Influence of multiplicity of infection and protein stability on retroviral vector-mediated gene expression in hematopoietic cells.

Using retroviral vectors encoding enhanced green fluorescent protein (EGFP), we addressed to what extent expression of retroviral transgenes in hematopoietic cells depends on the multiplicity of infection (MOI) and on the half-life of the encoded protein. We show that an elevation of the MOI not only elevates the frequency of transduced cells, but also increases transgene expression levels and reduces interanimal variability in vivo (hematopoietic cells of C57BL/6J mice analyzed 13 weeks after transplantation). This suggests that the MOI has to be carefully controlled and should be adapted as desired for clinical studies when evaluating vector performance in preclinical models. The impact of protein stability is demonstrated by comparing vectors expressing EGFP or a destabilized variant with a C-terminal PEST-sequence, d2EGFP. The loss of expression with d2EGFP was more pronounced in terminally differentiated cells of the peripheral blood (>30 fold) than in progenitor cells (five- to 10-fold), indicating a stronger transcription of the retroviral promoter in progenitor cells and a predominant role of protein inheritance over de novo synthesis of transgenic protein in mature blood cells. This analysis reveals an important and differentiation-dependent contribution of protein half-life to the expression of retroviral vectors in hematopoietic cells, establishes d2EGFP as a more accurate reporter for determination of vector transcription, and also suggests that preclinical data obtained under conditions of high transduction rates or with vectors expressing stable reporter proteins require careful interpretation.

Detoxification of cyclophosphamide by human aldehyde dehydrogenase isozymes.

In in vitro studies, no turnover of aldophosphamide and mafosfamide was observed with the tumor-specific aldehyde dehydrogenase 3 isozyme (ALDH3) isolated from human stomach mucosa as well as from lung (A549) and pharynx (UMSCC2) carcinoma cell lines. Only the human liver cytosolic ALDH preparation (ALDH1) showed any significant oxidation of aldophosphamide and mafosfamide.

Distribution of ADH2 and ALDH2 genotypes in different populations.

The distribution of the human liver alcohol dehydrogenase, ADH2, and aldehyde dehydrogenase, ALDH2, genotypes in 21 different populations comprising Mongoloids, Caucasoids, and Negroids was determined by hybridization of the amplified genomic DNA with allele-specific oligonucleotide probes. Whereas the frequency of the ADH1(2) allele was found to be relatively high in the Caucasoids, Mexican Mestizos, Brazilian Indios, Swedish Lapps, Papua New Guineans and Negroids, the frequency of the ADH2(2) gene was considerably higher in the Mongoloids and Australian Aborigines. The atypical ALDH2 gene (ALDH2(2)) was found to be extremely rare in Caucasoids, Negroids, Papua New Guineans, Australian Aborigines and Aurocanians (South Chile). In contrast, this mutant gene was found to be widely prevalent among the Mongoloids. Individuals possessing the abnormal ALDH2 gene show alcohol-related sensitivity responses (e.g. facial flushing), have the tendency not to be habitual drinkers, and apparently suffer less from alcoholism and alcohol-related liver disease.

Effect of acute ethanol drinking on alcohol metabolism in subjects with different ADH and ALDH genotypes.

The effect of different amounts of orally ingested ethanol on plasma alcohol dehydrogenase (ADH) and erythrocyte aldehyde dehydrogenase (ALDH), as well as on the blood ethanol and acetaldehyde levels, was examined in healthy nonalcoholic subjects. The genotypes at ADH2 and ALDH2 locus were identified in enzymatically amplified blood DNA by hybridization with allele-specific oligonucleotides. While the Japanese subject was found to be genotypically heterozygous for both ADH2 and ALDH2, the Caucasian subjects were genotypically homozygous normal for these alleles. A faster ethanol elimination associated with a higher blood acetaldehyde level was observed in the Japanese subject as compared to Caucasian subjects. However, no significant change in ADH and ALDH enzyme activities was detected as the result of acute ethanol intake.

Human liver aldehyde dehydrogenase: subcellular distribution in alcoholics and nonalcoholics.

Activity assay and isoelectric focusing analysis of human biopsy and autopsy liver specimens showed the existence of two major aldehyde dehydrogenases (ALDH I, ALDH II). Subcellular distribution of these isozymes was determined in autopsy livers from alcoholics and nonalcoholics. Nearly 70% of the total ALDH activity was recovered in the cytosol which contained both the major isozymes. Densitometric evaluation of isozyme bands showed that about 65% of the cytosolic enzyme activity was due to ALDH II and the rest due to ALDH I isozyme. Only about 5% of the total ALDH activity was found in the mitochondrial fraction (70% ALDH I and 30% ALDH II). Significantly reduced total and specific ALDH activities were noted in all the subcellular fractions from cirrhotic liver specimens. Apparently, ALDH I isozyme from cytosol and mitochondria is primarily responsible for the oxidation of small amounts of acetaldehyde normally found in the blood of nonalcoholics after drinking moderate amounts of alcohol. However, in alcoholics who exhibit higher blood acetaldehyde concentrations after drinking alcohol, ALDH II isozyme may be of greater physiological significance.

