S-Adenosylhomocysteine hydrolase (AdoHcyase) deficiency: enzymatic capabilities of human AdoHcyase are highly effected by changes to codon 89 and its surrounding residues

Recently, S-adenosylhomocysteine hydrolase deficiency was confirmed for the first time in an adult. Two missense mutations in codons 89 (A>V) and 143 (Y>C) in the AdoHcyase gene were identified [N.R.M. Buist, B. Glenn, O. Vugrek, C. Wagner, S. Stabler, R.H. Allen, I. Pogribny, A. Schulze, S.H. Zeisel, I. Bari?, S.H. Mudd, S-Adenosylhomocysteine hydrolase deficiency in a 26-year-old man, J. Inh. Metab. Dis. 29 (2006) 538-545]. Accordingly, we have proven the Y143C mutation to be highly inactivating [R. Beluzi?, M. Cuk, T. Pavkov, K. Fumi?, I. Bari?, S.H. Mudd, I. Jurak, O. Vugrek, A single mutation at tyrosine 143 of human S-adenosylhomocysteine hydrolase renders the enzyme thermosensitive and effects the oxidation state of bound co-factor NAD, Biochem. J. 400 (2006) 245-253]. Now we report that the A89V exchange leads to a 70% loss of enzymatic activity, respectively. Circular dichroism analysis of recombinant p.A89V protein shows a significantly reduced unfolding temperature by 5.5 degrees C compared to wild-type. Gel filtration of mutant protein is almost identical to wild-type indicating assembly of subunits into the tetrameric complex. However, electrophoretic mobility of p.A89V is notably faster as shown by native polyacrylamide gel electrophoresis implicating changes to the overall charge of the mutant complex. 'Bioinformatics' analysis indicates that Val(89) collides with Thr(84) causing sterical incompatibility. Performing site-directed mutagenesis changing Thr(84) to 'smaller' Ser(84) but preserving similar physico-chemical properties restores most of the catalytic capabilities of the mutant p.A89V enzyme. On the other hand, substitution of Thr(84) with Lys(84) or Gln(84), thereby introducing residues with higher volume in proximity to Ala(89) results in inactivation of wild-type protein. In view of our mutational analysis, we consider changes in charge and the sterical incompatibility in mutant p.A89V protein as main reason for enzyme malfunction with AdoHcyase deficiency as consequence.     

Cyclosporine a induces growth arrest or programmed cell death of human glioma cells

Human malignant gliomas are highly resistant to current therapeutic approaches. We previously demonstrated that cyclosporine A (CsA) induces an apoptotic cell death in rat C6 glioma cells. In the present study, we found the induction of growth arrest or cell death of human malignant glioma cells exposed to CsA. In studied glioma cells, an accumulation of p21Cip1/Waf1 protein, a cell cycle inhibitor, was observed following CsA treatment, even in the absence of functional p53 tumour suppressor. CsA induced a senescence-associated growth arrest, in U87-MG glioma cells with functional p53, while in U373 and T98G glioma cells with mutated p53, CsA treatment triggered cell death associated with alterations of cell morphology, cytoplasm vacuolation, and condensation of chromatin. In T98G cells this effect was completely abolished by simultaneous treatment with an inhibitor of protein synthesis, cycloheximide (CHX). Moreover, CsA-induced cell death was accompanied by activation of executory caspases followed by PARP cleavage. CsA treatment did not elevate fasL expression and had no effect on mitochondrial membrane potential. We conclude that CsA triggers either growth arrest or non-apoptotic, programmed cell death in human malignant glioma cells. Moreover, CsA employs mechanisms different to those in the action of radio- and chemotherapeutics, and operating even in cells resistant to conventional treatments. Thus, CsA or related drugs may be an effective novel strategy to treat drug-resistant gliomas or complement apoptosis-based therapies.     

1,2,3,4-Tetrahydroisoquinoline Derivatives as a Novel Deoxyribonuclease I Inhibitors

Herein we report an assessment of 24 1,2,3,4-tetrahydroisoquinoline derivatives for potential DNase I (deoxyribonuclease I) inhibitory properties in vitro. Four of them inhibited DNase I with IC₅₀ values below 200 μM. The most potent was 1-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)propan-2-one ( 2 ) (IC₅₀=134.35±11.38 μM) exhibiting slightly better IC₅₀ value compared to three other active compounds, 2-[2-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]-1-phenylethan-1-one ( 15 ) (IC₅₀=147.51±14.87 μM), 2-[2-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]cyclohexan-1-one ( 18 ) (IC₅₀=149.07±2.98 μM) and 2-[6,7-dimethoxy-2-(p-tolyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]cyclohexan-1-one ( 22 ) (IC₅₀=148.31±2.96 μM). Cytotoxicity assessment of the active DNase I inhibitors revealed a lack of toxic effects on the healthy cell lines MRC-5. Molecular docking and molecular dynamics simulations suggest that interactions with Glu 39, His 134, Asn 170, Tyr 211, Asp 251 and His 252 are an important factor for inhibitors affinity toward the DNase I. Observed interactions would be beneficial for the discovery of new active 1,2,3,4-tetrahydroisoquinoline-based inhibitors of DNase I, but might also encourage researchers to further explore and utilize potential therapeutic application of DNase I inhibitors, based on a versatile role of DNase I during apoptotic cell death.     

