Genetic changes of MLH1 and MSH2 genes could explain constant findings on microsatellite instability in intracranial meningioma

Postreplicative mismatch repair safeguards the stability of our genome. The defects in its functioning will give rise to microsatellite instability. In this study, 50 meningiomas were investigated for microsatellite instability. Two major mismatch repair genes, MLH1 and MSH2, were analyzed using microsatellite markers D1S1611 and BAT26 amplified by polymerase chain reaction and visualized by gel electrophoresis on high-resolution gels. Furthermore, genes DVL3 (D3S1262), AXIN1 (D16S3399), and CDH1 (D16S752) were also investigated for microsatellite instability. Our study revealed constant presence of microsatellite instability in meningioma patients when compared to their autologous blood DNA. Altogether 38% of meningiomas showed microsatellite instability at one microsatellite locus, 16% on two, and 13.3% on three loci. The percent of detected microsatellite instability for MSH2 gene was 14%, and for MLH1, it was 26%, for DVL3 22.9%, for AXIN1 17.8%, and for CDH1 8.3%. Since markers also allowed for the detection of loss of heterozygosity, gross deletions of MLH1 gene were found in 24% of meningiomas. Genetic changes between MLH1 and MSH2 were significantly positively correlated (p = 0.032). We also noted a positive correlation between genetic changes of MSH2 and DVL3 genes (p = 0.034). No significant associations were observed when MLH1 or MSH2 was tested against specific histopathological meningioma subtype or World Health Organization grade. However, genetic changes in DVL3 were strongly associated with anaplastic histology of meningioma (χ2 = 9.14; p = 0.01). Our study contributes to better understanding of the genetic profile of human intracranial meningiomas and suggests that meningiomas harbor defective cellular DNA mismatch repair mechanisms

First evidence of the presence of Multixenobiotic Resistance Mechanism activity in freshwater invasive species, signal crayfish Pacifastacus leniusculus (Dana, 1852)

Background and Purpose: The signal crayfish Pacifastacus leniusculus (Dana, 1852) is one of the most successful invasive species of crayfish in European freshwaters, an extremely diverse though endangered group of ecosystems. The main goal of this study was to functionally characterize multixenobiotic resistance (MXR) mechanism defense activity in P. leniusculus tissues for the first time. MXR mechanism protects the cell from a wide variety of toxic compounds, and it is mediated by the transport activity of ATP-binding cassette (ABC) proteins. Materials and Methods: MXR transporter activity dye assay was performed by using fluorescent model substrate rhodamine B (RB) in combination with inhibitors of MXR efflux pumps: MK571 and Verapamil, known to inhibit multidrug resistance-associated proteins (MRP) and P-glycoprotein (P-gp), respectively. In this assay, the increase in intracellular fluorescence of the substrate dye, indicates inhibition of MXR efflux protein pumps. The assay was performed in three different tissues (gills, hepatopancreas, tail muscle). Additionally, tissues were exposed to selected heavy metals – mercury (HgCl2) and zinc (ZnCl2), known to occur in open freshwaters as pollutants. Results: Optimal time for RB accumulation in gills and hepatopancreas was determined to be 30 minutes. RB efflux in gills was inhibited by MK571 and in hepatopancreas by Verapamil, suggesting that multidrug resistanceassociated proteins are dominant in gills of P. leniusculus, and P-glycoprotein in hepatopancreas. Finally, inhibitory effect of mercury (HgCl2: 10 and 20 μM) and zinc (ZnCl2: 5–20 μM) on multixenobiotic resistance mechanism activity in gills, and only mercury in hepatopancreas, was detected. Conclusions: The results for the first time demonstrate the presence of multixenobiotic resistance mechanism efflux activity as an important tissue specific defense mechanism in P. leniusculus and provide the basis for future molecular and toxicological studies of this invasive and adaptable species

