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Open access

Joanna Zeyland, Daniel Lipiński and Ryszard Słomski

Abstract

Mechanisms regulating the activity of the complement system responsible for the rejection of transplant organs are balanced so that the attack is instantaneous but is restricted to the infected cells of the organism. The most important components regulating its activity comprise CD55 and CD46 factors as well as the CD59 anchored in the cell membrane. Hyperacute response of the immunological system appears to be the key in the xenotransplant rejection and the elaboration of methods preventing its occurrence will give a real chance for the development of xenotransplantation.

Gene constructs containing coding sequences of human CD46, CD55 and CD59 were prepared and used to transfect porcine fetal fibroblasts. Stable lines were molecularly characterized for an integration of transgenes by PCR. Lines with a stable integration of transgenes were subjected to further characterization of expression by RT-PCR and vitality test. Molecular characteristics of the transgenic cell lines obtained revealed a steadfast integration and, in the majority of cases, expression of the introduced transgenes. The performed cytotoxicity analysis demonstrated that transgenic lines were characterised by a higher survivability rate than non-transgenic cells in the presence of human serum which proved their protective influence in relation to the activity of the complement system.

Open access

Krzysztof Waliszewski, Ryszard Słomski, Grzegorz Oszkinis, Wacław Majewski and Katarzyna Pawlaczyk

Increased Frequency of Ace Gene DD Genotype in Normotensive Male Smokers with Abdominal Aortic Aneurysm

The aim of the study was to assess whether the association between abdominal aortic aneurisms (AAAs) and the angiotensin-converting enzyme (ACE) DD genotype is changed in male smokers.

Material and methods. The ACE I/D polymorphism was examined by PCR in 112 subjects with AAA and in 50 control subjects. The frequency of ACE genotypes (II, ID and DD) in the patient group was assessed in subgroups divided on the basis of blood pressure, smoking and sex.

Results. A significant increase in the frequency of ACE DD genotypes has been found in all AAA patients studied (odds ratio OR=2.6; range 1.3-5.6, p <0.0002) when compared to the remaining ACE genotypes (ID and II) and the control group. The respective values were higher when all 74 male smokers with AAA were assessed (OR=3.6; p <0.006). No increase in ACE DD genotype frequency was seen in all females (38) and male nonsmokers when compared to the respective control group (n=16) (OR=1.4; p <0.75).

In the group of normotensive AAA patients, the proportion of ACE DD genotype in relation to the remaining ACE genotypes (ID and II) was found to be higher than in the general AAA patient population (odds ratio OR=6.14; range 2.5-14.9, p <0.0001). In 32 normotensive male smokers with AAA, the respective values were higher (OR=8.3).

Conclusions. An increased frequency of the ACE DD genotype in normotensive male smokers may account in large part for the increase in DD genotype occurrence noted in all AAA patients.

Open access

Daniel Lipiński, Joanna Zeyland, Andrzej Pławski and Ryszard Słomski

Determination of the Absolute Number of Transgene Copies in CMVFUT Transgenic Pigs

The aim of this research was to determine the number of transgene copies in the DNA of transgenic pigs. The copy number of the transgene was analysed in the transgenic animals with introduced pCMVFUT genetic construct containing a coding sequence of human H transferase under a control of CMV promoter. The copy number of the transgene that had integrated with the genome of the transgenic animals was analysed by qPCR with SYBR Green dye, which enabled nonspecific double-stranded DNA detection. CMVFT-2F and CMVFT-2R primers were used to amplify a 149 bp fragment of DNA. Forward primer had a sequence complementary to a promoter sequence and reverse primer to a coding sequence of H transferase. The copy number of the transgene in the examined samples was established by plotting the CT values obtained on a standard curve, which had been set by the usage of the CT values for the successive standard dilutions with known copy number (1.438-1.431 copies). As a standard we used pCMVFut genetic construct hydrolyzed with Not I restriction enzyme to a linear form. The real-time PCR results helped to establish the range of 3 - 4 as the number of the transgene copies that had integrated to the swine genome.

Open access

Angelika Andrzejewska, Klaudia Staszak, Marta Kaczmarek-Ryś, Ryszard Słomski and Szymon Hryhorowicz

Abstract

The endocannabinoid system (ECS) consists of the endocannabinoids, cannabinoid receptors and the enzymes that synthesize and degrade endocannabinoids. The whole EC system plays an important role in the proper functioning of the central and autonomic nervous system. ECS is involved in the regulation of the body energy and in the functioning of the endocrine system. It can affect on the regulation of emotional states, motoric movement, operations of the endocrine, immune and digestive system. Many of the effects of cannabinoids are mediated by G coupled –protein receptors: CB1, CB2 and GPR55 but also of transient receptor potential channels (TRPs) which not only induce the sensation of pain but also support inflammation via secretion of pro-inflammatory neuropeptides. In this review work we briefly summarize the role and action of cannabinoid receptors CB1 and CB2, protein-coupled receptor 55 (GPR55) and transient receptor potential vanilloid 1 (TRPV1).

Open access

Jacek Jura, Zdzisław Smorąg, Barbara Gajda, Daniel Lipiński and Ryszard Słomski

Abstract

Possible influence of a transgene on life functions of embryos makes it reasonable to confirm or deny it for a particular gene construct. In vitro development of an embryo is a widely used criterion of its competence. The aim of the study was to compare in vitro developmental capacity of transgenic and non-transgenic pig embryos. The results showed a statistically significant difference in in

vitro developmental capacity of embryos obtained from transgenic and non-transgenic pigs. Developmental competence of embryos (morula and blastocyst stage) produced from zygotes obtained from transgenic sows decreased compared to that obtained from non-transgenic sows.

