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One of the key questions of biology is the nature and mechanisms of gene function. It has been 60 years since proposing the right-handed model of DNA double helix in 1953. This discovery was honored with Nobel Prize in 1962 and become a breakthrough in knowing and understanding mechanisms of heredity and genetic code. Since that time a great deal of data have been gathered considering functions, structure and DNA application. It became the basis of modern molecular biology, chemical biology and biotechnology. Today we know, that double helix is characterized by its dynamics and plasticity, which depend on its nucleotide sequence. Chromatin structure and DNA mediated charge transport have a crucial role in understanding mechanisms of its damage and repair. Progress in epigenetics allowed to identify new DNA bases, such as 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine. Design of new catalytic nucleic acids and the nanotechnology field of DNA origami reveal its application potential.

In the Salmonella antigenic pattern, more than one phase of flagellar antigen is observed. The phase of flagellar antigen depends of the gene which encodes the protein building the filament of flagella. The fliC gene encodes the 1st phase of flagellar antigen and the fljB gene encodes the 2nd phase of flagellar antigen. The third phase of flagellar antigen is encoded by one of the genes localized on the plasmid. Expression of the fljB gene (part of the hinfljBA operon) is regulated by a mechanism of DNA fragment sequence inversion. The hin gene, which encodes Hin invertase, flanked by two regions - hixL and hixR - is inverted by Hin invertase together with Fis protein. This process turns on or turns off of the hinfljBA operon. When this operon is turned on, FljB protein is produced (structural protein of flagella filament), and also FljA protein, which is a transcriptional repressor of the fliC gene. This means that one Salmonella cell could have only one phase flagellar antigen--1st or 2nd phase. Sometimes, due to mutation in one of the mentioned genes, naturally diphasic Salmonella strains have the ability to produce only one phase of flagellar antigen. Mostly monophasic Salmonella with an active fliC gene are observed. In recent years such a strain, Salmonella enterica with the antigenic formula 1,4,[5],12: i: -, is one of the most often isolated strains from human cases in many European countries.

Introduction: Infections caused by Staphylococcus epidermidis form frequent complications after implantation of foreign bodies, including orthopedic implants. The ability of adhesion to synthetic surfaces is influenced by both the properties of the microorganisms under concern as well as physicochemical properties of the implant surface. In this study, biofilm formation abilities by S. epidermidis strains on orthopedic implants made of different materials have been evaluated., Methods: Researches have been performed on 60 S. epidermidis strains. The implants for osteosynthesis--cortical bone screws: steel, steel coated with titanium and steel coated with nanocrystalline diamond were used for the analysis. Slime production ability was assessed using Congo Red Agar method. Cell surface hydrophobicity was determined by salt aggregation test (SAT) and bacterial adhesion to hydrocarbon test (BATH). Presence of the icaA, icaD, icaC and icaB genes was detected by PCR. Biofilm formation of S. epidermidis strains was analysed according to Richards method., Results: Out of the 60 strains 43% were labeled as slime producing. 48% of S. epidermidis strains formed biofilm on steel screws, 43% on titanium implants and 45% of strains on implants coated with nanocristalline diamond. IcaA, icaD, icaC and icaB genes have been detected in 31%, 25%, 20% and 27% of the strains respectively., Conclusions: The degree of biofilm formation was the lowest on the steel screws coated with titanium.

Unlabelled: OBJECTIVES. The ST-17 clone of Streptococcus agalactiae (Group B Streptococcus, GBS) identified by Multi Locus Sequence Typing (MLST) is recognized as a hypervirulent international clone mainly associated with invasive neonatal infections. There is no data on the occurrence of the ST-17 clone of GBS in pregnant women and newborns in Poland., Methods: 169 strains of GBS, isolated from pregnant women's carriage, and 42 from newborns with invasive infection or asymptotic carriage, which were derived from the collection of the Chair of Microbiology Jagiellonian University Medical College, collected between 2006-2010, were genetically characterized. The PCR with specific primers ST17S and ST17AS was adopted to characterize the gbs2018 gene variant specific to ST-17 clone. All positive results in direction of ST-17 were confirmed by MLST. The genetic relatedness of the ST-17 clone was determined by PFGE., Results: The hypervirulent ST-17 clone was detected in 2.4% of women with GBS asymptotic carriage, whereas it was detected in 18.2% newborns with invasive infection. No ST-17 was isolated from healthy neonates. Statistically significant correlation between the presence of ST-17 and neonatal GBS infection was showed, in comparison to healthy neonates colonized by GBS (p = 0.0398) and to GBS-positive pregnant women (p <0.00001). The ST-17 strains belonged to 2 clearly distinguishable PFGE type, but were closely related to one another at a level of 60% similarity. CONCLUSIONS. The results emphasize the necessity of a regular study of the ST-17 GBS clone occurrence among pregnant women and newborns in Poland.

