dehydrogenase.

Aldehyde dehydrogenase can catalyze the hydrolysis of esters such as p-nitrophenyl acetate as well as oxidize aldehydes to acids. It has not been proven unequivocally that the two reactions occur at the same active site. In the accompanying paper (Tu, G. C., and Weiner, H. (1988) J. Biol. Chem. 263, 1212-1217) evidence was presented which showed that cysteine at position 49 was at the active site for the dehydrogenase reaction. Evidence also was presented which showed that cysteine located at position 162 was susceptible to modification by N-ethylmaleimide. It was shown here that the two activities of the enzyme can be differently protected from inactivation by substrate analogs. Furthermore, aldehydes were found to be poor inhibitors against the esterase reaction while ester was a good inhibitor against the dehydrogenase reaction. In addition, it was possible to modify cysteine 49 with N-ethylmaleimide but not find inhibition of the esterase reactivity until cysteine 162 was modified. It appears that horse liver aldehyde dehydrogenase has two separate active sites per subunit. The data fit a model where ester can be hydrolyzed at both sites but that aldehyde oxidation occurred only at position 49.

Aldehyde Dehydrogenase↗
PubMed1988

Purification and characterization of beef and pig liver aldehyde dehydrogenases.

Beef liver cytosolic, mitochondrial, and pig liver mitochondrial aldehyde dehydrogenases (ALDH) had been purified to homogeneity. The two mitochondrial enzymes as with other mammalian mitochondrial enzymes had properties very similar to that of the corresponding human enzyme. These include immunological as well as basic kinetic properties such as low Km for aldehyde, activation by Mg2+ ions, and lack of inhibition by disulfiram. A major difference between these two enzymes and the human mitochondrial enzyme was that they contained an N-terminal-blocked amino acid. Cytosolic ALDHs from human and horse liver have been shown to possess an N-acetyl serine as the N-terminal residue; beef cytosolic ALDH was also found to be blocked. Tissue preparations and subcellular fractions from beef or pig liver could be used to study acetaldehyde oxidation. This is the subject of the accompanying paper (Cao Q-N, Tu G-C, Weiner H, Alcohol Clin Exp Res 12:xxx-xxx, 1988).

Aldehyde Dehydrogenase↗
PubMed1988

Mitochondria as the primary site of acetaldehyde metabolism in beef and pig liver slices.

Aldehyde dehydrogenase (ALDH) is the major enzyme involved in the oxidation of acetaldehyde. It has been shown that the liver enzyme is located in both cytosol and mitochondria. It has not been established where the subcellular oxidation of acetaldehyde occurs in species other than rat. Using slices isolated from beef and pig livers and selectively inhibiting the mitochondria enzyme with cyanamide or the cytosolic enzyme with disulfiram, it was possible to address this question. It was found that with both beef and pig liver slices 60% of the oxidation was catalyzed by the mitochondrial ALDH and 20% by the higher Km cytosolic enzyme. The remainder of the metabolism was the result of non-ALDH involvement. Furthermore, any decrease in the level of the low Km mitochondrial aldehyde dehydrogenase activity resulted in a decreased rate of acetaldehyde oxidation showing that its activity governed the rate of acetaldehyde oxidation. These were the same conclusions previously reached using rat liver tissue slices. Thus, it appears that for all mammalian tissue, mitochondria is the primary location of acetaldehyde oxidation.

Acetaldehyde↗
PubMed1987

Use of polyclonal antibodies to study properties of mammalian aldehyde dehydrogenases.

Polyclonal antibodies prepared against horse liver mitochondrial aldehyde dehydrogenase (ALDH) crossreact with both cytoplasmic and mitochondrial ALDH. Antibodies prepared against the cytosolic enzymes were more specific in that they only precipitated enzymes derived from cytosol. These polyclonal antibodies were used to determine the subcellular localization of aldehyde dehydrogenase isozymes isolated from other species and other tissues. A sequential precipitation technique was developed which allowed for the determination if samples contain both antigens. The polyclonal antibodies contained antibodies which recognize peptide fragments of mitochondrial ALDH. These antibodies can be used to probe for structural homology between similar regions in different forms of ADLH.

Aldehyde Dehydrogenase↗