The aim of this study was to improve detection of heterozygous samples and loss of heterozygosity
using a new type of gel polymer. A total of 60 samples of normal and tumor colon tissue
were tested. ...Elctrophoresis of amplified loci was performed on a standard acrylamide / N,Nmethylenebisacrylamide
gel, and the same gel containing Spreadex polymer, NAB (native acrylamide-
bis). Standard gel displayed 36 samples of normal tissue as homozygous. Electrophoresis
of the same samples on gel with addition of polymer revealed 12/36 heterozygous samples.
Five of 12 matched tumor samples displayed loss of heterozygosity that could not be detected
on standard gel, because of its lower resolving power. Electrophoresis on a new polymer presents
a simple, inexpensive, reproducible and non-radioactive method for loss of heterozygosity
detection, with improved performances such as increased sensitivity, decreased amount of a
sample and improved separation of closely spaced bands. Application of the polymer may contribute
to a vide variety of diagnostic procedures.
In nonobese diabetic (NOD) mice, T cells play a major role in mediating autoimmunity against pancreatic islet beta-cells. We and others previously reported that age-related alterations in the thymic ...and peripheral T cell repertoire and function occur in prediabetic NOD mice. To study the mechanism responsible for these T cell alterations, we examined whether a defect exists in the thymus of NOD mice at the level of TCR-mediated signaling after activation by Con A and anti-CD3. We found that thymocytes from NOD mice respond weakly to Con A- and anti-CD3-induced proliferation, compared with thymocytes from control BALB/c, BALB.B, (BALB.B x BALB.K)F1, C57BL/6, and nonobese non-diabetic mice. This defect correlates with the onset of insulitis, because it can be detected at 7 to 8 weeks of age, whereas younger mice displayed a normal T cell responsiveness. Thymic T cells from (NOD x BALB/c)F1 mice, which are insulitis- and diabetes-free, exhibit an intermediate stage of unresponsiveness. This T cell defect is not due to a difference in the level of CD3 and IL-2R expression by NOD and BALB/c thymocytes, and both NOD CD4+ CD8- and CD4- CD8+ mature thymic T cells respond poorly to Con A. BALB/c but not NOD thymic T cells respond to Con A in the presence of either BALB/c or NOD thymic APC, suggesting that the thymic T cell defect in NOD mice is intrinsic to NOD thymic T cells and is not due to an inability of NOD APC to provide a costimulatory signal. The defect can be partially reversed by the addition of rIL-2 to NOD thymocytes. To determine whether a defect in signal transduction mediates this NOD thymic T cell unresponsiveness, we tested whether these cells elevate their intracellular free Ca2+ ion concentration in response to Con A. An equivalent Con A-induced increase in Ca2+ ion concentration in both NOD and BALB/c thymocytes was observed, suggesting a normal coupling between the CD3 complex and phospholipase C in NOD thymocytes. In contrast to their low proliferative response to Con A or anti-CD3, NOD thymocytes respond normally (i.e., as do BALB/c thymocytes) to the combinations of PMA plus the Ca2+ ionophore ionomycin and PMA plus Con A but weakly to Con A plus ionomycin. Our data suggest that the age-related NOD thymocyte unresponsiveness to Con A and anti-CD3 results from a defect in the signaling pathway of T cell activation that occurs upstream of protein kinase C activation.
Aim: To assess whether alterations in the K-ras, p53, and DPC4 genes are present in pancreatitis, a potential precancerous condition that can progress to pancreatic adenocarcinoma. To investigate the ...alterations occurring at hot spots of K-ras (exon 1), p53 (exons 5 and 7), and DPC4 (exons 8, 10 and 11).
Methods: In 10 patients with acute and 22 with chronic pancreatitis, without pancreatic intraepithelial neoplasia (PanIN), DNA was isolated from paraffin embedded tissue samples. The extracted DNA was analyzed by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis, single-strand conformation polymorphism (SSCP) analysis, and DNA sequencing.
Results: In acute pancreatitis samples no mutations were found in any of the investigated genes. In 7 out of 22 samples of chronic pancreatitis nucleotide substitution at exon 1 of K-ras (five at codon 12 and two at codon 13) were found. No mutations in p53 (exons 5 and 7) were detected. Two samples had nucleotide substitutions at exons 8 and 11 of DPC4, introducing STOP signal and change in the amino acid sequence, respectively. One chronic pancreatitis sample displayed simultaneous mutations in K-ras (exon 1, codon 12) and DPC4 (exon 8, codon 358).
Conclusion: Mutations of K-ras and Dpc4 genes can accumulate already in non-malignant, inflammatory pancreatic tissue, suggesting its applicability in monitoring of further destruction of pancreatic tissue and progression into malignancy.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cilj: Malondialdehid (MDA) je jedan od toksičnih produkata lipidne peroksidacije, a koristi se u evaluaciji oksidativnog stresa tijekom šećerne bolesti. Cilj ovog rada bio je ispitati učinak akarboze ...(inhibitora α-glukozidaza) na koncentraciju glukoze u serumu i MDA u homogenatu jetre NOD (engl. non-obese diabetic) miševa.
Materijali i metode: U NOD miševa šećerna bolest inducirana je i.v. aplikacijom aloksan-monohidrata (75 mg/kg t. mase). NOD miševi podijeljeni su u 4 skupine (n=6): Kontrolni, zdravi NOD miševi (K), Kontrolni, zdravi NOD miševi tretirani 7 dana akarbozom (K/A), dijabetični NOD miševi (D) te dijabetični NOD miševi tretirani 7 dana akarbozom (D/A).