Introduction: Mycoplasma pneumoniae is a common etiologic agent of community-acquired respiratory infection. In serological diagnosis of M. pneumoniae infections tests have been described based on the purified P1 protein, which is the most important virulence factor of this pathogen, as antigen. The aim of his study was to express and purify a recombinant protein P1 M. pneumoniae and next evaluate this protein as high specific antigen in serodiagnosis of mycoplasmosis., Methods: Protein P1 M pneumoniae was expressing in E. coli BL21 (DE3) using the pET-30 Ek/LIC expression vector. Based on published literature, we decided to express C-terminal region [P1-C1] encompassing amino acid residues 1160 to 1521. Purification was accomplished by immobilized metal (Ni2+) affinity column chromatography (His-trap). Serum samples collected from 221 patients with mycoplasmosis, positive in complement fixation test (CFT), 87 patients with other then mycoplasmosis bacterial infections and 80 blood donors were screened for anti-P1 recombinant protein IgA, IgG and IgM antibodies by using the home-made ELISA., Results: SDS-PAGE and Coomassie brilliant blue staining confirmed a high purity of the recombinant P1 protein preparation with an expected molecular mass of 39,7 kDa. The specificity of the recombinant protein was confirmed by western blot analysis using serum samples from rabbits immunized by M pneumoniae. The results of ELISA revealed that more then 70.0% of patients with mycoplasmosis confirmed by CFT, had antibodies to recombinant P1 protein in diagnostically significant level (x + 2SD). The antibodies were found only sporadically in sera obtained from patients with other then mycoplasmosis bacterial infections and clinically healthy persons. A comparison of results obtained in home-made ELISA with results of commercial western blot (Virotech) showed similar, ranged from 84.2% to 97.4%, compatible of results. The IgM antibodies to recombinant P1 protein were found in 87.2% sera obtained in acute phase of disease, in 80.0% sera obtained 2-4 weeks after onset of clinical symptoms and only in 43.8% sera obtained in chronic mycoplasmosis., Conclusions: The present study confirmed the earlier observations of the high usefulness of recombinant P1 protein for reliable serologic diagnosis of M. pneumoniae infection.

Introduction: Varicella-zoster virus (HHV-3) is spread by the respiratory route and disseminates to lymph nodes and then via lymph back to the skin, resulting with the rash of chickenpox. Like other alpha-herpesviruses, HHV-3 infects the neurons of the dorsal root ganglia, where it causes lifelong latency. Virus reactivation causes episode of herpes zoster (shingles). During severe HHV-3 infections molecular methods, such as real-time PCR (qPCR) assay, are recognized as a method-of-choice for detecting viral DNA in clinical specimens. The aim of this study was to develop the qPCR assay for detection and quantification of varicella-zoster virus DNA in different clinical samples, using specific primers targeting a HHV-3 DNA ORF62 gene and a fluorescent TaqMan probe., Methods: The analytical sensitivity of assay was tested using serial dilutions of viral DNA in range between 100 and 3 125 000 copies/ml. Limit of detection (LOD) was calculated using probit analysis, and was determined as 750 copies/ml. In further studies 18 clinical samples (sera, whole blood, skin swabs), taken from a small group of 5 children with symptoms of chickenpox were tested for the presence of varicella-zoster virus DNA, using LightCycler 2.0 system., Results: Described in-house qPCR method detected viral DNA in all examined specimens. Detected viral load was between 5750 copies/ml for sera samples and 27 300 000 copies/ ml for skin swabs, respectively., Conclusions: The results of this work showed that developed real-time PCR assay based on TaqMan probe was very reliable and valuable for detection and quantification of varicella-zoster virus DNA in different clinical samples. The high level of sensitivity, specificity, accuracy, and rapidity provided by the developed method are favorable for its use for the detection of HHV-3 DNA in laboratory practice.

Introduction: Among different laboratory techniques used in influenza surveillance, methods based on molecular biology, such as real-time PCR, play increasingly important role. They allow detection and identification of viruses currently circulating in human population and responsible for infections and diseases. The aim of the study was to develop duplex real-time PCR assay for detection and differentiation of influenza virus subtype A(H1N1) pdm09 and A(H3N2)., Methods: Specific primers targeting a highly conservative region ofhaemagglutinin gene and a dual-color TaqMan probes, labeled with fluorophore JOE (560 nm) and quencher BHQ-1 or CalFluor Red 610 (610 nm) and BHQ-2 were designed. The specificity of duplex reaction was evaluated using RNA isolated from reference strains of influenza virus A(H1N1)pdm09, A(H3N2), A(H1N1) and A(H5N1), B and other respiratory pathogens. Sensitivity of the assay was tested using serial dilutions ofplasmid constructs carrying fragment of specific viral HA gene, in range between 10 and 10(5) copies/sample. A number of 116 clinical samples were tested using developed duplex qPCR assay in comparison with the CDC monoplex assay,, Results: Positive duplex qPCR results were obtained only in samples containing RNA of influenza viruses of a specific subtype. The limit of detection (LOD) of developed method amounted up to 27 copies/sample for A(H1N1)pdm09 and up to 37 copies/sample for A(H3N2)., Conclusions: The results show that developed TaqMan-based duplex qPCR assay is a valuable diagnostic tool in influenza virus surveillance for using in direct study of clinical specimens as well as identification of isolated strains of influenza virus.

