In situ reverse transcriptase-nested polymerase chain reaction to identify intracellular nucleic acids without the necessity of DNAse pretreatment and hybridisation.

In the present study a protocol of in situ reverse transcriptase-nested polymerase chain reaction (in situ RT-nested PCR) was examined based on the following modifications. (i) To exclude false positive signals caused by "DNA repair mechanisms" and "endogenous priming", a two-step PCR was applied after reverse transcription. The first step was performed in the presence of extrinsic primers and unlabeled nucleotides with a maximum of PCR cycles possible without destroying the cell morphology. The second step consisted of only one annealing/elongation reaction, the target sequence marked by addition of digoxigenin-labeled nucleotides and intrinsic primers. (ii) In order to prevent amplifications of genomic DNA nested primer pairs were applied crossing intron sequences. (iii) To minimize the diffusion of PCR products in cells, the extrinsic primers were extended with complementary 5(prime, variant)-tails. This approach results in the generation of high molecular weight concatamers during PCR cycles. By applying this protocol, immunostainings specific for phospholipase A2 of type IIA mRNA were exclusively detectable in the cytoplasm of HepG2 hepatoma cells, which were used as a model system, whereas the nuclei were unstained. Multiple control experiments yielded completely negative results. These data suggest that the in situ RT-nested PCR, which in comparison to the method of in situ RT-PCR-in situ-hybridisation is simpler and less time-consuming, can be used as an alternative approach to identify intracellular nucleic acids.

Expression of secretory group IIA phospholipase A(2) in relation to the presence of microbial agents, macrophage infiltrates, and transcripts of proinflammatory cytokines in human aortic tissues.

Recent seroepidemiological and immunohistochemical studies have demonstrated an association between microbial infections and atherosclerosis. However, the mechanisms underlying this association are widely unknown. In the present study, arterial specimens obtained at autopsy after sudden death were analyzed concerning (1) the presence of Chlamydia pneumoniae, cytomegalovirus, herpes simplex virus, and Helicobacter pylori; (2) the expression of secretory group IIA phospholipase A(2) (sPLA(2)-IIA) and of proinflammatory cytokines; and (3) the stage of atherosclerosis. Genomic DNA of microbial pathogens was determined by the polymerase chain reaction technique. The expression of sPLA(2)-IIA was studied immunohistochemically by using monoclonal antibodies against human sPLA(2)-IIA. Transcripts specific for sPLA(2)-IIA, interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma were identified by reverse transcription-polymerase chain reaction. In 18 of 102 analyzed specimens, DNA of microbial pathogens was found. Thirteen sections were positive for C pneumoniae, whereas 2 specimens were positive either for cytomegalovirus or for herpes simplex virus. One section contained genomic DNA of all 3 pathogens simultaneously. None of the analyzed tissues exhibited nucleic acids specific for H pylori. In addition to macrophage infiltrates, the presence of microbial DNA was closely associated with the occurrence of transcripts specific for proinflammatory cytokines and sPLA(2)-IIA. Pathogens as well as sPLA(2)-IIA and cytokines were found to be present not only in advanced but also in early stages of atherosclerosis. In tissues negative for sPLA(2)-IIA and cytokine expression, none of the pathogens could be identified. Because macrophages exposed to phospholipase A(2)-treated lipoproteins are transformed into foam cells in vitro, the results of this study suggest an alternative mechanism by which microbial infections may act in a proatherogenic fashion in vessel walls.

Minimal oxidation and storage of low density lipoproteins result in an increased susceptibility to phospholipid hydrolysis by phospholipase A2.

In vitro-studies have shown that phospholipid hydrolysis of low density lipoproteins (LDL) by bee venom or porcine pancreatic phospholipase A2 (PLA2) leads to an increased uptake of these lipoproteins by macrophages transforming them into foam cells. Recently, a secretory phospholipase A2, group II, was detected in human atherosclerotic plaques. In order to investigate the role of this enzyme in the pathogenesis of atherosclerosis, a structurally identical human secretory PLA2 was purified from the medium of HepG2 cells stimulated with interleukin-6 and tumor necrosis factor-alpha. The activity of the purified enzyme towards the phospholipids of native and modified low density lipoproteins was compared with the activity towards Escherichia coli-membranes and other phospholipid substrates. Compared to E. coli-membranes, native LDL proved to be a poor substrate for group II PLA2. After mild oxidation induced by copper ions or by 2,2-azobis(2-amidinopropane) (AAPH), the susceptibility of LDL to phospholipid hydrolysis was found to be increased by 25 and 23%, respectively, whereas extensive copper-mediated oxidation caused a decreased hydrolysis. Aging of LDL at 6 degrees C for weeks or at 37 degrees C for hours resulted in an increase in PLA2-catalyzed phospholipid hydrolysis of up to 26-fold. LDL protected from oxidation by probucol during aging showed a lesser increase in susceptibility to phospholipid hydrolysis. Our results suggest that PLA2, group II, can increase the atherogenicity of LDL by its ability to hydrolyze the phospholipids of these lipoproteins, especially after modifications that are likely to occur in vivo.

