Desulfurization of 2-thiouracil nucleosides: conformational studies of 4-pyrimidinone nucleosides

4-Pyrimidinone ribofuranoside (H(2)o(4)U) and 4-pyrimidinone 2'-deoxyribofuranoside (dH(2)o(4)U) were synthesized by the oxidative desulfurization of parent 2-thiouracil nucleosides with m-chloroperbenzoic acid. The crystal structures of H(2)o(4)U and dH(2)o(4)U and their conformations in solution were determined and compared with corresponding 2-thiouracil and uracil nucleosides. The absence of a large 2-thiocarbonyl/2-carbonyl group in the nucleobase moiety results in C2'-endo puckering of the ribofuranose ring (S conformer) in the crystal structure of H(2)o(4)U, which is not typical of RNA nucleosides. Interestingly, the hydrogen bonding network in the crystals of dH(2)o(4)U stabilizes the sugar moiety conformation in the C3'-endo form (N conformer), rarely found in DNA nucleosides. In aqueous solution, dH(2)o(4)U reveals a similar population of the C2'-endo conformation (65%) to that of 2'-deoxy-2-thiouridine (62%), while the 62% population of the S conformer for H(2)o(4)U is significantly different from that of the parent 2-thiouridine, for which the N conformer is dominant (71%). Such a difference may be of biological importance, as the desulfurization process of natural tRNA 2-thiouridines may occur under conditions of oxidative stress in the cell and may influence the decoding process.     

From research to implementation: Nature conservation in the Eastern Rhodopes mountains (Greece and Bulgaria), European Green Belt

Nature conservation should ideally build on the scientific recommendations that are concluded from applied conservation research, as well as on monitoring schemes that evaluate the effectiveness of recommendations. We considered as a case study a system of six protected areas located in the Eastern Rhodopes mountains in the southern part of the European Green Belt (EGB). To investigate nature conservation effectiveness, we reviewed 196 articles from scientific journals and books, eight doctoral and master theses, and 39 scientific reports regarding the Greek (one protected area, 428 km²) and the Bulgarian (five protected areas, 904 km²) part of the study area. We extracted 743 conservation recommendations, and through questionnaires completed by 10 local experts, we found that 74% of the recommendations were familiar for the experts. In the Greek (GR) and the Bulgarian part (BG) only 52% and 16%, respectively, of the recommendations were implemented, and only 15% (GR) and 3.1% (BG) were implemented and evaluated regarding their effectiveness. According to the experts, the main reasons for non-implementation and non-evaluation were absence or incompetence of the responsible authorities. Some recommendations obtained a remarkable low rate of implementation, such as those regarding agriculture and livestock rearing practices (GR: 29%, BG: 16%) or mammal conservation (GR: 0%, BG: 16%). Some other recommendations obtained rather high rates at least for Greece, such as tourism and environmental education (GR: 57%, BG: 42%) and bird conservation (GR: 57%, BG: 11%). We found that researchers and conservation managers at both sides of the Greek-Bulgarian border face similar implementation problems, related often to the lack of political will for nature conservation and establishment of competent authorities. The role of the EGB is crucial in enhancing the established cross-border collaborations between stakeholders involved in nature conservation.     

Role of FSH and triiodothyronine in Sertoli cell development expressed by formation of connexin 43-based gap junctions

Follicle-stimulating hormone (FSH) and triiodothyronine (T3) are known regulatory factors of spermatogenesis initiation. Connexin 43 (Cx43) is the most ubiquitous constitutive protein of gap junctions in the testis. This study evaluates the effects of the hyperstimulation of FSH and T3 during testicular maturation on Cx43 expression in the testis. The newborn, male Wistar rats were divided randomly into four experimental groups: FSH group-daily injections of FSH 7.5?IU/animal; T3 group-100?μg T3/kg body weight; FSH+T3 group-both substances; A control group-received vehicles in the same volume. Proliferating cell nuclear antigen immunohistochemistry and toluidine blue staining were used to determine the germ cell proliferation and degeneration. Cx43 immunolocalization was evaluated to find Cx43 maturational changes. Under FSH treatment, the proliferation rate was high so the total number of Sertoli cells increased with a low level of degeneration and lumen formation. T3 stimulation evoked a reduction in the proliferation rate and a decrease in Sertoli cell number but with intensive formation of lumen. T3+FSH inhibited the proliferation rate and stimulated lumen formation together with degeneration, which negatively influenced the number of germ cells in the seminiferous epithelium. We conclude that T3 action seems to be particularly connected with the maturation of Cx43 gap junctions. FSH stimulates maturation of Sertoli cell function, but this effect may take place regardless of the presence of Cx43-dependent intercellular communication. The hyperstimulation of both FSH and T3 damages Cx43 connections and hence evokes regressional changes in the seminiferous epithelium.     