Introduction: Adhesion of pathogenic bacteria to host cells is a crucial step during infection. The presence of specific adhesion factors on the bacterial cell surface determines the tropism of the pathogen to the tissues expressing certain surface receptors. The adhesion is mediated primarily by filamentous structures called pili. Three distinct pilus structures can be produced by Corynebacterium diphtheriae: SpaA-, SpaD- and SpaH-type pili. Pilus genes encode a total of nine pilus proteins, named SpaA through SpaI, and six sortases, named SrtA through SrtF. All the pilus genes are located on pathogenicity islands and can be acquired and lost by different strains. The aim of presented studies was assessment of occurence of pili genes among C. diphtheriae strains isolated from different infections, including invasive infections, in Poland., Methods: Thirty-one toxigenic and nontoxigenic C. diphtheriae strains isolated from wounds, blood, nose and pharynx were investigated for presence of 15 pili genes. The studies were conducted using PCR. Gene specific primers were designed on the basis of the complete genome sequence of C. diphtheriae NCTC 13129., Result: All the nontoxigenic C. diphtheriae strains isolated from invasive infections possess every tested genes in contrast to toxigenic strains that revealed highly mosaic structure of pili gene clusters. Differences in gene content were detected in SpaA- and SpaH-type pili gene clusters. Complete set of genes in SpaD-type pili gene cluster was detected in all but two strains. The two strains did not possess any of SpaD-type pili genes., Conclusions: Invasiveness of C. diphtheriae strains could be related to adhesive factors. Results of our studies suggest that ability to express all types of pili is indispensable for causing invasive infections by nontoxigenic C. diphtheriae. Whereas full set ofpili genes is not necessary for causing classical diphtheria.

Aim: The aim of the study was the evaluation of clinical symptoms suggesting Epidermolysis bullosa dystrophica in a 5 years old boy and identification of an underlying molecular defect., Material and Methods: The patient was a 5 years old boy with the history of transient bullae and milla formation in the fi rst three years of life In the last two years such tendency has not been observed, with the exception of dystrophic toenails. The family history of bullous diseases is negative. Molecular analysis was performed using direct sequencing of COL7A1 gene encoding collagen type VII., Results: A novel mutation p.Gly2064&#95;Arg2069del was identified in the patient. The identified mutation does not appear in the patient's parents nor in the patient's brother. Molecular analysis in the family members revealed that the mutation occurred de novo. Biparental inheritance of genetic material for chosen genetic markers was confirmed., Conclusions: On the basis of clinical features observed, results of molecular studies and literature data, diagnosis of mild dominant Epidermolysis bullosa dystrophica can be established in the index case.

The human polyomavirus BK (BKV) is wide-spread pathogen, associated with urogenital tract disorders or even nephropathy in immunosuppressed patients. Nowadays molecular detection by real-time PCR (qPCR) is recognized as a method-of-choice for detecting human polyomaviruses in clinical samples. The aim of the study was development of real-time PCR assay for detection and quantification of polyomavirus BK DNA in clinical samples, using specific primers targeting a viral DNA VP3 gene and a TaqMan hydrolyzing probe. The analytical sensitivity of assay was tested using serial dilutions of BKV DNA in range between 13500 and 15 copies/ml. 27 urine samples and 23 plasma samples taken from a group of 22 adult recipients of allogeneic HSCT were tested for the presence of polyomavirus BK in the LightCycler system. Described in-house real-time PCR assay detected BKV DNA in 8 specimens (6 urine and 2 plasma). Detected average viral load was 170 copies/ml for plasma and 1250 copies/ml for urine samples, respectively. The results of this study show that developed TaqMan-based probe qPCR assay is very reliable and valuable for detection and quantification of BKV DNA, both in urine and plasma samples. These data, combined with its rapid turnaround time for results and decreased hands-on time, make the LightCycler PCR assay highly suitable for the rapid diagnostics of polyomavirus BK infections in the clinical laboratory.

Humanin (HN) is a newly discovered 24-amino acid peptide, which may suppress neuronal cell death. HN cDNA includes the open reading frame (HN-ORF) of 75 bases, located 950 bases downstream of the 5' end of the HN cDNA. It was demonstrated that HN cDNA is 99% identical with mitochondrial DNA (mtDNA) sequence. HN homologues have been identified as expressed sequence tags (ESTs) in rat and nematode. Certain regions homologous to the HN cDNA exist on human chromosomes. HN forms homodimers and multimers and this seems to be essential for the peptide functions. HN acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2). It was demonstrated that HN plays a protective role by an antiapoptotic activity interfering with Bax activation, and suppressing Bax-dependent apoptosis. HN is also shown to suppress the c-Jun N-terminal kinase (JNK) and ASK/JNK-mediated neuronal cell death. Several studies also confirm that HN could be important in prevention of angiopathy-associated Alzheimer's disease dementia, diseases related to mitochondrial dysfunction (MELAS), and other types of beta-amyloid accumulation associated neurodegeneration. A very recent study demonstrated a pluripotent cytoprotective effect and mechanisms of HNs in cells other than from the CNS, such as germ cells, or panreatic b-cells, and potent physiological consequences that result from HN interaction with IGFBP3 and STAT3. The in vivo studies suggest that humanin may protect against cognitive impairment, also due to ischemia/reperfusion injury.

