Analysis of proliferative activity in colorectal mucosa by immunohistochemical detection of proliferating cell nuclear antigen (PCNA). Methodological aspects and application to routine diagnostic material.

Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) has been suggested as a new approach for determining proliferative activity in paraffin-embedded tissue. In a prospective study PCNA immunostaining was performed in 284 colorectal biopsies using monoclonal antibodies 19F4 (Ogata et al. 1987) and PC10 (Waseem and Lane 1990) and compared with the Ki67 method. From each site three biopsies were taken and a variety of fixation regimens for frozen and paraffin-embedded samples tested. For frozen biopsies methanol fixation at -20 degrees C proved best. In paraffin sections PCNA could be detected after methacarn fixation as well as after controled fixation at 4 degrees C in 4% paraformaldehyde for 1 h and in most biopsies routinely fixed with 10% formalin. However, the latter fixation regimens revealed additional PCNA-positive cells in the normal superficial colonic mucosal epithelium. Although the percentage of cells positive for PCNA was generally lower than for Ki67, the rates correlated in a highly significant fashion, both in frozen methanol-fixed biopsies, and in paraformaldehyde-fixed paraffin-embedded samples. PCNA immunohistochemistry revealed a similar proliferative activity in different parts of the large bowel. A higher proliferative activity was found in inflamed mucosa, adenomas, carcinomas and even in normal mucosa from patients with colorectal neoplasms. In routinely fixed biopsies, the monoclonal antibody PC10 was superior to 19F4 because of considerably less background staining. However, in the routine material only a rough estimate of the proliferative activity was possible by PCNA immunohistochemistry using these antibodies, because unpredictable numbers of non-S-phase cells were also stained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of a cytotoxic substance produced by the cyanobacteriumMicrocystis aeruginosa strain PCC 7806: Isolation and differentiation from the peptide toxin microcystin-LR by cytotoxicity assays

Exposure of four permanent cell lines to crude aqueous extracts of the cyanobacteriumMicrocystis aeruginosa (strain PCC 7806) resulted in rounding and lysis of the cells within a few minutes. Cell damage was quantified by determination of lactate dehydrogenase (LDH) activity in the cell culture supernatants. By gel filtration, cation exchange chromatography and SDS-PAGE, the cytotoxic effects could be related to a defined substance with an apparent molecular weight of about 35 kDa, which was not heat resistant. The physicochemical properties very clearly mark off this compound from the peptide toxin microcystin-LR, which did not show any detectable cytotoxic effects.     

Deletion ΔF508 and haplotype analysis of CFTR gene region in Slovak CF patients

Summary Analysis of a sample of 50 unrelated cystic fibrosis (CF) patients and 46 nuclear families from Slovakia (Czechoslovakia) by the polymerase chain reaction and Southern hybridization revealed that the proportion of the F508 mutation was 58% in this population, and that the frequency of the B (i.e., KM19/XV2c [1–2]) haplotype was increased in both F508 and nonF508 CF chromosomes (98% and 46%, respectively). These results support the view that the trans-European gradient of the F508 frequency is of a geographical rather than of an ethnic origin, and that in Slavonic populations, there exists an as yet unidentified but frequent CF mutation other than F508, associated with the B haplotype.     

Differences in anti-oncogene p53 expression in human monocytes and lymphocytes in vitro]

The p53 gene has been associated with malignant transformation as well as "anti-oncogene" activity. In the present report expression of p53 in resting and activated human blood monocytes and lymphocytes is analyzed. It is found that human monocytes freshly isolated by continuous percoll gradient centrifugation contained detectable level of p53 mRNA. Stimulation of monocytes by potent activation inducer Staphylococcus Aureus Cowan I for 3-5 hr caused disappearance of r53 mRNA. In contrast, induction of high level of TNF-alpha mRNA was detected. Addition of cycloheximide had no effect on p53 mRNA content in stimulated monocytes, and caused disappearance of mRNA in resting cells. In lymphocytes cultures p53 mRNA was absent in freshly isolated cells and in resting lymphocytes cultured for 20 hr. Activation of lymphocytes by lectin caused accumulation of p53 mRNA. We suggest that r53 gene regulation and functions might be different in human monocytes and lymphocytes.     

Use of commercial highly sensitive tests for detecting HbsAg in healthy persons]

The study has revealed the possibility of the contamination of serum with HBsAg under laboratory conditions during its treatment and preparation for analysis, which may be the cause of false positive results of HBsAg detection by the enzyme immunoassay (EIA). Excluding the factors of contamination, the authors demonstrate the efficacy of commercial systems, such as the passive hemagglutination test system "Gorky" and the EIA systems "Gorky" and "DIAplus", for the detection of HBsAg in the blood of donors in Moscow and Tashkent.     

