New approaches for genotyping of Helicobacter pylori based on amplification of polymorphisms in intergenic DNA regions and at the insertion site of the cag pathogenicity island.

The population of the gastric pathogen Helicobacter pylori shows a high degree of genetic diversity. It is well established that heterogeneity at the isolate level is caused by nucleotide transitions within genes, differences in the gene order, and by genetic instability of single genes as well as of a large virulence-associated genomic DNA region, the cag pathogenicity island (PAI). Analysis of intergenic regions with specific PCR-assays developed in this study, revealed that DNA polymorphisms in the noncoding DNA localized in front of the genes ribA and vacA and at the insertion site of the cag PAI contribute to the genetic diversity of H. pylori and are useful for differentiation of individual isolates. Thirteen individual genotypes were identified by PCR analysis of these polymorphic loci in 487, 241, and 182 clinical H. pylori isolates. Sequence analysis revealed that genetic variability in front of genes ribA and vacA, and in the intergenic region at the PAI insertion site is caused by insertion and deletions of so-far-unknown DNA sequences as well as by parts of the H. pylori IS elements IS605 and IS606, respectively. The new genotypes identified could be used to differentiate antrum and corpus isolates from the same patients. Their combination with vacA allele subtypes and with the cagA status allowed to differentiate 140 isolates in 51 subtypes. In 36 cases the corresponding genotype patterns were isolate specific. In summary, the results confirm that DNA polymorphisms in intergenic regions contribute to the genetic diversity of H. pylori. Although individual H. pylori genotypes were not associated with peptic ulcer disease, the PCR-based approaches for their detection developed here should be of use for further investigation of genetic diversity in H. pylori and for epidemiological purposes.

Mutant p53 can provoke apoptosis in p53-deficient Hep3B cells with delayed kinetics relative to wild-type p53.

Wild-type (wt) p53 frequently induces apoptosis when expressed in tumor cells whereas mutant p53 acts as an oncoprotein and consequently, stimulates cell proliferation. We report here exceptions to that rule. p53 conformational mutant 175H and DNA contact mutant 273H provoke apoptosis in human p53-deficient Hep3B hepatoma cells with delayed kinetics relative to wt p53. Similarly, c-Myc strongly stimulates apoptosis in these cells. In contrast, viral oncoproteins E1A and E7, and the cellular oncoprotein MDM-2, fail to elicit cytocidal responses. Efficient apoptotic cell death by mutant p53 requires oligomerization as 175H and 273H with deletions between amino acid residues 326 and 347 of the oligomerization domain are nontoxic. Apoptosis by mutant or wt p53 was significantly inhibited by the serine protease inhibitor AEBSF but not by the inactive analog AEBSA. Together, these results suggest that a wt p53-independent control mechanism is operational in Hep3B cells that eliminates cells upon sensing illegitimate proliferation signals originating from certain oncoproteins, including mutant p53 and Myc. We suggest that some tumor cell types lack p53 altogether because they tolerate neither wild-type nor mutant forms of the protein.

[A fully enzymatic triglyceride determination in a continuous flow 12-channel analyzer (author's transl)]. / Vollenzymatische Triglyceridbestimmung im Continuous Flow System am 12-Kanal-Analysator.

A fully enzymatic triglyceride determination utilizing enzymatic hydrolysis with a lipase-esterase mixture and subsequent enzymatic glycerol determination, has been adapted for use in a continuous flow 12-channel-analyzer. The method is linear up to 7.9 mmol/l (700 mg/100 ml). The analytical precision in the concentration range of 1.5 to 5.4 mmol/l (133 to 478 mg/100 ml) is characterized by relative standard deviations of 0.8 to 4.8%. In the lower measuring range at concentrations around 0.7 mmol/l (62 mg/100 ml) a mean relative standard deviation of 7.2% is found for 1140 measurements under routine conditions. For triglyceride concentrations of 0.9 to 7.7 mmol/80 to 680 mg/100 ml) a mean relative coefficient of verspill Q = 2.0 is determined. Bilirubin caused no observable interference in the determination. In comparison with the manual method by Eggstein & Kreutz (1966) Klin. Wochenschr. 44, 262-267) the results from the fully enzymatic method on the 12-channel-analyzer were lower by approximately 16%, corrected by an additive factor of 0.029 mmol/l (2.59 mg/100 ml). The accuracy controls with controls sera showed a difference of 10%.

[Enzymatic determination of total cholesterol in serum: accuracy and comparison with other methods (author's transl)]. / Enzymatische Bestimmung von Gesamt-Cholesterin im serum. Richtigkeit und Methodenvergleich

Cholesterol can be specifically measured without difficulty and without complex reagents by means of a newly developed enzymatic colour test. Intensive technical evaluation confirmed its accuracy. Manual use gave a day-to-day coefficient of variation of 2-3 per cent; the sensitivity at a cholesterol concentration of 200 mg/dl was E equals 0.153 (gamma equals 405 nm), with a linearity up to 1000 mg/dl. Recovery of added pure cholesterol solution was 100 plus or minus 2 percent. A quantitative study of 53 representative drugs, anti-coagulants and metabolites was performed both in test tube and on patients. Accuracy of the result was unaffected by any of the substances (alpha equals 0.05). Comparison with the multi-step extraction method used at present as a reference (Abell-Kendall) gave a regression equation of y equals 0.99 times plus 1.1 (x axis: extraction method; y axis: enzymatic colour test). The direct chemical method after Liebermann and Burchard gave, in part, markedly differing results because of considerable systematic and accidental errors.