Nanomolar concentrations of human amylin promote death of RINm5F cells in a time- and concentrationdependent manner. Morphological changes of chromatin integrity suggest that cells are predominantly ...undergoing apoptosis. Human amylin induces significant activation of caspase-3 and strong and sustained phosphorylation of stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38, that precedes cell death. Extracellular signal-regulated kinase (ERK) activation was not concomitant with JNK and/or p38 activation. Activation of caspase-3 and mitogen-activated protein kinases (MAPKs) was detected by Western blot analysis. Addition of the MEK1 inhibitor PD 98059 had no effect on amylin-induced apoptosis, suggesting that ERK activation does not play a role in this apoptotic scenario. A correlative inhibition of JNK activation by the immunosuppressive drug FK506, as well as a selective inhibition of p38 MAPK activation by SB 203580, significantly suppressed procaspase-3 processing and the extent of amylin-induced cell death. Moreover, simultaneous pretreatment with both FK506 and SB 203580, or with the caspase-3 inhibitor Ac-DEVD-CHO alone, almost completely abolished procaspase-3 processing and cell death. Thus, our results suggest that amylin-induced apoptosis proceeds through sustained activation of JNK and p38 MAPK followed by caspase-3 activation.
Due to its capability for high-throughput screening
H nuclear magnetic resonance (NMR) spectroscopy is commonly used for metabolite research. The key problem in
H NMR spectroscopy of multicomponent ...mixtures is overlapping of component signals and that is increasing with the number of components, their complexity and structural similarity. It makes metabolic profiling, that is carried out through matching acquired spectra with metabolites from the library, a hard problem. Here, we propose a method for nonlinear blind separation of highly correlated components spectra from a single
H NMR mixture spectra. The method transforms a single nonlinear mixture into multiple high-dimensional reproducible kernel Hilbert Spaces (mRKHSs). Therein, highly correlated components are separated by sparseness constrained nonnegative matrix factorization in each induced RKHS. Afterwards, metabolites are identified through comparison of separated components with the library comprised of 160 pure components. Thereby, a significant number of them are expected to be related with diabetes type 2. Conceptually similar methodology for nonlinear blind separation of correlated components from two or more mixtures is presented in the Supplementary material. Single-mixture blind source separation is exemplified on: (i) annotation of five components spectra separated from one
H NMR model mixture spectra; (ii) annotation of fifty five metabolites separated from one
H NMR mixture spectra of urine of subjects with and without diabetes type 2. Arguably, it is for the first time a method for blind separation of a large number of components from a single nonlinear mixture has been proposed. Moreover, the proposed method pinpoints urinary creatine, glutamic acid and 5-hydroxyindoleacetic acid as the most prominent metabolites in samples from subjects with diabetes type 2, when compared to healthy controls.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Acute, subchronic and chronic effects of the P-9801091 plant mixture extract at a dose of 20 mg/kg body mass were assessed in serum of healthy CBA/HZg mice at 24 hours, 7 days, 3 months and 6 months ...of treatment (experimental group), and compared with the values obtained in the control group of untreated healthy CBA/HZg mice. The P-9801091 plant mixture extract is an antihyperglycemic preparation containing Myrtilli folium (Vaccinium myrtillus L.), Taraxaci radix (Taraxacum officinale Web.), Cichorii radix (Cichorium intybus L.), Juniperi fructus (Juniperus communis L.), Centaurii herba (Centaurium umbellatum Gilib.), Phaseoli fructus sine semine (Phaseolus vulgaris L.), Millefolii herba (Achillea millefolium L.), Mori folium (Morus nigra L.), Valerianae radix (Valeriana officinalis L.) and Urticae herba et radix (Urtica dioica L). Toxic effect of the P-9801091 plant mixture extract was assessed by the following biochemical parameters: urea, creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and cholesterol. Also, histopathological examination of the kidneys, liver, spleen, pancreas, testes and lungs was performed. Results of biochemical testing performed at specified time points generally showed no statistically significant differences from control values, with the only exception of the catalytic concentration of AST in the experimental group measured on day 7, which was significantly increased as compared with the control group (p<0.05). Pathohistological examination including characteristic organ and tissue structure, and parenchyma relationship to the adjacent blood vessels and connective tissue in the examined organs revealed no major pathologic changes.