Human placental aldehyde dehydrogenase. Subcellular distribution and properties.

Freshly obtained human term placentae were subjected to subcellular fractionation to study the localization of NAD-dependent aldehyde dehydrogenases. Optimal conditions for the cross-contamination-free subcellular fractionation were standardized as judged by the presence or the absence of appropriate marker enzymes. Two distinct isozymes, aldehyde dehydrogenase I and II, were detected in placental extracts after isoelectric focusing on polyacrylamide gels. Based on a placental wet weight, about 80% of the total aldehyde dehydrogenase activity was found in the cytosolic acid and about 10% in the mitochondrial fraction. The soluble fraction (cytosol) contained predominantly aldehyde dehydrogenase II which has a relatively high Km (9 mmol/l) for acetaldehyde and is strongly inhibited by disulfiram. The results indicate that cytosol is the main site for acetaldehyde oxidation, but the enzyme activity is too slow to prevent the placental passage of normal concentrations of blood acetaldehyde (less than 1 mumol/l) produced by maternal ethanol metabolism.

Plasma alcohol dehydrogenase in normal and alcoholic individuals.

Determination of plasma alcohol dehydrogenase (ADH) in patients with alcohol-related disorders may be a valuable diagnostic and prognostic parameter. By using a sensitive spectrophotometric method we have measured the plasma ADH activity in chronic alcoholics, non-alcoholic psychiatric patients and healthy blood donors from Germany. Serum alanine aminotransferase (GPT) and serum gamma-glutamyltransferase (gamma-GT) values were included for the comparative study to assess the extent of hepatic complications. The results indicate that plasma ADH activity in alcoholics and non-alcoholic psychiatric patients was significantly raised only when higher GPT and gamma-GT values were observed, which may hint at a generalized liver damage. It is suggested that plasma ADH determination alone is not useful as a diagnostic marker of alcoholism but could be of value when measured in combination with other biochemical parameters.

Aldehyde dehydrogenase isozymes in stomach autopsy specimens from Germans and Chinese.

Human stomach aldehyde dehydrogenase (ALDH) isozymes were screened in random autopsy specimens from 66 North German and 33 Chinese individuals. Three ALDH isozymes were identified, which differed in their electrophoretic mobility, affinity to acetaldehyde, propionaldehyde, furfural, and to NAD+ as coenzyme as well as in inhibition by disulfiram, pH optimum and heat stability. While all the German stomach specimens showed ALDH I, ALDH II, and ALDH III isozymes, these isozymes were found in 68, 88, and 99% of the Chinese samples, respectively. The electrophoretic resolution of ALDH III into different activity bands is apparently due to the artefactual formation of secondary isozymes rather than to the existence of genetic variants.

Population genetic studies on aldehyde dehydrogenase isozyme deficiency and alcohol sensitivity.

Population genetic studies on aldehyde dehydrogenase polymorphism using hair-root samples were performed on Europeans, Liberians, Sudanese, Egyptians, Kenyans, Vietnamese, Japanese, Indonesians, Chinese, Thais, and South American Indians. A possible correlation between ALDH I deficiency and sensitivity to alcohol in Oriental populations is discussed.

Physiological role of aldehyde dehydrogenase isozymes.

Our studies confirm that impaired acetaldehyde metabolism via a deficiency in aldehyde dehydrogenase isozyme composition plays an important role in the sensitivity to alcohol and the pathophysiology of alcoholism. Individuals with decreased activity of ALDH having less affinity to acetaldehyde in their liver, may be at a higher risk of acetaldehyde-related organ damage. In the case of Oriental subjects, the lack of ALDH I activity may also result in alcohol aversion. A protection of these individuals against alcoholism is suggested.

A search for the Indianapolis-variant of human alcohol dehydrogenase in liver autposy samples from North Germany and Japan.

Human liver alcohol dehydrogenase (ADH) variants were screened in random autopsy specimens from 53 North German and 34 Japanese individuals. Based on pH-activity profile and electrophoretic pattern, only ADH2 and ADH3 variants were detected. In relatively fresh specimens, an "anodic band" or "pi-ADH" band was also observed. The recently reported new molecular forms collectively called "ADH Indianapolis" (Bosron et al. 1980) could not be demonstrated and therefore may be confined hitherto only to the American black population.