Koncentracija glukoze u krvi izmjerena je glukoza oksidaza-peroksidaza metodom, a koncentracija MDA u homogenatu jetre određena je upotrebom metode s tiobarbiturnom kiselinom.
Rezultati: Nakon sedmodnevnog tretmana akarbozom zabilježeno je statistički značajno smanjenje koncentracije glukoze u krvi u skupini D/A u odnosu na skupinu D (p<0.05), a isto tako je uočen i statistički značajan pad koncentracije MDA (p<0.05).
Zaključak: Ovi rezultati potvrđuju pozitivan antioksidacijski učinak akarboze što se može objasniti njezinim antihiperglikemijskim djelovanjem.
The metabolism of tryptophan (TRP) was studied in diabetic and insulin-treated diabetic rats throughout a five-month period. In alloxan diabetic rats the serum and brain TRP levels were decreased ...(serum: 38 to 48 mmol/l, brain: 8.6 to 9.2 mmol/g) in comparison to the values of control rats (serum: 59 to 64 mmol/l, brain: 11.3 to 12.6 mmol/g). Daily long-term (for 45, 75, 90 or 135 days) treatment with intermediately acting insulin (4 IU/rat, s.c.) was not able to restore brain concentration of TRP. On the contrary, the serum TRP concentrations were totally or partially restored. The concentrations of branched chain amino acids (BCAA) were increased in serum (valine = 361.2 to 461.0 mumol/l or leucine + isoleucine = 431.0 to 520.3 mumol/l) throughout the entire five-month examination period. Insulin treatment did not return serum concentration of BCAA to normal level in the observation period either.
We determined whether disulfide-linked insulin peptides that are immunogenic in vitro for CD4+T cells bind to major histocompatibility complex class II in vivo. Radiolabeled recombinant human insulin ...(rHI) was injected into BALB/ c mice, and processed rHI peptides bound to I-Admolecules on different thymic antigen-presenting cells were characterized. The A6-A11/B7-B19 and A19-A21/B14-B21 disulfide-linked I-Ad-bound rHI peptides were isolated from thymic epithelial cells but not dendritic cells. While both thymic epithelial cells and dendritic cells present rHI to HI/I-Ad-specific T cells, these antigen-presenting cells do not present the reduced or nonreduced forms of the disulfide-linked rHI peptides. Thus, a naturally processed disulfide-linked peptide can bind to major histocompatibility complex class II in vivo. The potential role of these peptides in immunological tolerance is discussed.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The structure, complementary structure and cytogenetic/proliferative effects of the Met-enkephalin on human peripheral blood lymphocytes were analyzed. Met-enkephalin, i.e. Peptid-M (LUPEX®), is a ...low molecular weight synthetic pentapeptide that corresponds to thymus Met-enkephalin. Its structure was examined by means of NMR spectroscopy. The influence of Met-enkephalin on in vitro normalization of chromosomally aberrant lymphocytes of patients suffering from different immune-mediated diseases, was analyzed by the sensitive cytogenetic tests for screening and detection of genome damages in human lymphocytes. The tests showed that in vitro stimulation of human lymphocytes with the Met-enkephalin led to dissapearance of different types of chromosome aberrations, reduction in the number of micronuclei, decrease in the frequency of sister chromatid exchange (SCE) and apoptosis as well as a cytostatic effect on mitosis cycles. They have also confirmed normalization of chromosomally aberrant cell findings in patients suffering from different immune-mediated diseases. These results suggest a possible role of Met-enkephalin (Peptid-M) in immunotherapy of different diseases which involve chromosomal aberrations as well as abnormal cell proliferation and offer new approaches to immunotherapy by the use of Peptid-M. Based on the molecular recognition theory and the SCA method, peptide complementary to Peptid-M was designed, synthesized and denoted Peptide-D. Peptide-D is a calpastatin fragment. Predicted ligand-receptor interaction between both peptides is confirmed by the results showing that Peptide-D blocked the Peptid-M induced lymphocyte proliferation in a dosedependent manner.
Svrha ovog rada bila je ispitati kratkotrajni učinak 0.1% (m/m) akarboze u suhoj hrani na katalitičku koncentraciju specifičnih jetrenih enzima: alanin aminotransferaze (ALT) i aspartat ...aminotransferaze (AST) u jetri kontrolnih i dijabetičnih CBA miševa. Dijabetes je bio izazvan i.v. injekcijom aloksan-monohidrata u dozi od 75 mg kg -1 tjelesne mase miša sedam dana prije početka ishrane s akarbozom. U pokusu su ispitane četiri skupine CBA miševa: kontrolna (C) (n = 6) i dijabetična (D) (n = 8) skupina bile su sedam dana na standardnoj ishrani, te kontrolna (C / A – 100) (n = 8) i dijabetična (D / A-100) (n = 8) skupina koje su hranjene 0.1 % akarbozom umiješanom u standardnu hranu. U skupini D katalitička koncentracija ALT-a bila je značajno snižena u usporedbi s kontrolnom skupinom C. Sličan pad katalitičke koncentracije ALT-a zabilježen je i u jetri kontrolnih i dijabetičnih miševa hranjenih suhom hranom u koju je bila umiješana akarboza (0.1%). U ispitanim skupinama nisu zabilježene promjene u specifičnoj i ukupnoj aktivnosti AST-a.
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