Search

Background: The vacA genotypes and the cagA gene status were investigated in 80 Helicobacter pylori-infected patients with duodenal ulcer (DU) and 49 with gastritis only. Methods: Lysates of gastric biopsy specimens were used directly for polymerase chain reaction-based detection. Results: The m1 subtype was found in 36% and 31% and the m2 in 36% and 46% of specimens from patients with DU and gastritis, respectively (P > 0.05). In 15% of samples the midregion remained unclassified. The prevalence rate of s1 subtypes was higher in cases of DU (69%) than in gastritis (43%) (P < 0.0001); the opposite correlation was observed for s2. The cagA gene was detected in 80% of patients with DU and in 52% of those with gastritis (P < 0.0001). Infections with multiple H. pylori strains exceeded 50% in both groups. Conclusions: These results suggest that vacA s1 genotype and cagA[sup +] status are associated with higher DU prevalence and that mixed H. pylori infections are very common in our geographic region. [ABSTRACT FROM AUTHOR]

Background: Our previous work has shown peroxiredoxin-1 (PRDX1), one of major antioxidant enzymes, to be a biomarker in human breast cancer. Hereby, we further investigate the role of PRDX1, compared to its close homolog PRDX2, in mammary malignant cells., Methods: CRISPR/Cas9- or RNAi-based methods were used for genetic targeting PRDX1/2. Cell growth was assessed by crystal violet, EdU incorporation or colony formation assays. In vivo growth was assessed by a xenotransplantation model. Adenanthin was used to inhibit the thioredoxin-dependent antioxidant defense system. The prooxidant agents used were hydrogen peroxide, glucose oxidase and sodium L-ascorbate. A PY1 probe or HyPer-3 biosensor were used to detect hydrogen peroxide content in samples., Results: PRDX1 downregulation significantly impaired the growth rate of MCF-7 and ZR-75-1 breast cancer cells. Likewise, xenotransplanted PRDX1-deficient MCF-7 cells presented a retarded tumour growth. Furthermore, genetic targeting of PRDX1 or adenanthin, but not PRDX2, potently sensitised all six cancer cell lines studied, but not the non-cancerous cells, to glucose oxidase and ascorbate., Conclusions: Our study pinpoints the dominant role for PRDX1 in management of exogeneous oxidative stress by breast cancer cells and substantiates further exploration of PRDX1 as a target in this disease, especially when combined with prooxidant agents.

The family of testis-specific serine-threonine kinases (TSSKs) consists of four members whose expression is confined almost exclusively to testis. Very little is known about their physiological role and mechanisms of action. We cloned human and mouse TSSK3 and analysed the biochemical properties, substrate specificity and in vitro activation. In vitro TSSK3 exhibited the ability to autophosphorylate and to phosphorylate test substrates such as histones, myelin basic protein and casein. Interestingly, TSSK3 showed maximal in vitro kinase activity at 30 degrees C, in keeping with it being testis specific. Sequence comparison indicated the existence of a so-called 'T-loop' within the TSSK3 catalytic domain, a structure present in the AGC family of protein kinases. To test if this T-loop is engaged in TSSK3 regulation, we mutated the critical threonine residue within the T-loop to alanine (T168A) which resulted in inactivation of TSSK3 kinase. Furthermore, Thr168 is phosphorylated in vitro by the T-loop kinase phosphoinositide-dependent protein kinase-1 (PDK1). PDK1-induced phosphorylation increased in vitro TSSK3 kinase activity, suggesting that TSSK3 can be regulated in the same way as AGC kinase family members. Analysis of peptide sequences identifies the peptide sequence RRSSSY containing Ser5 that is a target for TSSK3 phosphorylation, as an efficient and specific substrate for TSSK3.

Changes of gel temperature during single-strand conformation polymorphism (SSCP) electrophoresis increase the sensitivity of mutation detection in polymerase chain reaction (PCR) products and significantly reduce the overall time and costs of analysis. Based on these findings, a new method for single nucleotide polymorphism (SNP) and point mutation detection--multitemperature single-strand conformation polymorphism (MSSCP) was devised. In order to control the gel temperature with 0.1 degrees C accuracy during electrophoresis, new equipment was developed. We demonstrated that increasing the gel temperature by 8 degrees C or decreasing it by 10 degrees C from 23 degrees C led to the disappearance of all electrophoretic differences between five alleles of exon 8 of the human p53 gene during the SSCP analysis. The interesting result was the detection of two additional SNPs (out of seven analyzed) in exon 7 of the human PAH gene during a one hour MSSCP electrophoresis. This result is better than that obtained by three classical SSCP analyses of the same samples at different but constant gel temperatures. We advocate the MSSCP technology as a fast, reliable, and cost-effective tool for the screening and preselection stage of genomics surveys, especially when a high variability of the analyzed DNA fragment is expected.