Structural alterations of selected genes in human testicular germ cell tumours

Tumori zametnih stanica testisa najčešće su maligne tvorbe u mladih muškaraca. Dvije histološke grupe, seminomi i neseminomi, razlikuju se morfološki i malignošću. Iako se uočava zajednička citogenetička promjena, amplifikacija 12p kromosomske regije, mehanizmi nastanka slabije agresivnih seminoma i izrazitije agresivnih neseminoma nisu poznati. Njihove molekularno-biološke studije često daju proturječne rezultate. Istražena je pojava strukturnih genskih promjena u 18 seminoma i 22 neseminoma u genima uključenim u maligni fenotip tumora: CDH1, APC (stanična diferencijacija i adhezija); NME1 (stanična pokretljivost); TP53, CDKN2A, RB1 (kontrola staničnog ciklusa i integriteta genoma); RAD51, MSH2, MLH1, BRCA1 (popravak oštecene DNA); BAX (apoptoza); ABCG2 (otpornost na ksenobiotike). Genske promjene, gubitak heterozigotnosti (LOH) i mikrosatelitna nestabilnost (MSI), istražene su koristeći polimorfizme u duljini restrikcijskih fragmenata ili duljini mikrosatelitnih ponavljanja. Uočena je razlika u strukturnim genskim promjenama seminoma i neseminoma. Profil genskih promjena ukazuje na njihovo zasebno podrijetlo, te jasno korelira s kliničkim ponašanjem tih tumora.Testicular germ cell tumors are the most common malignacies in young males. Two histological groups, seminoma and nonseminoma, differ morphologically and in malignant behaviour. Although a common cytogenetic feature is seen, amplification of 12p chromosomal region, development mechanisms of weakly aggressive seminoma and more aggressive nonseminoma are unknown. Molecular biology studies of these tumors are often contradictory. Occurence of structural genetic alterations was analysed in 18 seminoma and 22 nonseminoma for genes involved in a malignant tumour phenotype: CDH1, APC (cellular differentiation and adhesion); NME1 (cellular motility); TP53, CDKN2A, RB1 (cell cycle and genome integrity control); RAD51, MSH2, MLH1, BRCA1 (DNA damage repair); BAX (apoptosis); ABCG2 (resistance to xenobiotics). Genetic alterations, loss of heterozygosity (LOH) and microsatellite instability (MSI), were analysed using restriction fragments length or microsatellite repeats length polymorphisms. A difference betwen structural genetic alterations in seminoma and nonseminoma has been observed. Occurence of genetic alterations indicate an independent development of seminoma and nonseminoma, and clearly correlates with clinical behaviour of those tumours

Nucleotide variations of TP53 exon 4 found in intracranial meningioma and in silico prediction of their significance

The aim of the present study was to identify TP53 exon 4 mutations in patients with meningioma and to investigate their potential association with specific tumor pathology. Nucleotide alterations were investigated in 48 meningiomas via the direct sequencing of TP53 exon 4 in patient tumor and blood samples using the DNA Sanger method with the BigDyeTerminator v3.1 Cycle Sequencing kit and Applied Biosystems 3730XL apparatus. The results revealed that TP53 exon 4 was frequently altered in meningioma, occurring in 60.4% of the patients investigated. A total of 18 different alterations were detected in the meningioma samples assessed in the current study. The majority of these appeared more than once and some were repeatedly identified in several patients. Changes at codons 72 (c.215G>C) and 62 (c.186delA) were highly prevalent, occurring in 44.8% of patients. Other changes detected via frequency analysis included: Five substitutions on codon 105 (c.315C>T); four insertions on codon 70 (c.209_210insG); three insertions on codon 64 (c.190C>G), 82 (245C>T; 245delC; 243_244insA) and 104 (c.312G>A); and two insertions on codons 108 (c.322G>C), 71 (c.213C>A), 73 (c.217G>A), 91 (c.271T>C) and 100 (c.300G>T). Codons 68 (c.202_203insT), 77 (c.229C>T), 88 (c.263C>G) and 92 (c.276C>A) were altered once. Alterations on codons 82, 91, 108, 104, 105, 70 and 92 were characterized as possibly damaging by PolyPhen-2 and Mutation Taster2 tools. The current study also demonstrated that nucleotide alterations were significantly associated with the loss of p53 expression (P=0.04) and female patients (P=0.049), particularly codon 72. The results present novel data on the mutational spectrum of TP53 in meningeal brain tumors