The phages of halophilic vibrios and their use]

The range of the lytic activity of 46 phages of parahemolytic vibrios isolated from lysogenic strains, sea water samples, crabs and mussels has been studied. The phages are represented by virions belonging to morphological groups II, IV, V according to the phage classification currently used in Russia and to different serological groups. No relationship between the sensitivity of vibrio strains to the phages under study and the specificity of serotypes O and K has been established. The preparation of diagnostic phage [see text] suitable for the identification of 82% of strains of parahemolytic vibrios has been proposed.     

Cloning, organization and functional analysis of ilvA, ilvB and ilvC genes from Corynebacterium glutamicum.

Corynebacterium glutamicum is an industrially important bacterium for the manufacture of amino acids. We constructed genomic libraries of this Gram+ bacterium and screened for clones carrying isoleucine biosynthesis genes (ilv) by complementation of Escherichia coli mutants. Clones complementing ilvA, ilvB, and ilvC were isolated. As based on the functional analysis of the corresponding plasmids in C. glutamicum, the DNA fragments isolated encode threonine dehydratase, acetohydroxy acid synthase, and isomeroreductase, catalyzing three subsequent reactions in Ile synthesis. Subcloning and transposon mutagenesis revealed that ilvB and ilvC reside on a 7-kb chromosomal fragment and that these genes are transcribed in the same direction. A shuttle vector was constructed to allow exonuclease treatment and assay subsets of plasmids for gene expression in the original C. glutamicum background. These constructs and their enzyme activity determinations revealed that despite close linkage ilvC is expressed independently from ilvB. Using Southern blots, a 15-kb fragment of chromosomal DNA carrying the ilvBC cluster was characterized. This fragment does not contain ilvA, demonstrating the entirely different organization of the isoleucine biosynthesis genes in C. glutamicum from that in enterobacteria.     

Sequence of the 5-aminolevulinic acid dehydratase-encoding gene from the hyperthermophilic methanogen, Methanothermus sociabilis.

We report on the sequence of the Methanothermus sociabilis aladh gene, which encodes the 5'-aminolevulinic acid dehydratase. The identity of the enzyme was determined by sequence comparison and by expression in Escherichia coli.     

Mixed pituitary tumours--effects of bromocriptine treatment: Parlodel MR and Parlodel LAR.

Majority of pituitary tumours secrete one of the named hormones: PRL, GH, ACTH, proopiomelanocortine, alpha and beta subunit of TSH, LH, and FSH. Some of those tumours secrete two or more hormones. The aim of this study was to determine the effect of bromocriptine (Parlodel MR and LAR) upon secretion of hormones and tumour size in 10 patients with mixed pituitary tumours. In all patients pituitary and peripheral hormones, CT scan and visual fields were examined before and after treatment with bromocriptine: Parlodel MR and LAR. Bromocriptine treatment decreased PRL secretion in all 10 patients; GH--in all 6 in whom it was increased; TSH--in 2, FSH--in 2 and alpha-subunit in all 6 in whom they were increased. In 5 patients treatment resulted in shrinkage of the tumour mass by 20 to 35%. In all examined subjects clinical improvement was achieved. Our results demonstrate that bromocriptine (Parlodel MR and LAR) is very effective and well tolerated in the treatment of patients with mixed pituitary tumours particularly those with hyperprolactinemia.     

Plasma endothelin 1/2 levels in healthy blood donors as measured by RIA--a clinical application.

Normal endothelin 1/2 levels and their correlation with age were evaluated. For clinical application of the endothelin 1/2 RIA test, optimum storage conditions were investigated. Plasma endothelin 1/2 (ET) levels were measured in 36 healthy blood donors, mostly males, of mean age 36 +/- 8 years, subdivided into three age groups: 17-30, 31-40 and above 40 years old. The mean normal ET levels in the three age groups, and corresponding standard deviations, were: 0.58 +/- 0.19, 0.62 +/- 0.31, and 0.80 +/- 0.28 fmol/ml, respectively. The mean ET level for the whole normal population was 0.66 +/- 0.28 fmol/ml. Only differences between mean ET levels for the first and last groups were statistically significant (p < 0.05). Differences between mean ET levels in smokers (53% of total population) and non-smokers, women and men, irrespective of age, were found not to be statistically significant. At this stage of our work, we conclude that other factors than age alone play a role in enhancing ET levels above the age of 41 years. In our study of optimum storage conditions for endothelin 1/2, we found that after one week of storage at -24 degrees C the mean level of ET measured in frozen plasma dropped to 85% of the initial activity, while after the same period the respective decrease in ET activity in frozen extracts was 49%. Over the next two weeks, ET levels in plasma and extracts dropped to 57% and 32% of "zero time" activities, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)