The status of CDKN2a gene, coding for p16 and p19ARF proteins, was examined in 55 colorectal cancers. Polymerase chain reaction (PCR), single stranded conformational polymorphism and sequencing revealed 1 case of CDKN2a mutation. Methylation-specific PCR detected p16 locus methylation in 37 (73%) of 51 normal samples and 29 (53%) of 55 cancers (P=0.035). p16 transcript absence (assessed by reverse transcription-polymerase chain reaction) was noted in 10 (45%) of 22 normal samples and four (14%) of 29 cancers (P=0.012) and correlated with gene methylation (P=0.036). The decreasing frequency of p16 silencing in cancer comparing to normal mucosa does not support the postulated role of p16 in colorectal carcinogenesis.

Background/aims: Although signal transduction pathways activated by EGF have been extensively studied in cultured cells, few such studies have been done in whole animals. In this study, activation of hepatic kinases, phosphatases, and DNA-binding activity of AP-1 was examined after intraperitoneal injections of either EGF or sodium orthovanadate into mice., Methods: Cytoplasmic and nuclear proteins, extracted from isolated hepatocytes or whole liver tissue, were immunoprecipitated with either anti-ERK1/2, anti-70S6k, or anti-p90rsk antibodies and kinase activities were measured using specific substrates. Kinase protein levels was evaluated by Western blot analysis. AP-1 DNA binding activity was measured by electrophoretic mobility shift assay., Results: Systemic administration of EGF induced simultaneous increase in the activities of cytoplasmic and nuclear MAPK, p70S6k, and p90rsk. MAPK and p70S6k were more potently activated in the cytosol while p90rsk activation was more pronounced in the nucleus. Orthovanadate also activated these kinases but to a much lesser degree than EGF. In vitro phosphatase assays showed that neither EGF nor orthovanadate induced measurable changes in phosphatase activities. EGF, but not orthovanadate, activated nuclear AP-1 DNA-binding activity in intact liver, indicating that activation of MAPK, p70S6k, and p90rsk by orthovanadate is not sufficient to activate this transcription factor., Conclusion: These observations provide groundwork for future studies to examine the role of EGF-induced kinase cascades and transcription factors in liver regeneration and other growth factor-mediated hepatic processes.

Background: Prevention of Helicobacter pylori infection may help to control related gastritis, peptic ulcer and cancer. Of the possible preventive measures, immunization was successfully employed in various animal studies. However, no immunization protocol has been accepted for humans. A better characterization of the immune response against the pathogen may be required before a human vaccine is developed., Aim: To identify bacterial proteins which induce an immune response in infected humans or H. pylori-immunized rabbits., Methods: An expression library of H. pylori genes was screened with sera from infected humans and from immunized rabbits. Positive clones were partially sequenced and identified on the basis of a homology search of a H. pylori genome database. Encoded proteins were expressed directly from positive clones and analyzed by SDS-PAGE/Western blot techniques., Results: 114 positive clones were isolated: 79 by screening with human sera and 35 by screening with rabbit sera. Western blot analysis demonstrated that selected clones encoded one or more strongly immunoreactive proteins. 64 clones selected with human sera had no counterparts among clones from screening with rabbit serum. 13 of these clones encoded a total of 21 unknown H. pylori proteins. 17 clones selected with rabbit sera were not immunostained with human sera. They represent 2 various regions of the H. pylori genome which encoded 3 bacterial proteins of unknown function., Conclusions: Screening of H. pylori expression library identified immunogenic proteins - potential vaccine antigens., (Copyright 2000 S. Karger AG, Basel)

Comparison of immunohistochemical methods for detection of protein p53 accumulation and molecular techniques for analysis p53 gene mutation in colorectal cancer is presented. Thirty eight patients were included: all underwent surgery without preoperative treatment. Sex of patients, tumor localisation, macro and microscopic type of cancer and staging according to Astler-Coller and Jass classifications were evaluated. Protein p53 accumulation was detected by the streptavidin-biotin method using DO-7 (Dako) antibody. The number of cells stained were classified semiquanititatively according to a scoring system: (-)no positive cells, (+) : 10-30% positive cells, (++) : 40-70% positive cells, (+++) : >70% positive cells. For all cancer samples, exons 5 to 9 of p53 gene were amplified from isolated genomic DNA. PCR products were subjected to single standed conformational polymorphism analysis. All product were also directly sequenced on ABI PRISM 377 apparatus using fluorescent dideoxyterminators chemistry. The protein p53 accumulation was detected in 53% (20/38), whereas p53 gene mutation was seen in 55% (21/38). Among them, 15 patients (39%) with overexpression showed mutation in exon 5-8 gene p53. Discrepancies between results were noted in 29%. In conclusion, the necessity of both methods immunohistochemical and molecular is indicated for the objective evaluation of functional and structural status of p53 gene and protein.