Allogeneic hematopoietic stem cell transplantation (alloSCT) is a curative treatment for many patients suffering from malignant and non-malignant hematological disorders. Successful transplantation is a process that requires the engraftment of transplanted pluripotent hematopoietic stem cells which re-establish normal hematological and immunological systems. Distinguishing between host and donor origin of bone marrow and blood cells is vitally important for monitoring of the engraftment process. One of the most useful tools for engraftment monitoring is the assessment of hematopoietic chimerism. Which occurs after alloHSCT and describes the percentage of donor hematopoietic and lymphoid cells in a transplant recipient. 38 adult patients, after alloSCT performed in Katedra i Klinika Hematologii Collegium Medicum UJ entered the study and the total number of transplantations was 43. The evaluation of hematopoietic chimerism was based on PCR amplification of polymorphic non-coding DNA sequences--short tandem repeats (STR-PCR). The main tool was a semiquantitative method--fragment length analysis. The product of amplification was analyzed using the sequencer. The second method was based on a quantitative Real Time PCR technique (RQ-PCR) based on SYBRgreen chemistry. There were performed amplification of biallelic non-coding DNA sequences with short insertions or deletions. Hematopoietic chimerism evaluations were performed on +30, +60, +90, +120, +150, +180, +270 and +360 day and then every 6 months post alloSCT on peripheral blood or bone marrow samples. STR-PCR and RQ-PCR chimerism assays were compared and results evidenced the greater sensitivity of RQ-PCR method. There were not crucial differences in the results of chimerism evaluation obtained by means of these two methods. The analysis of chimerism kinetics after allogeneic stem cell transplantation allowed to modify the post-transplantation-treatment in 3 patients after alloNMSCT leading to increase of donor-origin hematopoiesis in transplant recipients (in 2 pts decision of DLI, 1 of them withdrawal of immunosuppression, 1 pt giving G-CSF). The results of chimerism monitoring confirmed that the failure of achieving a CC or lost of CC can predict the relapse of the disease.

In our study we analyzed the nucleotide sequence of the C- terminal 256 bp fragment of the myfA gene encoding MyfA protein, the major subunit of Yersinia enterocolitica Myf fimbriae. We examined ten representative strains of major Y. enterocolitica pathogenic bioserotypes belonging to European (4/O3; 2/O9; 3/O5,27) and American (1B/O8) phylogenetic lineages. DNA sequencing revealed that consensus nucleotide sequences of the tested myfA fragment were indistinguishable in all the tested strains. The resulting common consensus sequence found in our study was identical to the corresponding fragment of reference sequences Z21953 and NC008800 deposited in GenBank database for pathogenic Y. enterocolitica strains. In contrast, 18 point mutations leading to 13 amino acid substitutions were found when the common consensus sequence was aligned to sequence AY966879 determined for the myfA homologue detected by PCR in Y. enterocolitica 1A strain. The strong conservation of the nucleotide and amino acid sequence of myfA gene among virulent bioserotypes of Y. enterocolitica indicate that fimbriae MyF could play important role in pathogenesis, even before the divergence of European and American lineages.

The aim of study was to evaluate the applicability of the PCR multiplex method for the identification of Streptococcus agalactiae (group B streptococci; GBS) serotypes and to indicate the participation of serotypes in the group of pregnant women. 1178 pregnant women at 35-37 weeks of pregnancy were studied between 2007-2009. Vaginal and rectal swabs collected from these women were tested for the presence of GBS. S. agalactiae identification was done basing on the culture method according to the CDC recommendations. For all isolated GBS strains the surface polysaccharides which conditioning serotypes were determined using commercially available serological latex kit and multiplex PCR method with two sets of specific primers. The frequency of GBS carriage among 1176 pregnant women was 30% (353 cases). Simultaneous colonization of the vagina and anus was demonstrated in 80% women, whereas GBS carriage only in the vagina or only in the anus was indicated in 9% and 11% cases, respectively. In pregnant women colonized by GBS serotype III (35%) dominated. The other serotypes were detected with the following frequency: Ia (20%), V (17%), II (15%), Ib (8%) and IV (5%). In the analyzed women population, GBS strains belonging to the VI, VII, VIII serotypes were not found. The results of our study show a great diagnostic usefulness of multiplex PCR method for identification of genes encoding GBS surface polysaccharides that are typical for individual serotypes.

Human herpesvirus 7 (HHV-7) is a beta-herpesvirus widely spread within a population and has been recognized as a potential pathogen in immunocompromised hosts. The goal of the study was development of real-time PCR assay for detection of human herpesvirus 7 DNA in clinical samples, using primers targeting a conserved region of the viral DNA major capside proteine gene and a specific TaqMan hydrolyzing probe. Sixty four plasma samples taken from a group of adult recipients of allogeneic HSCT, during detectable CMV viremia or neutropenic fever, were tested for the presence of viral DNA in the LightCycler system with method described above. HHV-7 DNA was detected in 40 specimens (62.5%). The conclusion is that developed TaqMan-based probes real-time PCR test is very reliable and valuable tool for detection of HHV-7 viremia in plasma samples. The high level of sensitivity and accuracy provided by the LightCycler instrument is favorable for the use of this method in the detection of human herpesvirus 7 DNA in clinical specimens.

Rapid detection and identification ofmethicillin-resistant staphylococci in patient's blood is essential for the prompt antimicrobial therapy of infection. Molecular-based diagnosis, in comparison to conventional methods, allows to increase sensitivity and to reduce time necessary to achieve positive results and also enable to test susceptibility. The aim of the study was the use of multiplex PCR to detect region of the 16S rRNA that is unique to staphylococci, the S. aureus-specific clfA gene and the mecA gene which is a determinant of methicillin resistance, in a medium of the BACTEC blood culture system. Three different extraction methods were examined in order to ascertain the most suitable method to isolate staphylococcal DNA. The only method which removed PCR inhibitors contained in BACTEC blood culture material use Triton X-100 and lysostaphyin to lysis bacterial cells. The use of High Template Preparation Kit failed to yield a positive result in all investigated probes. Third of the methods, based on alkali lysis, resulted in DNA which could be amplified by PCR only in case of S. aureus blood culture. The findings of our study suggest that not all DNA extraction methods are appropriate because some of them may not remove potent amplification inhibitors found in blood and medium of system BACTEC.

