Mirjana Suver

Association of vitamin D receptor gene 3′-variants with Hashimoto's thyroiditis in the Croatian population

Hashimotos thyroiditis (HT) is the most frequent autoimmune thyroid disease with strong genetic background. Vitamin D receptor (VDR) endocrine system affects immunosuppressive, regulatory and tolerogenic decisions required for induction and maintenance of peripheral immune tolerance. With respect to the biological function of the VDR and functionally plausible gene‐expression data, we sought to test whether particular 3′‐restriction fragment length polymorphisms (RFLP) and haplotypes previously directly or indirectly associated with VDR mRNA 3′‐allelic imbalance phenotype and differences in total VDR mRNA expression are implicated in HT susceptibility. Thus, 145 Croatian HT patients and 145 age‐, sex‐ and ethnically matched euthyroid controls were genotyped for VDR rs1544410 (BsmI), rs7975232 (ApaI) and rs731236 (TaqI) polymorphisms by polymerase chain reaction‐RFLP method. Covariate‐adjusted single‐locus and haplotype–phenotype regression analyses were performed. Permutation corrections (Pc) and Akaike Information Criteria were used for model comparisons. The best‐fit [global Pc = 7.2 × 10−4]BsmI‐TaqI BT haplotype was found significantly more often in subjects without HT [12.2% vs. 3.7%; odds ratio (OR, 95% confidence intervals) = 0.28 (0.14–0.56), Pc = 8 × 10−4], whereas the bT haplotype was significantly more frequent in individuals with HT [45.7% vs. 61.8%; OR = 1.91 (1.37–2.65), Pc = 4 × 10−4]. Two extended BsmI‐ApaI‐TaqI RFLP haplotypes, the common baT [35.7 vs. 47.3%, OR = 1.63 (1.17–2.27), Pc = 0.012] and rare BaT variants [6.5 vs. 1.2%, OR = 0.17 (0.06–0.55), Pc = 1.2 × 10−3] were associated with HT, representing predisposing and protective haplotypes, respectively. In single‐RFLP association analyses, only rs1544410 polymorphism was associated with HT phenotype (allelic Pc = 0.0078) and appeared to function under the recessive model, with decreased risk of HT among the BB homozygotes [OR = 0.39 (0.21–0.7), Pc = 0.0052] when compared to the reference b+‐genotypes. These data suggest that common haplotypic variants within the VDR gene 3′‐region previously linked to VDR mRNA expression and allelic imbalance could be associated with HT in the general population, and thus, may be involved in the pathogenesis of HT.

9-Deazaguanine derivatives connected by a linker to difluoromethylene phosphonic acid are slow-binding picomolar inhibitors of trimeric purine nucleoside phosphorylase

Genetic deficiency of purine nucleoside phosphorylase (PNP; EC 2.4.2.1) activity leads to a severe selective disorder of T‐cell function. Therefore, potent inhibitors of mammalian PNP are expected to act as selective immunosuppressive agents against, for example, T‐cell cancers and some autoimmune diseases. 9‐(5′,5′‐difluoro‐5′‐phosphonopentyl)‐9‐deazaguanine (DFPP‐DG) was found to be a slow‐ and tight‐binding inhibitor of mammalian PNP. The inhibition constant at equilibrium (1 mm phosphate concentration) with calf spleen PNP was shown to be  = 85 ± 13 pm (pH 7.0, 25 °C), whereas the apparent inhibition constant determined by classical methods was two orders of magnitude higher ( = 4.4 ± 0.6 nm). The rate constant for formation of the enzyme/inhibitor reversible complex is (8.4 ± 0.5) × 105 m−1·s−1, which is a value that is too low to be diffusion‐controlled. The picomolar binding of DFPP‐DG was confirmed by fluorimetric titration, which led to a dissociation constant of 254 pm (68% confidence interval is 147–389 pm). Stopped‐flow experiments, together with the above data, are most consistent with a two‐step binding mechanism: E + I ↔ (EI) ↔ (EI)*. The rate constants for reversible enzyme/inhibitor complex formation (EI), and for the conformational change (EI) ↔ (EI)*, are kon1 = (17.46 ± 0.05) × 105 m−1·s−1, koff1 = (0.021 ± 0.003) s−1, kon2 = (1.22 ± 0.08) s−1 and koff2 = (0.024 ± 0.005) s−1, respectively. This leads to inhibition constants for the first (EI) and second (EI)* complexes of Ki = 12.1 nM (68% confidence interval is 8.7–15.5 nm) and  = 237 pm (68% confidence interval is 123–401 pm), respectively. At a concentration of 10−4 m, DFPP‐DG exhibits weak, but statistically significant, inhibition of the growth of cell lines sensible to inhibition of PNP activity, such as human adult T‐cell leukaemia and lymphoma (Jurkat, HuT78 and CCRF‐CEM). Similar inhibitory activities of the tested compound were noted on the growth of lymphocytes collected from patients with Hashimoto’s thyroiditis and Graves’ disease. The observed weak cytotoxicity may be a result of poor membrane permeability.

Inhibitory Properties of Nucleotides with Difluoromethylenephosphonic Acid as a Phosphate Mimic versus Calf Spleen Purine Nucleoside Phosphorylase and Effect of These Analogues on the Viability of Human Blood Lymphocytes

Several cyclic and acyclic 6-keto purine nucleotides with difluoromethylenephosphonic acid as phosphate mimic are proved to be potent inhibitors of mammalian purine nucleoside phosphorylase (PNP). Antiproliferative activity of these analogues on the growth of human blood lymphocytes was tested by MTT assay. Compared to inhibitory effects on the growth of human blood T-lymphocytes isolated from healthy donors, all analogues significantly slow down proliferation of T-lymphocytes isolated from patients with autoimmune thyroid disease—Hashimotos thyroiditis.

Antiproliferative Activity of Purine Nucleoside Phosphorylase Multisubstrate Analogue Inhibitors Containing Difluoromethylene Phosphonic Acid against Leukaemia and Lymphoma Cells

Potent inhibitors of purine nucleoside phosphorylase (PNP) are expected to act as selective agents against T‐cell tumours. Five compounds with guanine, three with hypoxanthine, and five with 9‐deazaguanine, all connected by a linker with difluoromethylene phosphonic acid, were studied on their inhibitory potential against human and calf PNPs. Antiproliferative activity of these analogues against lymphocytes as well as lymphoma and leukaemia cells has been also investigated. All tested compounds act as multisubstrate analogue inhibitors of PNP with the apparent inhibition constants in the range 5–100 nm, and also show a slight antiproliferative activity. Analogues with 9‐deazaguanine aglycone have better anti‐leukaemic and anti‐lymphoma activities compared to the guanine and hypoxanthine analogues, and applied in the concentration of 100 μm, caused a statistically significant decrease in the cell viability in all human leukaemia and lymphoma cells used. Despite the high PNP inhibitory potential of tested analogues, no differences were observed between the effects on the growth of tumour cells sensible to the inhibition of PNP, such as human adult T‐cell leukaemia and lymphoma cells, and other investigated cells. Obtained poor effects on cell proliferation could be explained probably by a poor ability of tested compounds to penetrate cell membranes.