TGF-beta1 is a multifunctional regulatory peptide (25 kDa) inducing growth arrest and apoptosis in many normal and neoplastic cells. In the present study, the involvement of proapoptotic (bax) and antiapoptotic (bcl-2) genes in the molecular mechanism of TGF-beta1-induced apoptosis of L1210 leukemic cells was investigated. Bax transcript was measured using the RT-PCR method with GAPDH as a "housekeeping gene" control, whereas Bcl-2 protein was determined using flow cytometry (FITC-conjugated monoclonal anti-Bcl-2 antibody and FITC-conjugated mouse anti-IgG1 antibody as a negative control). Apoptosis was evaluated using fluorescence microscopy and flow cytometry after cell staining with DAPI and sulforhodamine or propidium iodide and Hoechst 33342. ROS generation was assessed by flow cytometry using the oxidation-sensitive fluorescent marker C-DCDHF-DA. The response of L1210 leukemic cells to TGF-beta1 was two-directional: 1) partial arrest of the cell cycle at G1-S transition, and 2) induction of apoptotic cell death. TGF-beta1 increased the number of leukemic cells with typical morphological features of apoptosis: cell shrinkage, condensation of chromatin, pyknosis and fragmentation of nuclei, followed by secondary necrosis. DNA cleavage led to a decrease of the nuclear DNA content and the appearance of a hypodiploid peak sub-G1 in the DNA histogram. The extraction of low-molecular weight DNA fragments from apoptotic cells showed that TGF-beta1-induced apoptosis concerned first of all the cells from G1 phase. Two phases of intracellular ROS generation in TGF-beta1-treated cultures were observed: the first (rapid, 60 min after TGF-beta1 administration), and the second (slow, occurring between 24 and 48h of experiment, respectively). The increase of apoptotic cell number in TGF-beta1-treated cultures (2% FCS/RPMI 1640) was associated with the decrease of cell number expressing bcl-2, and with a significant drop of Bcl-2 level in the remaining cells after 24 h. The dose-dependent relationship between TGF-beta1 concentration and Bcl-2 level was nonlinear and described by power series regression. The lowest Bcl-2 level was noted at 1 ng/ml of TGF-beta1 concentration. The increase of Bax mRNA:GAPDH mRNA ratio was observed 3h after TGF-beta1 (1 ng/ml) administration to both the maintenance (2% FCS/RPMI) and growth promoting (10% FCS/RPMI) medium. Regardless of TGF-beta1 treatment, the quantity of Bax transcript was dependent on FCS concentration, being higher in the growth promoting medium. The results of this study indicate that bax may function as a primary response gene and together with lowered Bcl-2 level may determine the induction of apoptotic process in L1210 leukemic cells exposed to TGF-beta1.

Orotic acid (OA), a known promoter of carcinogenesis, significantly stimulated proliferation of K 562 leukemic cells even at as high a concentration as 0.1 mM. This effect was accompanied by a significant increase of the activity of two key enzymes of the polyamine pathway, i.e. ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC). The induction of ODC activity was associated with increased expression of the ODC gene. The participation of ODC in early events evoked by OA in leukemic cells was confirmed by the decrease of the stimulatory effect of OA on cell proliferation in the presence of alpha-difluoromethylornithine (DFMO)--an irreversible inhibitor of ODC. The involvement of protein kinase C (PK-C) and cyclic nucleotide-dependent kinases in OA action on K 562 leukemic cells was demonstrated by a significant reduction of cell proliferation by addition of H-7 (1-(5-isoquinolinesulphonyl)-2-methylpiperazine). Since PK-C is involved both in induction of ODC activity and in membrane transport of polyamines, H-7 significantly inhibited the proliferation of K 562 leukemic cells even in the presence of OA and exogenous putrescine. The importance of extracellular sources of polyamines for leukemic cell growth was shown by supplementation of the incubation medium with putrescine. Exogenous putrescine significantly enhanced the concentration of spermidine and spermine within the cell and increased the number of cells. The effect of OA on natural killer (NK) cell cytotoxicity was also examined. Rat peripheral blood mononuclear cells were used as effector cells and K 562 cells as targets. OA, progressively with dose, significantly decreased specific lysis when targets were preincubated with it. On the other hand, pretreatment of PBMC effector cells with OA, regardless of the applied concentration, did not affect the amount of 51Cr released from lysed cells. OA as a promoter of carcinogenesis stimulates proliferation of leukemic cells and impairs their responsiveness to NK activity. ODC/polyamine system and PK-C appear to be involved in OA action on K 562 cells. The presented observations are important from a practical point of view, since an elevated blood concentration of OA resulting from the impaired kidney function in hematological proliferative diseases may accelerate their progression.

