A novel "TAZ" gene mutation and mosaicism in a Polish family with Barth syndrome

Barth syndrome (BTHS) is an X‐linked recessive disease primarily affecting males. Clinically, the disease is characterized by hypertrophic or dilated cardiomyopathy, skeletal myopathy, chronic/cyclic neutropenia, 3‐methylglutaconic aciduria, growth retardation and respiratory chain dysfunction. It is caused by mutations in the TAZ gene coding for the tafazzin protein which is responsible for cardiolipin remodeling. In this work, we present a novel pathogenic TAZ mutation c.83T>A, p.Val28Glu, found in mosaic form in almost all female members of a Polish family. Sanger sequencing of DNA from peripheral blood and from epithelial cells showed female mosaicism in three generations. This appears to be a new mechanism of inheritance and further research is required in order to understand the mechanism of this mosaicism. We conclude that BTHS genetic testing should include two or more tissues for women that appear to be noncarriers when blood DNA is initially tested. The results of our study should not only be applicable to BTHS families, but also to families with other X‐linked diseases

Effect of caloric restriction on liver function in young and old ApoE/LDLr-/- mice

Background. Caloric restriction (CR) leads to decrease metabolic intensity, which results in a reduction of oxygen consumption and the amount of free radicals. This can affect the function of the liver. Studies show that caloric restriction does not alter or significantly increase the enzyme activity associated with gluconeogenesis, but the effect was different according to the age of the model animals. Objective. The aim of the study was to determine the effect of caloric restriction on liver function in young and old ApoE/ LDLr-/- mice. Material and methods. Dietary experiments were performed on 2 and 5 month old male ApoE/LDLr-/- mice. Animals were divided into 3 experimental groups (n=6) and fed AIN’93G diet for 8 and 5 weeks, respectively. Control animals were fed ad libitum (AL) and housed in a colony cages. These animals were checked for dietary intake. The second group were also fed ad libitum but the animals were kept individually in cages (stress AL- sAL). Similarly to sAL group, the animals from the CR group were kept individually but received a 30% less diet compared to AL group. At the end of the experiment animals were euthanized and the blood, liver and adipose tissue have been collected. Alanine aminotransferase (ALT) as well as aspartate aminotransferase (AST) were measured in plasma. Fatty acid profile was evaluated (relative %) in adipose tissue (GC-MS). Liver’s stetosis was assessed. Results were analyzed statistically (ANOVA, STATISTICA v.10.0). Results. CR ApoE/LDLr-/- mice showed significantly lower body weight compared to animals, both AL and sAL. There were no significant differences between ALT and AST in both younger and older animals. However, negative tendencies were more pronounced in younger animals. In young animals CR significantly increased liver weight compared to AL (4.14 vs 3.73g/100g). In adipose tissue fatty acid profile differed in CR mice compared to control in young animals. Conclusions. Caloric restriction did not affect liver enzymes in mice. Caloric restriction showed similar but not identical metabolic activity in young and old mice.Wprowadzenie. Restrykcje kaloryczne (CR) prowadzą do spadku intensywności metabolizmu, co wiąże się ze zmniejszeniem zużycia tlenu i ilości powstających wolnych rodników. Tym samym mogą mieć wpływ na funkcjonowanie wątroby. Badania wykazują, że ograniczenia kaloryczne nie zmieniają lub znacząco zwiększają aktywność enzymów związanych z glukoneogenezą, w tym kluczowego enzymu jakim jest aminotransferaza alaninowa (ALT). Obserwowany efekt był różny w zależności od wieku modelowych zwierząt. Cel badań. Celem pracy było określenie wpływu restrykcji kalorycznej na czynność wątroby u młodych i starszych myszy ApoE/LDLr-/-. Materiał i metody. Doświadczenie żywieniowe przeprowadzono na 2. i 5. miesięcznych samcach myszy ApoE/LDLr-/-. Zwierzęta podzielono na 3 grupy doświadczalne (n=6) i żywiono dietą AIN’93G przez okres 8 i 5. tygodni. Zwierzęta z grupy kontrolnej (AL) żywione były ad libitum i przetrzymywane zbiorowo w klatkach. Spożycie diety było sprawdzane. Grupa druga otrzymywała dietę ad libitum przy czym zwierzęta przetrzymywano w klatkach indywidualnie (sAL). Analogicznie do grupy drugiej zwierzęta z grupy restrykcji kalorycznych (CR) były trzymane indywidualnie, jednak otrzymywały 30% mniej diety w porównaniu do grupy kontrolnej AL. Po zakończeniu doświadczenia zwierzęta poddano eutanazji i pobrano wątrobę w celu oceny histologicznej (barwienie H&E i ORO) oraz krew, w której metodą spektrofotometryczną przeprowadzono oznaczenie enzymów aminotransferazy alaninowej (ALT), jak również aminotransferazy asparaginowej (AST). W tkance tłuszczowej oznaczono profil kwasów tłuszczowych (GC-MS). Wyniki poddano analizie statystycznej (ANOVA , STATISTICA v.10.0). Wyniki. Zarówno młodsze, jak i starsze myszy ApoE/LDLr-/- poddane restrykcji kalorycznej wykazały istotnie niższą końcową masę ciała w porównaniu do zwierząt z grup kontrolnych, zarówno AL, jak i sAL. Nie stwierdzono istotnych statystycznie różnic pomiędzy grupami w poziomie ALT, jak również AST zarówno u zwierząt młodszych, jak i starszych, przy czym negatywne tendencje były wyraźniej widoczne u zwierząt młodszych poddanych restrykcji kalorycznej. Również u młodszych zwierząt CR miało istotny niekorzystny wpływ na masę wątroby w porównaniu do kontroli (4,14 vs 3,73 g/100g). Zaobserwowano istotny wpływ CR na profil kwasów tłuszczowych w tkance tłuszczowej u zwierząt młodszych. Wnioski. Restrykcje kaloryczne nie miały wpływu na próby czynnościowe wątroby u myszy. Restrykcje kaloryczne wykazały podobną, ale nie identyczną aktywność metaboliczną u młodszych i starszych zwierząt