Aim: The paper focuses on candidate gene polymorphism and on the role of dopamine transporter gene polymorphism DAT 1 in the pathogenesis of alcoholism. We investigated this polymorphism in the association study in a whole group of alcoholics (n = 150), fathers (n = 84) and mothers (n = 101) and patients with alcohol dependence (n = 103). The control group consisted of healthy volunteers with excluded psychiatric disorders, gender and age matched (n = 183). The transmission disequilibrium test (TDT) was used in the study. At the last stage of the study we screened the DNA sequence and compared 9 VNTR and 10 VNTR. METHOD; The history of alcoholism was obtained using the SSAGA (Semi-Structured Assessment for the Genetics of Alcoholism - Polish version). The DAT 1 polymorphism was determined using PCR. Screening for DNA sequence variation in the dopamine transporter gene polymorphism DAT 1 was determined using ABI 310., Results: We did not find significant differences in the case-controlled study. The alleles and genotypes distribution of the investigated polymorphism did not differ significantly between the alcoholics and the controls in the case-control study. We found significant differences in allele transmission in our patient group (n = 77) 10 VNTR 63% and 9 VNTR 37% (p = 0.033), from mothers to proband (p = 0.049) and a statistical trend to frequent 10 VNTR allele transmission from the fathers (p = 0.071). Screening for DNA sequence variation in the dopamine transporter gene polymorphism DAT 1 showed the number 9 repeat in 9 VNTR as missing., Conclusion: The results of this study suggest that DAT 1 gene polymorphism plays a role in alcohol dependence.

Objectives: Ovarian cancers still remain a significant worldwide epidemiological problem. The vast majority of newly diagnosed cases are in advanced clinical stages, and the five-year survival rate in all clinical stages amounts up to less than fifty percent. A better understanding of the biology of ovarian cancer can bring us closer to successful treatment and recovery. The aim of this study was to evaluate the expression of luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor (LH/hCGR) and the expression of hCG protein in the ovarian cancer tissue., Materials: Histologically proven primary ovarian cancer samples (n=20) were obtained from women undergoing gynecologic surgery. Frozen sections of ovarian cancer tissues were evaluated by means of indirect immunofluorescence., Results: The expression of hCG protein was ubiquitous throughout all samples, while LH/hCGR was detected in only sixty percent of samples. Co-expression of LH/hCGR and hCG protein was detected in some individual cells in tumor tissue. Some cancer cells expressed only hCG protein, but not LH/hCGR., Conclusions: Expression of LH/hCG receptor is a characteristic feature of various histological types of ovarian cancer. Co-expression of LH/hCGR and hCG protein in individual cancer cells may point to an autocrine or paracrine mechanism of &#95;hCG activity in the tumor microenvironment. Further studies are needed to evaluate LH/hCGR and hCG protein function in the course of neoplastic development.

Unlabelled: H. pylori is a well recognized pathogenic factor of chronic gastritis and class I carcinogen. Specimens are collected on gastroscopy performed due to clinical symptoms suggesting upper alimentary tract organic disease for pathomorphological examinations and for the estimation of the presence of H. pylori infection in urease test, histological examination in Giemsa staining method or in a culture. The aim of the project was to compare the detection of H. pylori in gastric mucosa in children by conventional method and polymerase chain reaction technique (PCR)., Material and Methods: The study comprised 137 children and adolescents, aged 5-18 years (mean 13.2 years), in whom basing on the evaluation of the level of serum anti-H.pylori antibodies (ELISA), urease test and histological examination (HE), H. pylori infection was diagnosed or excluded. H. pylori genes: ureA, vacA, cagA were identified in gastric mucosa specimens with PCR method., Results: On the grounds of standard methods H. pylori was detected in 86 (62.8%) and excluded in 51 (37.2%) children. UreA gene was detected in gastric mucosa in 82 (95.3%) children with H. pylori (Hp+) and in 8 (15.7%) without H. pylori (Hp-) infection (p < 0.001). CagA H. pylori gene was identified in 21 (24.4%) children from Hp+ group and in 1 (2%) from Hp- group (p < 0.001), and vacA gene in 23 (26.7%) and 6 (11.8%) (NS), respectively. H. pylori strains type I (cagA+/vacA+) were found in gastric mucosa of 15 (17.4%) Hp+ patients. CONCLUSIONS. Testing of H. pylori genes (particularly ureA gene) with PCR technique in mucosa bioptates enables to diagnose H. pylori infection even in patients with the infection not detected with standard methods.

Infections with human adenoviruses are common worldwide and cause a wide range of signs and symptoms. Nowadays in current diagnostics procedures older virological methods, such virus isolation in a cell cultures, are replaced with molecular biology tests. The aim of the study was development of real-time PCR assay for detection of human adenoviruses. DNA isolated from A549 cell line infected with five different HAdV strains was used for development of a qualitative real-time PCR assay for detection of all human adenoviruses using primers targeting a conserved region of the hexon gene and a specific TaqMan probe. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of HAdV7 DNA in range between 10(0) and 10(-6). For comparison typical end-point detected PCR for adenovirus detection with the same DNA dilutions was made. The sensitivity of novel method; was about thousand-fold higher than older one. The conclusion is that real-time PCR is very advisable in diagnostics of diseases caused with adenoviruses. The high level of sensitivity, specificity, accuracy, and rapidity provided by this assay are favorable for the use in the detection of adenoviral DNA in clinical specimens, especially from neuroinfections or immunocompromised hosts.