TGF alpha shows structural and functional homology to EGF, but TGF alpha's mitogenic potency is greater. Our previous study showed that EGF may inhibit parietal cell secretory response through the induction of ornithine decarboxylase (ODC). The aim of this study was to determine parietal cell acid production in vitro in response to stimulation by TGF alpha and EGF and to compare the effect of these two growth factors on ODC activity and ODC mRNA in isolated rat gastric glands. 45 min treatment with TGF alpha and EGF had no effect on basal acid production but did inhibit histamine-stimulated acid production in a dose dependent manner. The two growth factors did not inhibit histamine-stimulated aminopyrine (AP) uptake from incubation medium with concentration of KCl increased from 5 to 70 mM. In the presence of specific ODC inhibitor, alpha-difluoromethylornithine (DFMO), both EGF and TGF alpha failed to inhibit histamine-stimulated AP accumulation. Polyamine spermine also inhibited AP accumulation but this inhibitory effect was not affected by DFMO. After 1 h treatment with TGF alpha and EGF, ODC activities increased to an average 283 +/- 78% and 227 +/- 64% above the basal activity, respectively. 30 min treatment of gastric glands with TGF alpha and EGF resulted in, respectively, 2.9 +/- 0.4- and 2.7 +/- 0.5-fold increases of ODC mRNA level, as assessed by RT-PCR.(ABSTRACT TRUNCATED AT 250 WORDS)

Unlabelled: Transforming growth factor-beta 1 (TGF-beta 1) exerted growth-inhibitory effect on L1210 leukemic cell line, manifested by the decrease in viable and increase in dead cells. The cell death evoked by TGF-beta 1 was both necrotic and apoptotic, quantified by the trypan blue exclusion method and apoptotic index, respectively. The induction of programmed cell death by TGF-beta 1 was confirmed by gel electrophoresis of DNA, where the characteristic 'DNA ladder' resulting from the internucleosomal DNA cleavage was visualized. The enhancement of cell mortality by TGF-beta 1 was associated with the inhibition of ornithine decarboxylase (ODC) expression (measured by the reverse transcriptase-polymerase chain reaction method) and impaired activity of this key enzyme in polyamine synthesis. Orotic acid (OA)--a known tumor promoter--stimulated proliferation of L1210 leukemic cells and diminished the necrotic effect of TGF-beta 1, but it did not change the extent of apoptosis evoked by TGF-beta 1. OA increased the expression of ODC and diminished depressional influence of TGF-beta 1 on transcription and activity of ODC in leukemic cells., In Conclusion: OA is a bioactive compound stimulating the growth of leukemic cells and diminishing the growth-inhibitory effect of TGF-beta 1. ODC gene is probably one of the targets for both OA and TGF-beta 1 influences in L1210 leukemic cells.

To examine the role of protein kinase C (PKC) in induction of human colon adenocarcinoma cell line, DETA/W, by polypeptide growth-promoting factors, ornithine decarboxylase activity (ODC) and DNA synthesis were determined in cells depleted of PKC. PKC depletion was achieved by prolonged cultivation (more than 30 passages) with 10(-6) M phorbol 12-myristate 13-acelate. Lack of PKC in studied cells was proved by measurements of PKC activity and immunoreactivity. Although ODC activities and DNA syntheses in PKC-depleted cells were decreased by about 40-50% compared to normal DETA/W cells, the percentage increase of these mitogen-responsive reactions was quantitatively similar in both cell sublines. These results raise the possibility that not all of the biological responses to growth factors are connected with the activation of calcium-dependent PKC.

In 56 patients with various liver diseases and in 15 healthy controls fasting serum concentrations of caffeine (HPLC method) and total endogenous bile acids (enzymatic-spectrophotometric assay) were determined. Serum caffeine concentrations were significantly higher in patients with chronic hepatitis or liver cirrhosis than in controls but no differences was found between patients with obstructive jaundice and controls. Contrary to caffeine, fasting serum bile acids concentrations were higher in all patients groups than in controls. In all studied groups there was no correlation between caffeine and serum bile acids estimations. In patients with liver cirrhosis there was correlation between caffeine test and the Child's classification score. However, no correlation existed between the Child's classification and the serum bile acids concentration. Our data suggest that fasting serum caffeine concentration is more usefull indicator of liver injury than determination of total endogenous serum bile acids.