Correlates of pentraxin 3 serum concentration in men and women with type 2 diabetes

Elevated levels of plasma pentraxin 3 (PTX3), a marker of inflammation, are associated with the risk of developing cardiovascular diseases in the general population, as well as in patients with type 2 diabetes (DM2). In this study, we aimed to determine factors associated with PTX3 serum concentrations in men and women with DM2. The study included 116 consecutive patients (67 men and 49 women) with DM2 from an outpatient diabetic clinic. Men were characterised by lower age and higher uric acid, creatinine and bilirubin concentrations and waist/hip ratio than women. In women, low-density lipoprotein cholesterol (LDL-C) levels were higher than in men. In men, median (interquartile range) values of PTX3 concentration were 4.02 (1.99), and in women they were 4.53 (3.31) ng/ml (NS). In men, PTX3 concentrations correlated with total cholesterol (TC), triglycerides, apolipoprotein (Apo) C3, Apo B48, Glc and creatinine levels. In women, PTX3 correlated significantly with TC and LDL-C and Apo B100. Partial regression analysis revealed that after adjusting for age, PTX3 concentrations in men were significantly associated with TC, LDL-C, triglycerides, creatinine, Apo C3 and Apo B48, while in women they were associated with TC, LDL-C and Apo B100. The results could be of importance in sex-specific prevention of vascular complications in DM2 patients.     

The Oedipus complex in the contemporary psychoanalysis

In this article, authors have tried to answer the question: "Where is the place, and what is the meaning of the Oedipus complex in contemporary psychoanalysis?" The review of different theoretical standpoints was given, according to meaning and place of the Oedipus complex in human development. Although it depends on the resolving of preoedipal conflicts, the conflicts of phallic phases of the psychosexual development are universal to all human being, no matter how we call them--Oedipus, Electra or Persephone Complex.     

Morphological classification and sexual dimorphism of hyoid bone: new approach

Hyoid bone is a part of viscerocranium placed between the tongue root and thyroid cartilage to which it is connected by thyrohyoid membrane. Widely accepted morphological classification does not exist. Sexual dimorphism was analysed in this study and new guidelines for anatomical classification of hyoid bones based on anthropometric parameters were given. Total number of analysed bones was 70. The bones were classified into three groups: symmetrical U-type, symmetrical V-type and asymmetrical type according to the angle between greater horns and the proportion of greater horns length. In the females incidence of asymmetrical type is considerably higher than in the males, while the incidence of symmetrical V-type is lower The angle value that is on average higher in males may be the parameter indicating that in puberty hyoid bone, still not completely ossified, to some extent follows development of thyroid cartilage because of their close anatomical relation.     

Evolution of the tetraploid Anemone multifida (2n = 32) and hexaploid A. baldensis (2n = 48) (Ranunculaceae) was accompanied by rDNA loci loss and intergenomic translocation: evidence for their common genome origin

Background and Aims

In the genus Anemone two small groups of taxa occur with the highest ploidy levels 2n = 6x = 48, belonging to the closely related clades: the montane/alpine Baldensis clade and the more temperate Multifida clade. To understand the formation of polyploids within these groups, the evolution of allohexaploid A. baldensis (AABBDD, 2n = 6x = 48) from Europe and allotetraploid Anemone multifida (BBDD, 2n = 4x = 32) from America was analysed.

Methods

Internal transcribed spacer and non-transcribed spacer sequences were used as molecular markers for phylogenetic analyses. Cytogenetic studies, including genomic in situ hybridization with genomic DNA of potential parental species as probe, fluorescence in situ hybridization with 5S and 18S rDNA as probes and 18S rDNA restriction analyses, were used to identify the parental origin of chromosomes and to study genomic changes following polyploidization.

Key Results

This study shows that A. multifida (BBDD, 2n= 4x = 32) and A. baldensis (AABBDD, 2n = 6x = 48) are allopolyploids originating from the crosses of diploid members of the Multifida (donor of the A and B subgenomes) and Baldensis groups (donor of the D subgenome). The A and B subgenomes are closely related to the genomes of A. sylvestris, A. virginiana and A. cylindrica, indicating that these species or their progeny might be the ancestral donors of the B subgenome of A. multifida and A and B subgenomes of A. baldensis. Both polyploids have undergone genomic changes such as interchromosomal translocation affecting B and D subgenomes and changes at rDNA sites. Anemone multifida has lost the 35S rDNA loci characteristic of the maternal donor (B subgenome) and maintained only the rDNA loci of the paternal donor (D subgenome).

Conclusions

It is proposed that A. multifida and A. baldensis probably had a common ancestor and their evolution was facilitated by vegetation changes during the Quaternary, resulting in their present disjunctive distribution.     

Environment-related variations of the composition of the essential oils of rosemary (Rosmarinus officinalis L.) in the Balkan Penninsula

Composition of the essential oils of Rosmarinus officinalis of ten populations from the Balkan Peninsula were determined by GC/FID and GC/MS. The main constituents were 1,8-cineole, camphor, α-pinene, and borneol. Multivariate statistical analysis (UPGMA cluster analysis and principal-component analysis (PCA)) revealed two major types of rosemary oil, i.e., 1,8-cineole and camphor-type, and two intermediate types, i.e., camphor/1,8-cineole/borneol type and 1,8-cineole/camphor type. The regression analyses (simple linear regression and stepwise multiple regression) have shown that, with respect to basic geographic, orographic, and 19 bioclimatic characteristics of each population, bioclimatic factor temperature of habitat represented the dominant abiogenetic factor, which, in chemical sense, led to differentiation of populations in the studied region. Also, the regression analysis have shown that some constituents of essential oils are independent of any single bioclimatic factors. However, some constituents display statistically significant correlations with some abiotic factors.