In the last decade the abnormalities in male infertility became the main problem of more than 50% couples. The main reasons of male infertility are abnormal gonadotropin releasing hormone (GnRH) secretion, pituitary gland diseases and presence of testicular genetic defects. The male infertility also may result from chemotherapy, radiotherapy and viral infections. The main genetic factors responsible for male infertility encompass the mutations of genes, which encode important factors of spermatogenesis. Recently mRNAs of numerous genes have been identified in spermatozoa. The first transcripts found in spermatozoa included protamine 1 and 2 (PRM1 and PRM2). Protamines are basic polypeptides, which form complex with DNA in spermatids and spermatozoa. Structure of PRM1 and PRM2 genes and function of these proteins suggest the possible relationship between of protamines expression disorders and male infertility. The PRM1, PRM2 and transition proteins 1 and 2 (TP1 and TP2) play important role in DNA condensation. We attempted to present current knowledge regarding structure and expression regulation of PRM1 and PRM2 genes. We also discussed the effect of disorders of PRM1 and PRM2 expression on male infertility.

Ribosomal RNA (rRNA) is a component of the ribosomes. Eukaryotic ribosomes contain four different rRNA molecules: 18S, 5,8S, 28S and 5S rRNA. rRNA is the most conserved (least variable) gene in all cells. For this reason, genes that encode the rRNA (rDNA) are sequenced to identify an organism's taxonomic group, calculate related groups, and estimate rates of species divergence. Especially the internal transcribed spacers (ITS) are very useful for molecular diagnostic of parasite. They are noncoding regions of DNA sequence that separate genes coding for the 28S, 5.8S, and 18S ribosomal RNAs. These ribosomal RNA (rRNA) genes are highly conserved across taxa while the spacers between them may be species-specific. In this paper authors describe practical using of rRNA gene to parasite diagnostic.

In a paternity test with 21 short tandem repeats (STRs) an isolated exclusion for the TH01 locus was observed. The probability of paternity or maternity in this case turned out greater than 99,999%. The analysis of sequence indicates only the deletion character of the observed mutation event without any changes in the conservative sequence.

Many Salmonella Enteritidis virulence factors are encoded by genes localized on plasmids, especially large virulence plasmid, in highly conserved fragment, they create spv plasmid gene group. The aims of realized researches were spv genes occurrence evaluation and composition analysis among Salmonella Enteritidis strains caused infection in chickens. Researches were realized on 107 isolates, where in every cases large virulence plasmid 59 kbp size were detected. Specific nucleotides sequences of spv genes (spvRABCD) were detected in 47.7% of isolates. In the rest of examined bacteria spv genes occurred variably. Most often extreme genes of spv group, like spvR and spvD were absent, what could indicate that factors encoded by them are not most important for Salmonella Enteritidis live and their expressed virulence.

In many biological systems substantial roles are played by interactions between amino acids and RNA. Among amino acids L-arginine seems to be particularly relevant, because the guanidinium group of arginine side chain can potentially form five hydrogen bonds with appropriately positioned acceptor groups of RNA. Extensive studies reveal that specific arginine recognition is achieved by many different RNAs over a broad range of binding affinities. Arginine is frequently found among amino acids in the nucleic acid-binding motifs in various proteins. For example, specific binding of the HIV-1 Tat protein to its RNA site (TAR) is mediated by a single arginine residue. Free arginine can be also bound by the guanosine site in the group I Tetrahymena ribosomal RNA intron catalytic centre, as well as by numerous RNA motifs, called arginine aptamers, which have been selected in vitro.

The aim of this study was to investigate the mutations in gyrA gene at Thr-86 position in fluoroquinolone resistant C. jejuni clinical isolates (2003-2005). The change of Thr to Ile at 86 position is associated with high-level resistance to fluoroquinolone in C. jejuni. Thirty five (58%) of 65 C. jejuni strains were found to be resistant to ciprofloxacin using E-test method. PCR-RFLP technique with the RsaI enzyme was used for the identification of mutation in gyrA gene. The primers spanning a part of the fluoroquinolone resistance determining region (QRDR) were designed based on the article of Alonso et al. and the gyrA sequence of C. jejuni (Gen Bank accession number LO4566). One of this primer had mismatch introduced at the second nucleotide from 3' end of the primer what gives an artificial RsaI cleavage site. All of the ciprofloxacin-resistant isolates contained a single)point mutation in the gyrA gene: the replacement of Thr 86 by Ile. The results showed that PCR-RFLP is a rapid and simple method for the detection of the high-level fluoroquinolone resistance in C. jejuni.

Post-transcriptional processes influencing mRNA stability are an important control point in gene expression regulation. The best-studied regulatory elements controlling mRNA stability are AU-rich sequences (AURE) found in 3'UTRs of many genes coding cytokines and stress response proteins. The presence of these elements results in short half-life of mRNA, but such mRNA half-life is prolonged upon cell stimulation or exposition to stress factors. Research on AURE-mediated mRNA decay resulted in discovery of AURE-binding proteins and in elucidating signal transduction pathways involved in the process of mRNA stability regulation. Defects in mRNA stability regulation can result in diseases such as lymphomas, neuromas or immunological and inflammatory disorders.

