Polymorphism of the Y-chromosome diallelic loci in the ethnic populations of the Altai-Sayan region

Using the data on five biallellic Y-chromosome loci (DYS199, 92R7, SRY1532, RBF5 and DYS287) polymorphism, genetic structures of the five Turkic-speaking ethnic groups of the Altai-Sayan highland (Tuvinians, Sojots, Shorians, Khakassians, and Southern Altaians (Altai-Kizhi), were described. The gene pools of the populations examined were characterized by the presence of pronounced paleo-Caucasoid component (92R7-T-lineages). The frequency of this component increased westward, reaching more than 70% in Shorians and Southern Altaians. Haplotype TAT-C (RBF5 locus) was observed in all populations, except Shorians, with the frequencies varying from 5.4% in Altai-Kizhi to 18.8% in Khakassians. The Alu-insertion in the DYS287 locus was revealed only in the Altaian sample with the frequency of 3.3%. It was established that the Altai-Sayan populations studied split into two statistically significantly different groups. One of the groups was represented by Tuvinians, Sojots, and Khakassians, while another one was comprised of Shorians and Altaians.     

Volume-activated chloride currents from human fibroblasts: blockade by nimodipine

The whole-cell patch clamp technique was used to identify and to characterize volume-activated Cl- current (ICl(vol)) in fibroblasts derived from human periodontal ligament. During osmotic cell swelling, the cells exhibited an outwardly rectifying current, which was dependent upon the concentration of external Cl-. The anion permeability sequence of the chloride channel for anions was as follows: SCN- > I- > Br- > Cl- > F- > methanesulphonate > gluconate. Being an inhibitor of Cl- channels and Cl-/HCO exchanger, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) inhibited the currents with a voltage-dependence (EC50 57 micromol/l at +80 mV), and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), a carboxylate analogue Cl- channel blocker, showed the reversible suppression of the currents in a dose-dependent manner (EC50 = 59 micromol/l). Nimodipine, a selective dihydropyridine Ca2+ channel blocker suppressed ICl(vol) (EC50 = 66 micromol/l) and the effects were quite similar to those of NPPB. Nifedipine, another DHP blocker also inhibited the currents but with lesser efficacy (EC50 = 139 micromol/l). The removal of external Ca2+ or the addition of Cd2+ in the bath solution did not affect the blocking effects of nimodipine on ICl(vol). These findings demonstrate that the human fibroblasts ICl(Vol) was suppressed by nimodipine in an extracellular Ca2+-independent way. These results may provide, at least in part, an explanation for the Ca2+-independent decrease in Cl-/organic osmolytes efflux and RVD responses by nimodipine in some cell types.     

Potassium-controlled synthesis of heterotopic macrocycles based on isothiosemicarbazide

The macrocyclisation reaction of 3,3′-(3,6-dioxaoctane-1,8-diyldioxy)-bis(2-hydroxybenzaldehyde) (1) with S-methylisothiosemicarbazide hydroiodide (H₂NNC(SCH₃)NH₂·HI) in the presence of potassium triflate, followed by addition of M(CH₃COO)₂·nH₂O, where M=Ni, Cu, Zn, afforded [NiLKI₃] (2), [NiLK(CF₃SO₃)] (3), [CuLK(CF₃SO₃)(CH₃OH)] (4) and [(ZnILK)₂CH₃OH] (5), respectively. Compounds 2-5 have been characterised by X-ray crystallography. IR, electronic, mass, ¹H, ¹³C{¹H} and ¹⁹F{¹H} NMR spectra are reported. Magnetic susceptibility measurements and ESR spectra of 4 indicate weak intermolecular spin-spin interactions, which are mostly dipolar in origin.     

First structural evidence of a specific inhibition of phospholipase A2 by alpha-tocopherol (vitamin E) and its implications in inflammation: crystal structure of the complex formed between phospholipase A2 and alpha-tocopherol at 1.8 A resolution

