Frequency of Galactose-1-phosphate Uridyl Transferase Gene Mutations in Healthy Population of Croatia

Galactosemia is a human disease caused by deficient activity of each one of the three enzymes involved in galactose metabolism, galactokinase (GALK), galactose-1-phosphate uridyl transferase (GALT) and UDP-galactose-4-epimerase (GALE). Absence or deficiency of GALT activity results in classical galactosemia. This disorder exhibits allelic heterogeneity in different populations and ethnic groups. The aim of this study was to search for galactosemia mutations Q188R, N314D, and K285N in healthy population of Croatia. DNA samples from 221 subjects were analyzed by the polymerase chain reaction, followed by digestion with restriction endonucleases (PCR-RFLP procedure). Allele frequencies for Q188R, N314D, and K285N were found to be 0.2 %, 7.5 % and 0 %, respectively, and correlate well with those published for most other healthy Caucasian populations

Frequency of galactose-1-phosphate uridyl transferase gene mutations in healthy population of Croatia

Galactosemia is a human disease caused by deficient activity of each one of the three enzymes involved in galactose metabolism, galactokinase (GALK), galactose-1-phosphate uridyl transferase (GALT) and UDP-galactose-4-epimerase (GALE). Absence or deficiency of GALT activity results in classical galactosemia. This disorder exhibits allelic heterogeneity in different populations and ethnic groups. The aim of this study was to search for galactosemia mutations Q188R, N314D, and K285N in healthy population of Croatia. DNA samples from 221 subjects were analyzed by the polymerase chain reaction, followed by digestion with restriction endonucleases (PCR-RFLP procedure). Allele frequencies for Q188R, N314D, and K285N were found to be 0.2 %, 7.5 % and 0 %, respectively, and correlate well with those published for most other healthy Caucasian populations

Utjecaj različitih koncentracija peroksovanadijevog spoja bpV(phen) na preživljavanje PC12 stanica

Peroxovanadium compounds are potent insulinomimetic agents and protein tyrosine phosphatase inhibitors. In this study, the potential toxicity of peroxovanadium complex bpV(phen) on rat pheochromocytoma PC12 cells was examined, and the mechanism by which this compound influences cell survival and/or death was explored. BpV(phen) exerted a bimodal effect on PC12 cells: survival enhancing effect at lower and death-inducing effect at higher micromolar concentrations. 1 and 3 μmol dm–3 bpV(phen) intensely induced ERK activation. In contrast, 10 and 100 μmol dm–3 bp (phen) stimulated strong and sustained JNK and p38 MAPK activation as well as caspase-3 activation that preceded bpV(phen)-induced apoptotic cell death. It is suggested that bpV(phen) might exert its action on PC12 cell survival by modulation of MAPKs and caspase-3 activation.Spojevi peroksovanadija snažni su inzulinomimetički agensi i inhibitori tirozinskih fosfataza. U ovom istraživanju ispitivana je moguća toksičnost peroksovanadijevoga kompleksa bpV(phen) na PC12 stanicama feokromocitoma štakora te način na koji ovaj spoj utječe na preživljavanje i/ili umiranje stanica. BpV(phen) dvojako je djelovao na PC12 stanice: primijenjen u nižim mikromolarnim koncentracijama poticao je preživljavanje stanica, dok su više mikromolarne koncentracije poticale umiranje stanica. 1 i 3 μmol dm–3 bpV(phen) snažno je inducirao aktivaciju ERK kinaza. Suprotno tome, 10 i 100 μmol dm–3 bpV(phen) stimulirao je snažnu i dugotrajnu aktivaciju JNK i p38 MAPK kinaza te aktivaciju kaspaze-3, što je prethodilo umiranju stanica procesom apoptoze. Pretpostavlja se da bpV(phen) djeluje na preživljavanje PC12 stanica na način da modulira aktivaciju MAPK kinaza i kaspaze-3