Open access

Andrzej Pławski, Marta Podralska, Wojciech Cichy, Michał Drews, Piotr Krokowicz and Ryszard Słomski

DNA Bank for Polish Patients with a Predisposition for Intestinal Polyposis

Intestinal polyposis syndromes include a group of diseases conditioned by the occurrence of hereditary mutations. The current paper presents a collection of DNA samples derived from persons from families with a diagnosed adenomatous polyposes which comprise: familial polyposis coli together with its recessive form, Turcot's syndrome, inherited mixed polyposis as well as persons with recognised hamartomatous polyposes: juvenile polyposis, Peutz-Jeghers syndrome, Cowden syndrome and Proteus syndrome.

The aim of the study was to present current achievements associated with the establishment of the DNA Bank for intestinal polyposis.

Material and methods. Investigations were conducted on DNA isolated from cells of the peripheral blood. The search for mutations in APC, MUTYH, PTEN, BMPR1A, SMAD4 and STK11 genes preconditioning the occurrence of individual diseases was performed employing PCR-SSCP, PCR-HD, DHPLC as well as RFLP techniques and DNA sequencing.

Results. At the present time, the DNA Bank comprises the total of 1097 DNA samples derived from 449 families with intestinal polyposis of which 945 samples come from persons in whose families Familial Adenomatous Polyposis (FAP) occurred. In addition, the collected data also contain material for analyses derived from 25 families with Peutz-Jeghers syndrome and 20 families with juvenile polyposis as well as single cases with the Cowden syndrome, Proteus syndrome and desmoid tumors. The performed molecular investigations allowed identification of mutations ranging from 44 to 50%.

Conclusions. With regard to the quantity of the material collected for analyses and the efficacy level of the employed molecular methods, the obtained results are in keeping with the results found in the literature from the field of genetics and medicine and do not differ from world standards. The collection of data and materials for investigations in the case of rare diseases allows qualitative, organisational and economic optimisation of the performed investigations.

Open access

Anita Spychaj, Marlena Szalata, Ryszard Słomski and Edward Pospiech

Abstract

The aim of this study was to develop a method using PCR and self-designed primers on the basis of the mtDNA cytochrome oxidase subunit I (COI) gene sequence to enable direct identification of the meat of three species of animals, i.e. bovines, pigs and ducks, in the single type sample, in meat mixtures and meat products. The mixtures comprised up to six meat species including apart from beef, pork and duck also chicken, turkey and goose meat. The obtained results indicate the possibility of qualitative identification of the aforementioned meat species in all types of investigated food products. The maximum length of PCR products did not exceed 300 bp, which was supposed to favour the amplification of DNA from meat products which are usually thermally processed and/or exposed to high pressure. PCR primers hybridised selectively with bovine, pig and duck DNA, showing total species specificity.

Open access

Paweł Śledziński, Agnieszka Nowak, Joanna Zeyland and Ryszard Słomski

Abstract

Cannabinoids impact human body by binding to cannabinoids receptors (CB1 and CB2). The two main phytocannabinoids are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). THC interacts with CB1 receptors occurring in central nervous system and is responsible for psychoactive properties of marijuana. CBD has low affinity to CB1 receptor, has no psychoactive characteristics and its medical applications can be wider. CB receptors are part of a complex machinery involved in regulation of many physiological processes – endocannabinoid system. Cannabinoids have found some applications in palliative medicine, but there are many reports concerning their anticancer affects. Agonists of CB1 receptors stimulate accumulation of ceramides in cancer cells, stress of endoplasmic reticulum (ER stress) and, in turn, apoptosis. Effects of cannabinoids showing low affinity to CB receptors is mediated probably by induction of reactive oxygen species production. Knowledge of antitumor activity of cannabinoids is still based only on preclinical studies and there is a necessity to conduct more experiments to assess the real potential of these compounds.

Open access

Magdalena Hryhorowicz, Joanna Zeyland, Agnieszka Nowak-Terpiłowska, Jacek Jura, Wojciech Juzwa, Ryszard Słomski, Jan Bocianowski, Zdzisław Smorąg, Anna Woźniak and Daniel Lipiński

Abstract

The use of pigs as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human donors. Human NK cells play an important role in the cell-mediated rejection of pig-to-human xenografts. In this paper we report the generation and extensive characterization of three generations of transgenic pigs with HLA-E gene encoding the antigen which can inhibit the human NK cell-mediated response. The gene construct pHLAE-GFPBsd containing the human gene encoding the human leukocyte antigen under the promoter of the EF-1α elongation factor ensuring systemic expression was introduced by microinjection into a pronucleus of the fertilized porcine oocyte. PCR analysis revealed and FISH analysis confirmed that the pHLAE-GFPBsd gene construct was present in the genome of the founder female pig. As a result of inter-breeding, an additional 7 transgenic animals were obtained (one individual from F1 generation and six individuals from F2 generation). The transgene expression was shown by RT-PCR and flow cytometry. Real Time PCR analysis estimated the approximate number of transgene copies at 16–34. Karyotype analysis did not show any changes in the structure or the number of chromosomes. The expression level of the transgene was stable in the next generation of genetically modified pigs. An NK cell-mediated cytotoxicity assay showed the increased viability of the transgenic cells in comparison with the wild-type, which confirmed the protective influence of HLA-E expression.