This is the first structural evidence of alpha-tocopherol (alpha-TP) as a possible candidate against inflammation, as it inhibits phospholipase A2 specifically and effectively. The crystal structure of the complex formed between Vipera russelli phospholipase A2 and alpha-tocopherol has been determined and refined to a resolution of 1.8 A. The structure contains two molecules, A and B, of phospholipase A2 in the asymmetric unit, together with one alpha-tocopherol molecule, which is bound specifically to one of them. The phospholipase A2 molecules interact extensively with each other in the crystalline state. The two molecules were found in a stable association in the solution state as well, thus indicating their inherent tendency to remain together as a structural unit, leading to significant functional implications. In the crystal structure, the most important difference between the conformations of two molecules as a result of their association pertains to the orientation of Trp31. It may be noted that Trp31 is located at the mouth of the hydrophobic channel that forms the binding domain of the enzyme. The values of torsion angles (phi, psi, chi(1) and chi(2)) for both the backbone as well as for the side-chain of Trp31 in molecules A and B are -94 degrees, -30 degrees, -66 degrees, 116 degrees and -128 degrees, 170 degrees, -63 degrees, -81 degrees, respectively. The conformation of Trp31 in molecule A is suitable for binding, while that in B hinders the passage of the ligand to the binding site. Consequently, alpha-tocopherol is able to bind to molecule A only, while the binding site of molecule B contains three water molecules. In the complex, the aromatic moiety of alpha-tocopherol is placed in the large space at the active site of the enzyme, while the long hydrophobic channel in the enzyme is filled by hydrocarbon chain of alpha-tocopherol. The critical interactions between the enzyme and alpha-tocopherol are generated between the hydroxyl group of the six-membered ring of alpha-tocopherol and His48 N(delta1) and Asp49 O(delta1) as characteristic hydrogen bonds. The remaining part of alpha-tocopherol interacts extensively with the residues of the hydrophobic channel of the enzyme, giving rise to a number of hydrophobic interactions, resulting in the formation of a stable complex.     

Lack of an immune response against the tetracycline-dependent transactivator correlates with long-term doxycycline-regulated transgene expression in nonhuman primates after intramuscular injection of recombinant adeno-associated virus

We previously documented persistent regulation of erythropoietin (Epo) secretion in mice after a single intramuscular (i.m.) injection of a recombinant adeno-associated virus (rAAV) vector harboring both the tetracycline-dependent transactivator (rtTA) and the Epo cDNA (D. Bohl, A. Salvetti, P. Moullier, and J. M. Heard, Blood 92:1512-1517, 1998). Using the same vector harboring the cynomolgus macaque Epo cDNA instead, the present study evaluated the ability of the tetracycline-regulatable (tetR) system to establish long-term transgene regulation in nonhuman primates. The vector was administered i.m., after which 5-day induction pulses were performed monthly for up to 13 months by using doxycycline (DOX), a tetracycline analog. We show that initial inductions were successful in all individuals and that there was a tight regulation and a rapid deinduction pattern upon DOX withdrawal. For one macaque, regulation of Epo secretion was maintained during the entire experimental period; for the five remaining macaques, secreted Epo became indistinguishable from endogenous Epo upon repeated DOX inductions. We investigated the mechanism involved and showed that, except in the animal in which secretion persisted, delayed humoral and cellular immune responses were directed against the rtTA transactivator protein associated with the reduction of vector DNA in transduced muscles. This study provides some evidence that, when the immune system is not mobilized against the rtTA transactivator, the tetR-regulatable system is able to support long-term transgene regulation in the context of an rAAV in nonhuman primates. In addition, our results suggest potential improvements for vector design.     

Cathepsin B stability,but not activity,is affected in cysteine:cystine redox buffers

In order to test the hypothesis that the lysosomal cysteine protease cathepsin B may be redox regulated in vivo, cathepsin B activity and stability were measured in cysteine- and/or cystine-containing buffers. Cathepsin B activity in cysteine-containing buffers was similar at pH 6.0 and pH 7.0, over all thiol concentrations tested. In contrast, the stability of the enzyme was greater at pH 6.0 than at pH 7.0. This suggests that the enzyme's operational pH in vivo may be < pH 7.0. The activity of the enzyme was depressed in glutathione-containing buffers. When assessed in cysteine:cystine redox buffers (pH 6.0-7.0) cathepsin B was active over a broad redox potential range, suggesting that cathepsin B activity may not be redox regulated. However, at pH 7.0, the stability of cathepsin B decreased with increasing reduction potential and ambient cystine concentration. This suggests that the stability of the enzyme at neutral pH is dependent on redox potential, and on the presence of oxidising agents.     

Physical and functional interaction of the Arabidopsis K(+) channel AKT2 and phosphatase AtPP2CA

The AKT2 K(+) channel is endowed with unique functional properties, being the only weak inward rectifier characterized to date in Arabidopsis. The gene is expressed widely, mainly in the phloem but also at lower levels in leaf epiderm, mesophyll, and guard cells. The AKT2 mRNA level is upregulated by abscisic acid. By screening a two-hybrid cDNA library, we isolated a protein phosphatase 2C (AtPP2CA) involved in abscisic acid signaling as a putative partner of AKT2. We further confirmed the interaction by in vitro binding studies. The expression of AtPP2CA (beta-glucuronidase reporter gene) displayed a pattern largely overlapping that of AKT2 and was upregulated by abscisic acid. Coexpression of AtPP2CA with AKT2 in COS cells and Xenopus laevis oocytes was found to induce both an inhibition of the AKT2 current and an increase of the channel inward rectification. Site-directed mutagenesis and pharmacological analysis revealed that this functional interaction involves AtPP2CA phosphatase activity. Regulation of AKT2 activity by AtPP2CA in planta could allow the control of K(+) transport and membrane polarization during stress situations.     