The aim of this study was to investigate the alterations in the DPC4 tumor suppressor gene in renal cell carcinoma (RCC). The study included 32 tumor specimens from Croatian patients with a diagnosis ...of RCC. Loss of heterozygosity (LOH) was investigated using three specific oligonucleotide primers for the three DPC4 polymorphic markers. Our investigation of mutations in the DPC4 gene was focused on exons 2, 8, 10 and 11. These exons belong to the mad homology domains 1 (exon 2) and 2 (exons 8-11). The presence of previously documented mutation in exons 2 (codon 100), 8 (codon 358), 10 (codon 412), and 11 (codon 493) was investigated by restriction fragment length polymorphism (RFLP) analysis, as a first screening method. Finally, the study was extended to search for any other type of mutation in the four selected exons by single strand conformation polymorphism (SSCP) assay. To increase heterozygosity, all 32 tumor specimens were tested with primers for three polymorphic markers. A total of 30 (94%) were heterozygous (informative). LOH at any of these markers was only revealed in four (13%) of the 30 informative samples. No tumor samples were positive for mutation in the four investigated exons analyzed by RFLP. In addition, no samples showed other types of mutation in denaturing conditions. Genetic alterations were shown only in a minority of patients, probably because mutation analysis of the DPC4 gene has only been partially covered by our work. It seems that exon 2 (belonging to the MH1 domain) and exons 8, 10, 11 (belonging to the MH2 domain) are not altered in RCC. This investigation must be extended on other exons of DPC4 for a better understanding a role of this gene in renal cell carcinoma.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The aim of this study was to establish an in vitro model to confirm earlier observations on the role of the myc/ras oncogenes as promoting factors in the process of normal Langerhans islet β cell ...transformation. For that purpose we infected primary mouse Langerhans islets with a recombinant retrovirus containing the v-H-ras and v-myc oncogenes, before or after treatment with transforming growth factor α (TGFα). Normal Langerhans islets, when grown in culture, are viable for 2–3 weeks. After treatment with TGFα, viability was extended by 10 days, following which islets disintegrated. Langerhans islets transformed with v-H-ras and v-myc became immortal and insulin negative. Single infected β cells, liberated from a primary islet into the surrounding medium, gave rise to neo islet formation. Moreover, single infected β cells were able to grow and divide, even without fibroblast support. These results indicate that the myc and ras oncogenes are sufficient for commencement of β cell transformation and, therefore, could represent `early events' in the multistep carcinogenesis of insulinomas.
Abstract A case-control study was performed to establish a potential association of two TNF-α gene promoter SNPs (−238G>A and −308G>A) with occurrence of type 1 Diabetes mellitus (T1DM) in Croatian ...population (174 patients and 193 healthy controls). Genotypes (obtained by polymerase chain reaction–restriction fragment length polymorphism), and the clinical parameters of T1DM patients were statistically evaluated by SPSS 13 and Arlequin software, G*Power 3.0.10 program, and calculator for Hardy–Weinberg equilibrium. The frequency of the risk (A) allele, as well as the distribution of high-expression (GA, AA) genotypes were significantly higher ( p < 0.0001) in T1DM patients only at locus −308. The distribution of the −238G/−308A haplotype was also significantly higher in patients compared with controls (27.6% vs 9.6%, p < 0.0001). Gender-dependent analysis revealed that female T1DM −308GA genotype carriers exhibit considerably stronger association with T1DM (odds ratio = 6.37, 95% confidence interval = 3.16–12.85) than male −308GA patients (odds ratio = 2.71, 95% confidence interval = 1.31–5.59). Clinical parameter analysis of T1DM patients revealed significantly decreased level of hemoglobin A1 c (HbA1 c) in −238A allele carriers compared with −238G allele carriers (6.55% vs 7.17%, p = 0.022), as well as the tendency of the risk allele carriers at −238 or −308 locus to develop T1DM earlier in life compared with non–risk allele carriers. In conclusion, susceptibility to T1DM in the Croatian population is strongly associated with the TNF -α −308G>A polymorphism, especially in women. In addition, significantly lower HbA1c levels found in T1DM −238A allele carriers might indicate better glycemic control in these patients.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Diabetes mellitus succeeded by diabetic cataract was induced to experimental animals (Wistar rats) by applying an Alloxan injection. Eye properties deterioration were monitored from clinical ...standpoint and using Raman and infrared spectroscopies. All cases of developed cataract were followed by important changes in vibrational spectra, but Raman spectroscopy proved to be more useful because of larger number of resolved bands. Each
kth Raman spectrum of diseased lens (in our notation
k denotes disease age and cataract degree as described in chapter Alloxan diabetes) can be expressed as a sum of the Raman spectrum of healthy lens, I
R, multiplied by a suitable constant
c
k, and the fluorescent background spectrum, I
FB. We introduce the ratio of integrated intensities
I
FB and
c
k*
I
R as a physical parameter called fluorescent background index F
FB. It turns out that
F
FB grows as cataract progresses and has its maximum at approx. 4, whence it decreases.