Human gastric alcohol dehydrogenase activity: effect of age, sex, and alcoholism.

As various isoenzymes of gastric alcohol dehydrogenase exist and as the effect of sex and age on these enzymes is unknown, this study measured the activity of gastric alcohol dehydrogenase at high and low ethanol concentrations in endoscopic biopsy specimens from a total of 290 patients of various ages and from 10 patients with chronic alcoholism. Gastric alcohol dehydrogenase was also detected by immunohistological tests in biopsy specimens from 40 patients by the use of a polyclonal rabbit antibody against class I alcohol dehydrogenase. A significant correlation was found between the immunohistological reaction assessed by the intensity of the colour reaction in the biopsy specimen and the activity of alcohol dehydrogenase measured at 580 mM ethanol. While alcohol dehydrogenase activity measured at 16 mM ethanol was not significantly affected by age and sex, both factors influenced alcohol dehydrogenase activity measured at 580 mM ethanol. Young women below 50 years of age had significantly lower alcohol dehydrogenase activities in the gastric corpus and antrum when compared with age matched controls (SEM) (6.4 (0.7) v 8.8 (0.6) nmol/min/mg protein; p < 0.001 and 6.0 (1.3) v 9.5 (1.3) nmol/min/mg protein; p < 0.001). Over 50 years of age this sex difference was no longer detectable, as high Km gastric alcohol dehydrogenase activity decreases with age only in men and not in women. In addition, extremely low alcohol dehydrogenase activities have been found in gastric biopsy specimens from young male alcoholics (2.2 (0.5) nmol/min/mg protein), which returned to normal after two to three weeks of abstinence. The activity of alcohol dehydrogenase in the human stomach measured at 580 mM ethanol is decreased in young women, in elderly men, and in the subject with alcoholism. This decrease in alcohol dehydrogenase activity may contribute to the reduced first pass metabolism of ethanol associated with raised ethanol blood concentrations seen in these people.

Human liver glutamic gamma-semialdehyde dehydrogenase: structural relationship to the yeast enzyme.

The amino acid sequences of nine tryptic peptides (containing altogether 105 amino acids) from human liver glutamic gamma-semialdehyde of dehydrogenase (hitherto designated as ALDH4) were found to correspond, at 33-66% identity, to segments from the yeast 1-proline-5-carboxylate (P5C) dehydrogenase encoded by the PUT2 gene.

[Genetically-induced variability of alcohol metabolism and its effect on drinking behavior and predisposition to alcoholism]. / Genetisch bedingte Variabilität des Alkoholstoffwechsels und ihr Einfluss auf Trinkverhalten und Neigung zum Alkoholismus.

Alcoholism is one of the most challenging current health problems in the Western countries with far-reaching medical, social, and economic consequences. There are a series of factors that interact in predisposing or protecting an individual against alcoholism and alcohol-related disorders. This article surveys the state of our knowledge concerning the biochemical and genetic variations in alcohol metabolism and their implications in alcohol sensitivity, alcohol drinking habits, and alcoholism in different racial/ethnic groups. The major pathway for the degradation of ethanol is its oxidation to hydrogen and acetaldehyde--to which many of the toxic effects of ethanol can be attributed. Variations in alcohol and acetaldehyde metabolism via genetically determined polymorphisms in alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) seem to play an important role in individual and racial differences in acute and chronic reactions to alcohol, alcohol drinking habits, as well as vulnerability to organ damage after chronic alcohol abuse. Alcohol sensitivity and associated discomfort symptoms accompanying alcohol ingestion may be determinental for the significantly low incidence of alcoholism among the Japanese, Chinese and other Orientals of Mongoloid origin. An abnormal ALDH isozyme has been found to be widely prevalent among individuals of the Mongoloid race and is mainly responsible for the acute sensitivity to alcohol commonly observed in this race. Persons sensitive to alcohol by virtue of their genetically controlled ALDH isozyme deficiency may be discouraged from drinking large amounts of alcohol in their daily life due to the initial adverse reaction experienced after drinking alcohol. Indeed, a significantly low incidence of the mitochondrial ALDH isozyme deficiency has been observed in alcoholics as compared to psychiatric patients, drug dependents and healthy controls in Japan. How far any variation in ADH and/or ALDH activity among individuals of Caucasian origin will have similar effects has yet to be studied.