Okratoksin A izaziva apoptozu u LLC-PK1 stanicama aktivirajući JNK i p38 MAPK

Ochratoxin A (OTA) is a potential inducer of a tubular-interstitial nephropathy in humans and animals. In our study we addressed the question of involvement of apoptosis in the development of OTA-provoked nephrotoxicity. LLC-PK1 kidney cells were treated with nanomolar and micromolar concentrations of OTA for different lengths of time. The apoptotic process was estimated by morphological (haematoxylin/eosin staining, fluorescent staining of DNA free ends – TUNEL assay) and biochemical (MAPKs and Hsps) changes of cells. Forty-eight hours of treatment with 5 10–6 M OTA significantly decreased cell viability and induced apoptosis in 30.7 % of cells. In addition, a transient activation of ERK was observed as well as a strong and prolonged activation of stress kinases, JNK and p38 MAPK, after 12 and 48 hours of treatment. Expression of Hsp72 and Hsp27 was not affected by OTA. The results suggest that apoptosis mediated by activation of JNK and p38 MAPK might play an important role in OTA-induced nephrotoxicity in LLC-PK1 cells.Okratoksin A (OTA) može izazvati tubularno-intersticijsku nefropatiju u ljudi i životinja. Cilj našeg istra- živanja bio je ispitati ulogu apoptoze u nastanku nefrotoksičnosti koju uzrokuje OTA. Naš eksperimentalni model bile su LLC-PK1 bubrežne stanice izložene djelovanju OTA, od nanomolarnih do mikromolarnih koncentracija kroz različita vremenska razdoblja. Apoptoza je u stanicama utvrđena na temelju morfoloških (bojanje stanica hematoksilin/eozinom, fluorescentno bojanje slobodnih krajeva DNA tzv. TUNEL) i biokemijskih (MAP kinaze, Hsps) promjena. 48-satno izlaganje stanica 5 x 10–6 M OTA značajno je smanjilo staničnu vijabilnost i potaknulo apoptozu u 30,7 % stanica. Osim toga, utvrđena je kratkotrajna aktivacija ERK te snažna i dugotrajna aktivacija JNK i p38 MAPK nakon 6-, 12- i 48-satnoga tretiranja. OTA nije utjecao na razinu Hsp72 i Hsp27. Rezultati istraživanja ukazuju da je apoptoza posredovana aktivacijom JNK i p38 MAPK važan događaj u razvoju nefrotoksičnosti izazvanoj djelovanjem OTA na LLC-PK1 stanice

Gamma-Glutamyltransferase and C-Reactive Protein in Stable Chronic Obstructive Pulmonary Disease

Systemic inflammation and oxidative stress are the most important features of chronic obstructive pulmonary disease (COPD). The presence of oxidative stress in the airways of smokers, the largest population of COPD patients, is a consequence of direct inhalation of cigarette smoke and increased inflammation-related production of reactive oxygen species. On the other hand, oxidative stress appears to be the key component of many processes associated with chronic inflammation. We intend to examine whether serum C-reactive protein (CRP) concentration and gamma-glutamyltransferase (GGT) activity might be used as auxiliary markers in monitoring level of oxidative stress and inflammation in clinically stable COPD. We also investigated influence of cigarette smoking on these two systemic parameters. Catalytic activity of GGT and concentration of CRP were determined in sera of COPD patients (N=109) and in healthy controls (N=51) by using standard spectrophotometric method and immunoturbidimetric method, respectively. Concentration of CRP and activity of GGT were increased in COPD patients, as compared to healthy controls (p<0.05). We found a significant positive correlation between those two parameters in COPD patients (r=0.202, p=0.0371). Our results showed no difference in GGT activity (p=0.606) or CRP concentration (p=0.573) between groups of patients when subdivided according to the severity of the disease. Smoking did not have a significant impact on CRP and GGT values in COPD patients and healthy controls. We showed an increase of serum CRP and GGT values in COPD patients, and we suggest that serum GGT activity might also represent an inflammation/oxidative stress marker. It seems that COPD patients present higher serum CRP and GGT values than healthy subjects independently from their smoking habits

Genetic Frequencies of Paraoxonase 1 Gene Polymorphisms in Croatian Population

Paraoxonase 1 (PON1), a HDL-associated enzyme, is believed to contribute to the protective effects of HDL by decreasing the generation of lipid peroxidation products during the process of LDL oxidation. It has been reported that polymorphisms in promoter and coding regions of the pon1 gene could affect PON1 activity. The aim of this study was to determine the frequencies of pon1 polymorphisms Q192R, L55M and –108C>T and their influence on PON1 activity in healthy population of Croatia. The following genotype frequencies were determined: 60 % QQ, 34 % QR, 6 % RR for Q192R; 44 % LL, 43 % LM, 13 % MM for L55M; 30 % CC, 48 % CT, 22 % TT for –108C>T polymorphism. Genotypes RR, LL and CC were found to be associated with higher PON1 activity. The most frequent genotypes in healthy Croatian population were QQ, CT and equally present LL and LM

Germline variants of the genes involved in NF-kB activation are associated with the risk of COPD and lung cancer development