The problem of species recognition in relation to parasite organisms is discussed, based on literature data. Taking into account that over 20 species concepts have hitherto been formulated, the choice of characters, which should define a species differs from author to author. Lately the differences in some gene fragments are regarded as one of most important feature for species differentiation. On this basis some lineages, genotypes or strains have been differentiated within some species, but in most cases the respective authors did not decide to elevate these units to the species level. The present paper focuses on the studies on species representing three genera (Taenia, Echinococcus, Trichinella) as they show, especially the complex studies on the species within the genus Trichinella, how big material is necessary to formulate any proper conclusion and, on the other hand, that the differences in the DNA sequences cannot be the only characters defining species; some other features (morphological, biological, behavioral) should not be considered redundant.

Evolution and systematics of nematodes based on molecular investigation. The use of molecular phylogenetics to examine the interrelationships between animal parasites, free-living nematodes, and plant parasites versus traditional classification based on morphological-ecological characters was discussed and reviewed. Distinct differences were observed between parasitic nematodes and free-living ones. Within the former group, animal parasites turned out to be distinctly different from plant parasites. Using small subunit of ribosomal RNA gene sequence from a wide range of nematodes, there is a possibility to compare animal-parasitic, plant-parasitic and free-living taxa. Nowadays the parasitic nematodes expressed sequence tag (EST) project is currently generating sequence information to provide a new source of data to examine the evolutionary history of this taxonomic group.

Many laboratories have been involved in characterization of the estrogen receptors (ER-alfa, ER-beta) with special emphasis on the number of isoforms of this receptors and their role in the pathogenesis of many gynecological disorders. ER isoforms, generated by alternative mRNA splicing have been defined in several tissues and are postulated to play a role in modulating the estrogen response. These splicing variants were shown to act differently from the wild-type receptors and their biological significance remain unknown but same studies have implicated a role of specific isoforms in the pathogenesis of estrogen dependent tissues. Expression of the estrogen receptors, as well as their splicing variants may impact on the biologic properties of same tissues and affect their ability to respond to estrogen and antiestrogen therapies. From the clinical point of view it is importance to know what molecular characteristic of ERs and their isoforms occures in estrogen dependent tissues before histopathological and clinical changes. All of these developments have had a great impact on our understanding of the estrogen action.

Recently, the single strand conformation polymorphism (SSCP) analyses were shown to be useful for identification a variety of bacterial genes. Although, SSCP was successfully applied for detection of single nucleotide polymorphism (SNP), it was also considered a time consuming and insufficiently reliable technique. Therefore, the modified Multitemperature-SSCP method was introduced. It was shown to be reliable and time effective technique due to a stringent control of the gel temperature and utilization of a high voltage up to 1 kV. In this study the usefulness of MSSCP for differentiation of gene variants and detection of the single point mutations was evaluated, using Yersinia enterocolitica O3 and O8 ail alleles or genes blaCTX-M-15 and blaCTX-M-3, which differs by a single point mutation. The 425 and 251 bp fragments of O3 and O8 ail alleles containing 15 and 11 point mutations respectively, as well as 277 and 208 bp fragments of both blaCTX-M genes differing in positions 243 and 62 were PCR amplified, denatured and loaded onto 7% and 9% polyacrylamide nondenaturing gel. Electrophoresis was carried out in the DNA-Pointer apparatus (Kuchrczyk, Poland) at voltage ranging from 500 to 750 V. The thermal profile consisted of 50 min. at 35 degrees C, 40 min at 20 degrees C and 40 min at 5 degrees C. Obtained results showed, MSSCP was capable to differentiate ail alleles independently of the length of analyzed fragment. However, the 425 bp fragment profile consisted of three bands, whereas 251 bp revealed two bands. The single point mutation in blaCTM, genes was also successfully distinguished by MSSCP in both tested fragments. Surprisingly, 277 bp fragment profile showed differences more apparently than 208 bp. Summarizing, MSSCP was found to be useful, sensitive and time efficient tool for detection of multiple and single point mutations in DNA fragments ranging from 208-425 bp.

Vaccinology became interdisciplinary field of knowledge. It takes advantage of epidemiological and immunological researches and achievements of molecular biology, biotechnology and genetic engineering. Nowadays vaccinology remains mainly immunoprevention, but immunization of organism, as a result of vaccine injection, opens new possibilities in medicine. Directions of development present vaccinology are subordinated to one task--to invent an ideal vaccine. According to Edelman's criterions this vaccine has to be safe, effective in immunization, efficient in disease prevention and accessible for people all over the world. Realization of this aim causes modification of antigens, changes of adjuvant and preservatives and applying new technologies in production of vaccines.

Clostridium perfringens strains of A type capable of enterotoxin (CPE) synthesis may be a potential source of food-poisoning. Suitability of methods for CPE detection on the protein level is limited by difficulties in inducing sporulation in vitro. A number of unknown facts concerning coregulation the sporulation processes and CPE synthesis are recognised. The goal of the work was to determine the level of correlation between CPE synthesis and spores formation. Enterotoxin and cpe gen were detected by RPLA after sporulation induction test and by methods based on amplification on the DNA and mRNA levels. Sixty-four C. perfringens strains of A type isolated from patients with food poisoning symptoms and from food samples were analysed. Collection of isolates was differentiated as not enterotoxic, enterotoxic, and potentially enterotoxic strains based on appropriate strain profile: plc(+), cpe(-), CPE(-); plc(+), cpe(+), CPE(+); and plc(+), cpe(-), CPE(-), respectively. No significant difference between expression of cpe mRNA in vegetative and sporulation phase was found. The obtained results indicate that sporulation is not an essential factor for cpe gene expression.