Inheritance of mitochondrial aldehyde dehydrogenase: genotyping in Chinese, Japanese and South Korean families reveals dominance of the mutant allele.

Genotyping of mitochondrial aldehyde dehydrogenase (ALDH I) was performed in enzymatically amplified DNA of 20 Chinese, Japanese and South Korean families (85 individuals) and in 113 unrelated persons by employing allele-specific oligonucleotide probes and dot blot hybridization. Genotyping individuals with phenotypic deficiency of ALDH I activity always showed the presence of at least one mutant allele. The data are compatible with a model assuming dominant inheritance of the mutant allele, which we have previously suggested on the basis of a population study.

[New aspects of acetaldehyde metabolism in human tissues and erythrocytes]. / Neue Aspekte des Acetaldehydmetabolismus in menschlichen Geweben und Erythrozyten.

Acetaldehyde, the active metabolite of ethanol oxidation, has been implicated to be mainly responsible for the diverse toxic effects of alcohol drinking. Aldehyde dehydrogenase (ALDH) catalyzes the oxidation of acetaldehyde in human liver and other organs. Impaired acetaldehyde metabolism by way of reduced ALDH activity may be the primary cause of tissue injury. Erythrocyte ALDH activity has been found to be decreased in alcoholics and returns to normal values on reducing the alcohol intake. The purpose of this study was to determine the interindividual variation in ALDH activity in different human tissues and erythrocytes. Results of the present study show that significant interindividual variation occurs in ALDH activity in different tissues. This variability in ALDH activity might explain the widely observed interindividual and ethnic/racial differences in alcohol intolerance and susceptibility to ethanol- and acetaldehyde-related tissue and organ damage. The study also confirms earlier observations that erythrocyte ALDH activity significantly varies in healthy individuals without alcohol abuse. This fact should be taken into consideration while evaluating the erythrocyte ALDH activity as a potential marker of alcoholism.

Detection and partial characterization of a variant form of cytosolic aldehyde dehydrogenase isozyme.

A rare case of human liver cytosolic aldehyde dehydrogenase (isozyme II) variation discovered in a Chinese autopsy liver specimen is reported. While the major isozyme band was nearly absent, several additional minor bands were observed on isoelectric focusing gel. Rabbit antibodies to purified human liver ALDH II showed immunological cross-reactivity for the variant enzyme bands. The existence of additional minor bands indicates the presence of tetramer hybrid forms made up of normal and variant monomers. The observed abnormality may represent the heterozygous form of ALDH II variation. A similar variant was also detected in erythrocytes of a male Thai student.

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

Population genetic studies on the prevalence of aldehyde dehydrogenase isozyme I (ALDH I) deficiency in various Caucasian, Oriental, African, and American Indian subjects were carried out using hair roots as peripheral source of the enzyme activity. While a very high percentage of Orientals with Mongoloid origin were found deficient in ALDH I activity, no deficiency was detected in Caucasian and African populations. Native American Indians showed a relatively low incidence of ALDH I deficiency. A genetic model based on the phenotype determination using antisera against purified human liver ALDH I is proposed. Pedigree analysis of Japanese families suggests an autosomal codominant mode of inheritance.

Basis of aldehyde dehydrogenase deficiency in Orientals: immunochemical studies.

While most Caucasians have two main isozymes of liver aldehyde dehydrogenase, in about 50% of Orientals the ALDH I isozyme is missing. This isozyme, which has a faster electrophoretic mobility, is predominantly present in mitochondria and has a relatively low Km for acetaldehyde. The inherent deficiency of ALDH I is responsible for the impaired acetaldehyde oxidation leading to facial flushing and other cardiovascular symptoms of alcohol sensitivity commonly observed in Japanese and Chinese. Antibodies raised against apparently homogeneous liver ALDH I and ALDH II isozymes did not show an immunological similarity between the two isozymes which do not share common subunits. While erythrocyte ALDH II is also immunologically distinct from hepatic ALDH I, it showed an immunological similarity with hepatic ALDH II. On isoelectric focusing in agarose gel followed by immunoelectrophoresis, at least 4 components with an anti-ALDH I antibody were detected in extracts from Caucasian and Oriental livers. In Japanese livers deficient in ALDH I activity, the prominent ALDH component was missing. Apparently, more than one gene is responsible for the synthesis of ALDH isozymes reacting with an antibody against ALDH I. A deletion in one of the genes may be responsible for the loss of ALDH I enzyme activity and altered antigenic properties. However, at this stage, a point mutation in a structural gene coding for ALDH I resulting in a defective protein with altered electrophoretic and enzymatic properties is not ruled out.