F
FB values are larger for 200–1800
cm
−1 spectral interval than for 2500–4000
cm
−1 interval.
In the same manner another quantity called water band index
F
W is defined for each Raman spectrum of diseased lens in the 2800–3730
cm
−1 interval. It is the ratio of the integrated intensity from 3100 to 3730
cm
−1 (water band interval) divided by the integrated intensity of the 2800–3100
cm
−1 interval (C–H stretching region).
F
W increases monotonously with cataract progression with maximum at the end of monitored period (5 months).
These two indices helped us to formulate a model describing disease development from the earliest molecular changes to its macroscopic manifestation. As glucose and other small saccharide molecules enter the lens tissue, they bind to crystallin and other proteins via O- and S-glycosidic linkages which occur probably at tyrosine and cystein sites. In Raman spectrum this corresponds to broad bands at 540 and 1100
cm
−1 which grow together with the fluorescent background, because both contributions originate in nonenzimatically glycated proteins. The maximum of possible binding ends after approximately 4 months (cataract degree 4), but the water continues to enter the tissue and resides in water agglomerates.
The lens impairing caused by fluorescent light scattering on aberrant glycoproteins and other fluorescent centers appears first and is usually associated with the ageing cataract, while deterioration of lens properties caused by increased binding of water steadily rises with glucose and is characteristic of diabetic cataract. This interpretation is in agreement with electron microscopy results of other groups and with our preliminary findings obtained with light microscopy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
We investigated the prevalence of DPC4 loss of heterozygosity in sporadic colorectal cancer. Thirty-six cases of human sporadic colon carcinoma and corresponding normal tissue samples were examined ...to evaluate loss of heterozygosity at the DPC4 tumor suppressor locus using variable nucleotide tandem repeat (VNTR) analysis and three polymorphic markers. From 36 analyzed samples 35 (97%) were heterozygous or informative. Loss of heterozygosity at the DPC4 locus was detected in 18 (51%) of informative tumor DNAs. The DPC4 LOH was more frequent in smaller tumors (<5 cm) than in larger ones. There was no correlation between DPC4 LOH and age or sex of patients. There was a negative correlation between DPC4 LOH and histological grade or Dukes' stage of tumors, but without statistic significance. Observed results are in agreement with the view that malignant progression is consequence of many genetic changes. It can be concluded that inactivation of the DPC4 gene plays a role in a multistep process of outgrowth and progression of colon cancer.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The effects of ferric-sorbitol-citrate and ferric-citrate on the severity of experimental arthritis, TNF-α secretion and the immune status were examined in mice. Arthritis was induced by footpad ...injection of methylated BSA and intraperitoneal injection of
Bordetella pertussis. Joint and footpad swelling were measured weekly by a caliper. TNF-α serum levels were measured by ELISA. The immune status was determined by the response of mouse lymphocytes to ConA in vitro and by the antigen-presenting cell assay. Experimental arthritis was aggravated by ferric-citrate, whereas ferric-sorbitol-citrate did not promote it. If applied to normal (non-arthritic) mice three times a week for 4 weeks, ferric-sorbitol-citrate stimulated isolated splenocytes to increase production of TNF-α, the function of antigen-presenting cells and lymphocyte proliferation in response to ConA in vitro. TNF-α production by cultured splenocytes was also stimulated. In mice with antigen-induced arthritis, iron compounds did not additionally stimulate TNF-α production. Thus, we have shown that ferric-sorbitol-citrate stimulated TNF-α production, antigen-presenting cell activity and cellular immune response. Development of antigen-induced arthritis and TNF-α production in arthritic mice were not stimulated.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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