Priručnik za vježbe iz biologije 1

Sveučilišni priručnik za vježbe iz Biologije 1 autora: Lidija Šver, Ana Bielen, Ivana Babić, Tomislav Vladušić, Reno Hrašćan, Ksenija Durgo, Jasna Franekić detaljno je i pristupačno napisano djelo namjenjeno prvenstveno studentima prve godine Prehrambeno-biotehnološkog fakuteta, no zbog multidisciplinarnosti ovaj priručnik može biti koršten i na drugim srodnim fakultetima ili višim razredima gimnazije bilo kao izvor fundamentalnih znanja iz biologije, bilo kao vodič za izvođenje određenih vježbi koje su opisane u samom priručniku. Priručnik je napisan na 244 stranice, a uključuje poglavlja: Napuci za rad na praktikumu iz modula Biologija 1 gdje su date detaljne upute o pravilima rada i ponašanja u praktikumu, pisanju znanstvenih naziva rodova i vrsta te informacije o vođenju laboratorijskog dnevnika. Slijedeće poglavlje bavi se mikroskopom, te je objašnjena građa i funkcija svakog pojedinog dijela mikroskopa te su predložene vježbe koje se izvode kako bi se izvježbale tehnike pravilnog mikroskopiranja. Drugo poglavlje bavi se građom stanične membrane, objašnjeni su fenomeni prijenosa tvari kroz membranu te su navedene vježbe kojima se zakonitosti prijenosa tvari krozn membranu mogu dokazati. U trećem poglavlju detaljno je obrađena stanična stijenka te se predloženim vježbama dokazuje građa i funkcija stanične stijenke biljnih stanica, komunikacija među biljnim stanicama te sekundarne promjene stanične stijenke. Četvrto poglavlje se bavi citoskeletom eukariotske stanice, mikrotubulima i mikrofilamentima te aktinskim filamentima. U petom su poglavlju detaljno obrađeni plastidi, a predloženim praktičnim dijelom studenti se upoznaju s kromoplastima, leukoplastima te kloroplastima, njihovim smještajem i ulogom u stanici. U šestom poglavlju objašnjena je uloga i smještaj ulja i masti te ugljikohidrata u stanici, detaljno su opisani kvalitativni testovi za dokazivanje ugljikohidrata te su predloženi i detaljno objašnjeni testovi kojima studenti mogu dokazivati određene skupine. Sedmo je poglavlje posvećeno DNA molekuli, njenom otkriću, građi i replikaciji, osmo se poglavlje bavi RNA molekulama, mehanizmima njihovog nastanka, procesiranja mRNA u eukariotima te procesom translacije. Deveto se poglavlje bavi produktima translacije-proteinima te metodama kojima se protein mogu dokazati. U desetom i jedanaestom poglavlju detaljno su objašnjeni procesi mitoze i mejoze te su predložene vježbe kojima je moguće pratiti promjene stupnja kondenzacije, položaja i broja molekula DNA te broja kromosoma tijekom procesa mitoze i mejoze. U dvanaestom poglavlju objašnjeni su Mendelovi postulati nasljeđivanja te su opisane metode monohibridnog križanja, test križanja te dihibridnog križanja. U trinaestom poglavlju studenti se upoznaju s genomom, životnim ciklusom te tehnikama uzgoja vinske mušice koja se koristi kao eksperimentalni model višestaničnog eukariotskog organizma. Detaljno je opisan postupak dokazivanja mutacija i nasljeđivanja svojstava vezanih uz spol vinske mušice. Četrnaesto i petnaesto poglavlje obrađuje teme vezane za međudjelovanje gena i populacijsku genetiku

Variations in the Sporulation Efficiency of Pathogenic Freshwater Oomycetes in Relation to the Physico-Chemical Properties of Natural Waters

Oomycete pathogens in freshwaters, such as Saprolegnia parasitica and Aphanomyces astaci, are responsible for fish/crayfish population declines in the wild and disease outbreaks in aquaculture. Although the formation of infectious zoospores in the laboratory can be triggered by washing their mycelium with natural water samples, the physico-chemical properties of the water that might promote sporulation are still unexplored. We washed the mycelia of A. astaci and S. parasitica with a range of natural water samples and observed differences in sporulation efficiency. The results of Partial Least Squares Regression (PLS-R) multivariate analysis showed that SAC (spectral absorption coefficient measured at 254 nm), DOC (dissolved organic carbon), ammonium-N and fluoride had the strongest positive effect on sporulation of S. parasitica, while sporulation of A. astaci was not significantly correlated with any of the analyzed parameters. In agreement with this, the addition of environmentally relevant concentrations of humic acid, an important contributor to SAC and DOC, to the water induced sporulation of S. parasitica but not of A. astaci. Overall, our results point to the differences in ecological requirements of these pathogens, but also present a starting point for optimizing laboratory protocols for the induction of sporulation

Loss of heterozygosity of selected tumor suppressor genes in human testicular germ cell tumors

Human testicular germ cell tumors (TGCTs) are histologically heterogenous neoplasms with a variable malignant potential. Two main groups of germ cell tumors occur in men: seminomas and nonseminomas. In the present study, a set of four tumor suppressor genes was investigated in testicular cancers. CDH1, APC, p53, and nm23-H1 genes were tested for loss of heterozygosity (LOH). Thirty-eight testicular germ cell tumors (17 seminomas and 21 nonseminomas) were analyzed by PCR using restriction fragment length polymorphism or the dinucleotide/tetranucleotide repeat polymorphism method. An allelic loss of p53 at exon 4 was detected in five nonseminomas, whereas LOH of p53 at intron 6 occurred in one of the seminoma and two of the nonseminoma samples. Allelic losses of the APC gene were present in three seminomas and one nonseminoma, whereas one seminoma and three nonseminomas showed LOH of CDH1. The analysis of allelic losses showed no common structural genetic alterations in tumor tissues, although a different pattern of LOH was observed between the two main histological groups of TGCTs