Chronic obstructive pulmonary disease (COPD) and lung cancer (LC) are closely related diseases associated with smoking history and dysregulated immune response. However, not all smokers develop the disease, indicating that genetic susceptibility could be important. Therefore, the aim of this study was to search for the potential overlapping genetic biomarkers, with a focus on single nucleotide polymorphisms (SNPs) located in the regulatory regions of immune-related genes. Additionally, the aim was to see if an identified SNP has potentially an effect on proinflammatory cytokine concentration in the serum of COPD patients. We extracted summary data of variants in 1511 immune-related genes from COPD and LC genome-wide association studies (GWAS) from the UK Biobank. The LC data had 203 cases, patients diagnosed with LC, and 360 938 controls, while COPD data had 1 897 cases and 359 297 controls. Assuming 1 association/gene, SNPs with a p-value < 3.3 × 10–5 were considered statistically significantly associated with the disease. We identified seven SNPs located in different genes (BAG6, BTNL2, TNF, HCP5, MICB, NCR3, ABCF1, TCF7L1) to be associated with the COPD risk and two with the LC risk (HLA-C, HLA-B), with statistical significance. We also identified two SNPs located in the IL2RA gene associated with LC (rs2386841; p = 1.86 × 10–4) and COPD (rs11256442; p = 9.79 × 10–3) but with lower significance. Functional studies conducted on COPD patients showed that RNA expression of IL2RA, IFNγ and related proinflammatory cytokines in blood serum did not correlate with a specific genotype. Although results presented in this study do not fully support our hypothesis, it is worth to mention that the identified genes/SNPs that were associated with either COPD or LC risk, all were involved in the activation of the NF-κB transcription factor which is closely related to the regulation of the inflammatory response, a condition associated with both pathologies

Reduction in Peripheral Blood Leukocyte Heat Shock Proteins 27 and 70 Expression in Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is characterized by chronic local and systemic inflammation, and increased oxidative stress. Changes in peripheral blood leukocyte counts or in the fractions of leukocyte subsets might be implicated in the development of this disease. In this study, increased monocyte fraction was observed in COPD non-smokers. In light COPD smokers, the fraction of neutrophils was significantly increased along with significantly decreased lymphocyte fraction and lung function parameters. Heat shock proteins (Hsps) could stimulate antioxidant defence of cells by decreasing intracellular levels of reactive oxygen particles (ROS) and by neutralizing toxic effects of oxidized proteins. This study showed that the expression of Hsps in leukocytes was influenced by health and smoking status. Hsp70 and Hsp27 were significantly decreased in COPD ex-smokers and healthy smokers, but the most striking suppression in Hsps expression was detected in COPD smokers. It is suggested that this decline in Hsps intracellular levels could be implicated in the pathogenesis of COPD

Okratoksin A inducira apoptotičko i nekrotičko umiranje bubrežnih stanica

MDCK, LLC-PK1 and RK 13 renal cells were exposed to different OTA concentrations (2.5 to 25 μg OTA/mL of medium) for 24 hours. 2.5 μg OTA/mL significantly reduced the number of living RK 13 cells to 69.4 %, LLC-PK1 cells to 34.5 % and MDCK cells to 27.5 %, as compared to untreated control cells. Effects of OTA on cell morphology were examined by haematoxylin/ eosin or phalloidin-FITC/propidium iodide staining. 12 %, 11 % and 8.2 % apoptotic cells were observed in MDCK, LLC-PK1 and RK 13 cells treated with 2.5 μg OTA/mL, respectively. The structure of actin cytoskeleton showed significant changes in OTA-treated cells. Release of LDH into the culture medium was more abundant in MDCK and LLC-PK1 cells. OTA induced apoptotic and/or necrotic cell death in a cell type- and concentration-dependent manner. The results suggest that MDCK and LLC-PK1 cells are more sensitive to OTA than RK 13 cells.MDCK, LLC-PK1 i RK 13 bubrežne stanice tretirane su različitim koncentracijama OTA (od 2,5 do 25 μg OTA/mL medija) tijekom 24 sata. Koncentracije toksina od 2,5 μg/mL uzrokuju značajno smanjenje broja živih stanica u odnosu na netretirane kontrolne uzorke: kod RK 13 stanica broj je smanjen na 69,4 %, kod LLC-PK1 na 34,5 %, a kod MDCK na 27,5 %. Ispitan je učinak OTA na morfologiju stanica koje su bojane hematoksilinom/ eozinom odnosno faloidin-FITC/propidijevim jodidom. Kod MDCK, LLC-PK1 i RK 13 stanica tretiranih s 2,5 μg OTA/mL opaženo je 12 %, 11 % odnosno 8,2 % stanica u apoptozi. OTA je uzrokovao značajne promjene u strukturi aktinskoga citoskeleta u tretiranim stanicama. OTA je potaknuo istjecanje LDH iz stanica u okolni medij, što je osobito izraženo u MDCK i LLC-PK1 stanicama. Možemo zaključiti da način umiranja stanica (apoptoza ili nekroza) ovisi o vrsti samih stanica i o primijenjenoj koncentraciji toksina. Naši rezultati ukazuju da su MDCK i LLC-PK1 stanice osjetljivije na djelovanje OTA od RK 13 stanica