E. coli belonging to the O157 serological group are among the organisms isolated most frequently out of all the so called entero-hemorrhagic E. coli strains (EHEC). Since several years they have been isolated also in Poland. The purpose of the present study was determination on selected phenotypic and genotypic properties of E. coli O157 strains isolated in our country from clinical material samples and from food. The serotype of the strains was determined, together with the following properties regarded as pathogenicity markers of verotoxic E. coli strains such as absence of beta-glucuronidase activity and sorbitol fermentation ability, as well as production of verotoxins SLT I and/or SLT II and entero-hemolysin. Besides that, by the PCR method the fragments of the genes coding for verotoxins, intimin and enterohaemolysin were amplified. The products of PCR were analysed by the restriction enzyme analysis (RFLP). All verotoxic E. coli O157 strains isolated in Poland were analysed by the pulsed field gel electrophoresis of genomic DNA (PFGE). The studied group comprised E. coli O157 strains, among them 40 strains were isolated from human faeces and 5 from food. The remaining strains were the reference E. coli O157:H7 EDL 933 and G 5244 strains and strains from NIH collection. The obtained results showed that the tested strains were a very varying population. 21 of them (all isolated from food, 11 from faeces and 5 reference strains) belonged to serotype O157:H7, five were not peritrichous O157:NM and the remaining ones had other ciliary antigen than H7. All strains isolated from food, reference strains and only 3 O157:NM strains isolated from humans were verotoxic. The strains from food and two reference strains produced only SLT II, 2 of 3 strains isolated from humans and one reference strain also produced only SLT II and the other produced both verotoxins. Apart from these 13 verotoxic strains all remaining strains caused sorbitol fermentation.

Prognostic value of p53 gene mutation was determined in 95 radically operated non small cell lung cancer patients (78 males and 17 females, mean age 57.8 years). Study group included 62 cases of squamous cell carcinoma, 30--adenocarcinoma and 3--large cell carcinoma. There were 52 patients in stage I disease, 16--in stage II, 26--in stage IIIa and one--in stage IIIb. Paraffin-embedded samples of resected tumors were assayed for p53 mutations with the use of PCR/SSCP analysis. p53 mutation were present in 22 cases (23%). The median survival in patients with and without p53 mutations were 49 and 75 months (p = 0.46), respectively, and the five-year survival rate 53% and 50%, respectively. In stage I disease the median survival for patients with p53 mutation was 53 months and for those without mutations the median survival could not be determined as more then a half of them were alive. Median survival in stage II patients with and without mutations was 35 months and 44 months (p = 0.62), and in stage IIIA--9.5 months and 17 months, respectively (p = 0.37). The results of this study indicate that p53 gene mutation is not correlated with prognosis in non-small cell lung cancer patients.

Atrial natriuretic peptide (ANP) is involved in the pathogenesis of sodium-sensitive hypertension. The loss of Sca I restriction site in the ANP precursor gene abolishes the regular stop codon. The aim of our study was the analysis of the Sca I gene polymorphism in 23 patients with sodium-sensitive hypertension, the molecular characteristic of the mutation and the comparison of the blood pressure values, plasma renin activity, plasma ANP and aldosterone concentration between patients with or without mutation. Applying the polymerase chain reaction (PCR), followed by digestion with Sca I, the heterozygous mutation has been found in 9 (39%) patients. The sequencing of PCR products indicated that the loss of Sca I restriction site is caused by T2238-->C transition leading to the translation of ANP with two additional arginines. The higher concentration of ANP in plasma has been found in T2238-->C transition patients on normal and high sodium diet as compared with patients without mutation. These preliminary results suggest that the heterogeneity of sodium-sensitive hypertension is associated with the T2238-->C mutation of the ANP precursor gene.

Nephropathy is a frequent complication of long term diabetes. Diabetic nephropathy is the major determinant of premature morbidity and mortality both in insulin-dependent (IDDM) and in non-insulin dependent-diabetes mellitus (NIDDM). There is good evidence that genetic predisposition plays a major role in development of diabetic nephropathy. This hypothesis is based on the observation that diabetic nephropathy clusters within families, both in IDDM and NIDDM. Components of the renin-angiotensin system (RAS) are plausible candidate genes to examine for a association with microalbuminuria and diabetic nephropathy. In this study we compared the distribution of PstI melting polymorphism at the ACE locus among NIDDM patients with diabetic nephropathy and in patients who, despite long duration of NIDDM, remain without this complication. The 220 NIDDM patients for whom DNA was available were classified into two groups according to their renal status: normoalbuminuric control subjects (n = 80) who are NIDDM patients with an A/C ratio < 2.5 and nephropathy cases (n = 140) who are NIDDM patients with A/C ratio > 2.5. Albumin excretion rate was assayed by radioimmunoassay. HbA1c was assayed using HPLC methods, creatinine--using Jaffe methods and DNA analysis using PCR reaction, and then after the amplification product was digested with PstI enzyme. The study revealed that PstI sequence differences ("+/= and -") in the ACE gene do not contribute to genetic susceptibility to diabetic nephropathy in NIDDM.