A Comparative Structure–Function Analysis and Molecular Mechanism of Action of Endonucleases from Serratia marcescens and Physarum polycephalum

Structural and functional characteristics were compared for wild-type nuclease from Serratia marcescens, which belongs to the family of DNA/RNA nonspecific endonucleases, its mutational forms, and the nuclease I-PpoI from Physarum polycephalum, which is a representative of the Cys-His box-containing subgroup of the superfamily of extremely specific intron-encoded homing DNases. Despite the lack of sequence homology and the overall different topology of the Serratia marcescens and I-PpoI nucleases, their active sites have a remarkable structural similarity. Both of them have a unique magnesium atom in the active site, which is a part of the coordinatively bonded water–magnesium complex involved in their catalytic acts. In the enzyme–substrate complexes, the Mg²⁺ ion is chelated by an Asp residue, coordinates two oxygen atoms of DNA, and stabilizes the transition state of the phosphate anion and 3"-OH group of the leaving nucleotide. A new mechanism of the phosphodiester bond cleavage, which is common for the Serratia marcescens and I-PpoI nucleases and differs from the known functioning mechanism of the restriction and homing endonucleases, was proposed. It presumes a His residue as a general base for the activation of a non-cluster water molecule at the nucleophilic in line displacement of the 3"-leaving group. A strained metalloenzyme–substrate complex is formed during hydrolysis and relaxes to the initial state after the reaction.     

Immunodetection and clinico-pathological correlates of two tumour growth regulators in laryngeal carcinoma

Activation of telomerase, present in the vast majority of all human cancers, is associated with elongation of chromosomal telomeres and consequent cell immortalization. Telomere length homeostasis is a dynamic process governed by the negative feedback mechanism of the telomeric repeat binding factor 1 (TRF1) which inhibits the action of telomerase in telomerase-positive cells. In an attempt to investigate markers of tumour growth as possible prognostic indicators in laryngeal cancer, we studied the expression of TRF1 and of the proliferation marker Ki67 on 96 invasive squamous carcinomas of the larynx. A standard three step immunoperoxidase staining method was applied on paraffin sections incubated with appropriate polyclonal antibodies. The percentages of Ki67- and TRF1-immunopositive cancerous cells were calculated by image analysis. Univariate and multivariate statistical analysis of the staining results were performed in order to detect any association of the examined immunomarkers with the tumours' classical clinicopathological variables including nuclear morphometric features as well as with patients' disease-free survival. Ki67 immunostaining was positively linked with advanced patients' age, nodal involvement as well as presence of early recurrence. No relation was found between proliferative fraction and TRF1 immunoexpression. TRF1 was expressed in 55.2% of all cases and was positively linked only to tumour size. Multivariate statistical analysis revealed the presence of lymph nodal metastasis and Ki67 immunopositivity index > or = 20% as significant predictors of relapse. Increased Ki67 immunostaining appears to be a promising marker of tumour aggressiveness in laryngeal cancer. After one point at the tumour's natural history, the maintenance of tumour growth does not seem to depend on cell proliferation but on TRF1 immunoexpression. Whether the latter can be used for the identification of immortalized cells in every-day practice is worth investigating.     

Phenotypic heterogeneity associated with identical mutations in residue 870 of the androgen receptor

BACKGROUND/AIMS: Mutations in the androgen receptor (AR) gene result in an X-linked recessive form of male pseudohermaphroditism known as the androgen-insensitivity syndrome (AIS). The alterations most frequently observed are missense or nonsense point mutations in exons 4-8 of the AR gene that affect the steroid-binding domain of the receptor in subjects with various degrees of androgen resistance. Despite the increasing number of AR mutations identified, a reliable genotype-phenotype correlation has not been established and individuals with the same molecular defect may exhibit different phenotypes. Here, we studied a patients with an AIS characterized by bilateral gynecomastia, normal male external genitalia, and normal sperm counts. METHODS: Exon-specific polymerase chain reaction, single-stranded conformational polymorphism, and sequencing analysis of the subject's AR gene were performed in addition to hormone-binding assays in skin fibroblasts from the patient. RESULTS: A point mutation at codon 870 of the AR, changing alanine to valine, was detected. CONCLUSION: As AR missense mutations changing alanine 870 to valine have been previously described in 3 unrelated patients showing severe AIS phenotypes, we conclude that phenotypic heterogeneity associated to identical mutations in the AR gene is probably due to individual functional differences in AR coregulator molecules.