F. M. Van Den Berg

Helicobacter pylori and Epstein-Barr virus infection and the p53 tumour suppressor pathway in gastric stump cancer compared with carcinoma in the non-operated stomach

AIM: To evaluate similarities and differences between gastric stump cancer and conventional carcinoma in the non-operated stomach. METHODS: 26 stump carcinomas were compared with 24 conventional stomach cancers. Stage, histological type, and demographics were comparable in the two groups. Expression of p53 and p21-Waf1/Cip1 was evaluated by immunohistochemical staining. Helicobacter pylori infection was evaluated by examining haematoxylin-eosin stained slides and immunohistochemistry. Epstein-Barr virus infection was evaluated by RNA in situ hybridisation. RESULTS: Expression of p53 and p21-Waf1/Cip1 was similar in both groups and positive in more than half of the patients. H pylori infection was observed in six stump carcinomas and 17 conventional carcinomas in the intact stomach (p < 0.01). RNA in situ hybridisation (EBER1-ISH) for Epstein-Barr virus was positive in nine stump carcinomas and two carcinomas in the non-operated stomach (p < 0.05). CONCLUSIONS: There appear to be aetiological differences between stump carcinoma and cancer in the intact stomach. Further study of these differences may improve our understanding of gastric carcinogenesis in general.

Molecular markers for diagnostic cytology of neoplasms in the head region of the pancreas: Mutation of K-ras and overexpression of the p53 protein product

AIMS--To determine the potential efficiency of molecular markers specific for neoplastic change--mutations of the K-ras oncogene and the p53 tumour suppressor gene--in diagnosing pancreatic carcinoma. METHODS--Archival cytology samples obtained from 17 patients with established pancreatic carcinoma were assayed for alterations in K-ras and p53. To detect changes in p53 expression, immunocytochemistry with polyclonal antibody CM1 was performed on the archival cytology slides after destaining. Mutations in K-ras codon 12 were then analysed on the scrapings of the same slides using mutant enriched polymerase chain reaction (PCR) amplification and restriction fragment length polymorphism analysis with allele specific oligonucleotide hybridisation for confirmation and characterisation. RESULTS--False negative results were recorded for five of the cytology slides when compared with p53 immunostaining of the surgical resection specimen. These five cases had been stained previously with Giemsa which interacts adversely with the immunostaining in contrast to the Papanicolaou procedure. The K-ras codon 12 mutations followed the well established distribution frequency and spectrum for pancreatic cancer and corresponded with the findings in the resection specimens in all cases. Two scrapings yielded insufficient DNA for PCR. Importantly, for two cases with an inconclusive cytology diagnosis on routine light microscopy, the diagnosis was confirmed by one of the molecular markers. The application of the molecular markers increased the diagnostic accuracy of cytology in this small study from 76 to 89%. CONCLUSIONS--The study indicates that assessment of alterations in the K-ras and p53 genes may be a valuable adjunct to diagnostic cytopathology of the head region of the pancreas, although there are some difficulties which will have to be overcome.

Plasmodium berghei: in vivo generation and selection of karyotype mutants and non-gametocyte producer mutants.

We previously reported that karyotype and gametocyte-producer mutants spontaneously arose during in vivo asexual multiplication of Plasmodium berghei. Here we studied the rate of selection of these mutants in vivo. Gametocyte production and karyotype pattern were established at regular intervals during prolonged periods of asexual multiplication of clone 8417 of P. berghei. We found that karyotype mutants and mutants which do not produce gametocytes can replace the original high-producer parasites of clone 8417 within several weeks. The time at which mutants became predominant in the population in different experiments, however, differed greatly. Mutants with intermediate or low gametocyte production were not found. In experimentally mixed infections, containing parasites from two clones from different strains (clone 8417 of the ANKA strain; clone 1 of the K173 strain), high-producer parasites of clone 8417 were overgrown by parasites of the nonproducer clone. Nonproducer mutants from the originally high-producer clone 8417, however, were able to coexist with parasites of the nonproducer clone. These results demonstrate that in our experiments nonproducer parasites had a strong selective advantage during asexual multiplication compared to high producers. All karyotype mutants which became predominant in our experiments were nonproducers. In two experiments a change in karyotype coincided with the loss of gametocyte production which may suggest a causal relationship between these events.

Implausibility of an aetiological association between cytomegalovirus and Kaposi's sarcoma shown by four techniques.

The presence of cytomegalovirus (CMV) was analysed in either lymph node or skin and lung tissue necropsy specimens affected by Kaposis sarcoma, from 10 patients who had died of AIDS. The different detection techniques used were: (i) immunohistochemical demonstration of CMV immediate early antigen (IEA); (ii) in situ hybridisation with a biotinylated CMV DNA probe; (iii) Southern blot hybridisation of DNA extracted from sequential tissue sections; and (iv) polymerase chain reaction (PCR) with CMV specific primers on the DNA samples. The results of these analyses were compared with the postmortem data on CMV obtained by infectious particle assays and histological examination, especially of adrenal glands of the same patients. The results of the various detection methods correlated very well, yielding a combined score of six of 10 patients positive for CMV; there did not seem to be any association between the presence of CMV and the occurrence of Kaposis sarcoma in our patients.

Aminolaevulinate dehydratase in greening leaves of Phaseolus vulgaris L.