Plasma centrifugation does not influence thrombin-antithrombin and plasmin-antiplasmin levels but determines platelet microparticles count

Introduction: Centrifugation is an essential step for plasma preparation to remove residual elements in plasma, especially platelets and platelet-derived microparticles (PMPs). Our working hypothesis was that centrifugation as a preanalytical step may influence some coagulation parameters. Materials and methods: Healthy young men were recruited (N = 17). For centrifugation, two protocols were applied: (A) the first centrifugation at 2500 x g for 15 min and (B) at 2500 x g for 20 min at room temperature with a light brake. In protocol (A), the second centrifugation was carried out at 2500 x g for 15 min, whereas in protocol (B), the second centrifugation involved a 10 min spin at 13,000 x g. Thrombin-antithrombin (TAT) and plasmin-antiplasmin (PAP) complexes concentrations were determined by enzyme-linked immunosorbent assays. PMPs were stained with CD41 antibody and annexin V, and analyzed by flow cytometry method. Procoagulant activity was assayed by the Calibrated Automated Thrombogram method as a slope of thrombin formation (CAT velocity). Results: Median TAT and PAP concentrations did not differ between the centrifugation protocols. The high speed centrifugation reduced the median (IQR) PMP count in plasma from 1291 (841-1975) to 573 (391-1010) PMP/µL (P = 0.001), and CAT velocity from 2.01 (1.31-2.88) to 0.97 (0.82-1.73) nM/min (P = 0.049). Spearman’s rank correlation analysis showed correlation between TAT and PMPs in the protocol A plasma which was (rho = 0.52, P < 0.050) and between PMPs and CAT for protocol A (rho = 0.74, P < 0.050) and protocol B (rho = 0.78, P < 0.050). Conclusion: Centrifugation protocols do not influence the markers of plasminogen (PAP) and thrombin (TAT) generation but they do affect the PMP count and procoagulant activit

Apoptosis-related gene expression in glioblastoma (LN-18) and medulloblastoma (Daoy) cell lines

The expression of apoptosis genes in a commercial pre-designed low-density array from Applied Biosystems was evaluated in two human brain cancer cell models, LN-18 and Daoy (HTB-186™) in comparison to the reference human primary endothelial cells under basic conditions. Analysis of the gene expression in the cancer cell lines compared to the normal control revealed features reflecting anti-apoptotic and inflammatory characteristics of the former. There was an overall downregulation of apoptosis-stimulating genes in both cancer cell lines, along with an upregulation of certain apoptosis inhibitors. A number of genes demonstrated statistically significant changes in their expressions, including BAX (BCL2-associated X protein); the CARD4/NLR family, CARD domain containing 4; CASP10 (caspase 10, apoptosis-related cysteine peptidase); DAP1 (death-associated protein kinase 1), and BIRC5 (baculoviral IAP repeat-containing 5). Anti-apoptotic potential in both cell lines was demonstrated by changes in the Bax:Bcl-2 ratio and downregulation of the APAF1 gene in LN18 cells. There was also significant downregulation of extrinsic signals and the TNF/FADD/inflammatory cascade, and upregulation of caspase inhibitors (IAPs). These results provided a novel molecular characterization of important human cancer cell lines, which might provide a useful research tool for investigating the experimental model of the CNS cell