Summary In greening leaves of Phaseolus vulgaris cv. Prelude a 4-fold increase of the activity of aminolaevulinate dehydratase (ALAD, E.C. 4.2.1.24) was observed. Some properties of this enzyme, which is involved in the biosynthesis of chlorophyll and heme compounds, were examined. Studies with the specific inhibitors of protein synthesis, chloramphenicol and cycloheximide, showed that the increase of enzyme activity was due to de novo synthesis on cytoplasmic ribosomes. Experiments with chloroplasts isolated in a non-aqueous medium indicated that at least part of the enzyme is localized within these organelles. These results are discussed with reference to the known inhibition of chlorophyll synthesis by cycloheximide and to possible factors limiting this synthesis.

Human placental steroid sulphatase--purification and monospecific antibody production in rabbits

Human steroid sulphatase was purified 43-fold from placental microsomes using a four step procedure: solubilization with Miranol H2M, Bio-Gel A 1.5m chromatography, column chromatofocusing and Sephadex G-75 chromatography. The purified enzyme that appeared electrophoretically homogeneous was used to immunize rabbits. Protein blotting demonstrated that the resulting antiserum mainly reacted with a polypeptide of 63 000 dalton, which is about the size of placental steroid sulphatase. The antiserum was freed from minor impurities by absorbing it to Sepharose 4B with immobilized antigens prepared from a steroid sulphatase deficient placenta.

Detection of Campylobacter pylori in stomach tissue by DNA in situ hybridisation.

A non-radioactive DNA in situ hybridisation (DISH) protocol was developed. It requires the use of biotinylated Campylobacter pylori DNA as the probe to detect C pylori DNA in routinely embedded stomach biopsy specimens. In sequential tissue samples from a 58 year old woman with recurrent chronic active gastritis the C pylori probe hybridised with bacteria whenever they were histologically visible. When no bacteria were visible histologically, hybridisation was negative with one exception. In a single biopsy specimen without visible C pylori, hybridisation occurred with the surface of the antrum epithelium, while control hybridisation with a heterologous probe remained negative. From a parallel biopsy specimen taken at the same time the C pylori culture was positive. It is concluded that DISH, although more laborious than routine staining techniques, may be more sensitive in that it detects bacteria very easily, even when sections are not counterstained or when they are present in low numbers, and that bacteria which do hybridise are unequivocally identified as C pylori and not Campylobacter-like organisms.

Combined beta-galactosidase and immunogold/silver staining for immunohistochemistry and DNA in situ hybridization.

A combination of beta-galactosidase enzyme and the immunogold/silver staining method was studied for evaluation of double-staining experiments. Applications are shown for immunohistochemical double staining using two monoclonal antibodies and for combined immunohistochemistry and DNA in situ hybridization in one tissue section. The following advantages for the present double-staining method were evaluated: superior sensitivity of the immunogold/silver staining method for at least one epitope, which also allows detection of biotinylated DNA probes. The structure of the indolyl precipitate after revelation of beta-galactosidase activity did not show a concealing effect during a sequential double-staining method, as compared with the visualization of peroxidase with diaminobenzidine. These factors, and the sharply contrasting colored reaction products of beta-galactosidase (blue-green) and the immunogold/silver staining method including silver enhancement (brown-black), allow clear distinction of mixed-stained cell constituents.

Isolation and characterization of the oligo(dA-dT) clusters and their flanking DNA segments in the rabbit genome.

Abstract Duplex DNA containing (dA-dT) clusters † can be bound specifically to poly(U)-Sephadex in high-salt solutions by the formation of a (dA-dT)rU triple helix. By decreasing the salt concentration the DNA is fractionated predominantly on the basis of the length of the (dA-dT) cluster it contains, long clusters being bound tighter than short ones. A stepwise elution protocol was used to obtain three fractions containing (dA-dT) clusters of which the average length is approximately 12, 16 and 23 base-pairs, respectively. Each given fraction probably also contains impure (dA-dT) clusters of greater length than these but which have additional bases inserted in the (dA-dT) sequence. When bound to poly(U) these clusters should give triplexes of equivalent stability to those formed with shorter pure (dA-dT) tracts. Measurement of the distribution of rabbit DNA in these poly(U)-Sephadex fractions as a function of DNA size shows that the three (dA-dT) cluster length-classes are interspersed with one another in the rabbit genome. The short, intermediate and long clusters are spaced approximately once every 6000, 12,000 and 18,000 base-pairs throughout at least 80% of the rabbit genome. There is an average of one (dA-dT) cluster every 3300 base-pairs in rabbit DNA. When rabbit DNA is sheared to 400 base-pairs, 50% of the molecules containing a (dA-dT) cluster contain a sequence reiterated approximately 2 × 10 5 -fold in the rabbit genome although only 20% of unfractionated DNA of this size contains such repeated DNA. The remaining 50% of the clusters are linked to DNA sequences occurring one to five times per haploid genome.

DETECTION OF DIFFERENT DEVELOPMENTAL STAGES OF MALARIA PARASITES BY NON-RADIOACTIVE DNA IN SITU HYBRIDIZATION

SummaryA highly sensitive non-radioactive DNAin situ hybridization procedure is described that enables detection and unequivocal identification of various developmental stages of human and rodent malaria parasites. Using biotinylated species-specific DNA probes, erythrocytic parasites can be specifically stained in blood smears. Similarly exoerythrocytic stages can be visualized in cell culture and in sections of paraffin-embedded liver. In blood smears, the hybridization procedure provides a rapid detection of (low) parasitemia and species-determination for experienced microscopists at 100 to 400x magnification. Moreover, the procedure can be applied even after previous Giemsa staining of the preparation, enabling revision of patient smears which were difficult to read after routine Giemsa staining.