Effect of aminoguanidine and albendazole on inducible nitric oxide synthase (iNOS) activity in T. spiralis-infected mice muscles

The aim of this study was to provide evidence for the expression of iNOS in the cells of inflammatory infiltrates around larvae in skeletal muscles of T. spiralis infected mice. The BALB/c mice (n = 8) divided into subgroups, received either aminoguanidine (AMG)--a specific iNOS inhibitor or albendazole (ALB)--an antiparasitic drug of choice in trichinellosis treatment. Control animals (n = 2 in each subgroup) were either uninfected and treated or uninfected and untreated. Frozen sections of hind leg muscles from mice sacrificed at various time intervals after infection were cut and subjected to immunohistochemistry, using monoclonal anti-iNOS antibody. The ALB-treated mice revealed stronger iNOS staining in the infiltrating cells around larvae than the infected and untreated animals. On the contrary, in the AMG-treated animals, the infiltrating cells did not show any specific iNOS reaction. These data confirm the specificity of iNOS staining in the cellular infiltrates around T. spiralis larvae and shed some light on the role of nitric oxide during ALB treatment in experimental trichinellosis.     

Significance of MDM2 protein in the cell cycle

The basis of oncogenesis underlies the modification of the control of the cell cycle, which leads to disturb balance between proliferation and apoptosis. The MDM2 protein suppresses the ability of p53 to activate genes responsible for repairing or apoptosis, but also promotes p53 degradation by ubiquitination. MDM2 inhibits tumor suppressor property of pRb, by releasing E2F1, which stimulates DNA synthesis in S-phase. MDM2 influences on the neuronal and muscle differentiation. Quantity and stability of the MDM2 protein is regulated by p73, p53, TSG101, p14ARF and Ras-Raf-MEK-ERK pathway. Changes of the level of the MDM2 can disturb control of cell cycle and contribute to oncogenesis.     

Sodium orthovanadate exerts influence on liver Golgi complexes from control and streptozotocin-diabetic rats

The paper presents the effect of one-week 3mM sodium orthovanadate (Na3VO4) oral treatment of control and streptozotocin[STZ]-diabetic rats. The body weight decreased as compared with untreated control (C group) in both vanadate treated groups (C + V and D + V) and in diabetic untreated rats (D group)--in all cases p < 0.01. A similar tendency was demonstrated by the weight of the livers, which was statistically significant lower than in the controls (p < 0.01). The fluid and food intake were lower in comparison with control vanadium treated groups, in D + V as compared with D it was limited, however, not achieved control level. A high mortality rate, approx. 67%, after the administration of streptozotocin and vanadate (D + V group) was noted; such result had never been previously found within all study groups of rats. But the surviving rats show very good decreased (60%) free blood sugar levels, however euglycaemia was not achieved. The activity of galactosyltransferase, the Golgi complex marker enzyme in group D, was statistically lower than the controls (p < 0.001). Treatment of STZ-diabetic rats with orthovanadate did not increase the enzyme activity toward control level, in both diabetic groups (treated and untreated with Na3VO4) similar dispersion of individual results was found. Morphological study demonstrated, for the first time, no larger cellular lesion in C + V group. The Golgi complex was well developed; showed several cisterns at the trans side, which were grossly distended and contained electron-lucid floccular material. In D + V group typical, cylindrical forms of Golgi complexes predominated. These structures consisted of 3-4 almost practically non-distended cisterns. Also in this case, large, electron-dense vesicles were noted in the vicinity. In this group, small in size, myelin-like structures were also found. These structures might indicate a relatively small, but nevertheless clear damage of the internal membrane system. The external cistern of the cylindrical forms of Golgi complexes, which corresponded the trans side, was often markedly distended and formed a vacuole-like structure filled with electron lucent material; the structure itself sometimes looked empty. Multi-vesicular structures were observed also in this case, but they were seen much more rarely.     

Selenium level in benign and cancerous prostate

The dietary microelement selenium (Se) has been proposed as a potential chemopreventive agent for prostate cancer. This element is present in various amounts in all tissues. Little information is available on Se level in patients with prostate gland disorders. The levels of Se in prostatic gland of patients with prostate cancer, benign prostate hyperplasia, and healthy controls were examined. The Se level for benign prostate hyperplasia (156±30.6 ng/g) was the same as in the control group (157±26.0 ng/g), but in the gland of prostate cancer patients (182±34.1 ng/g wet weight), the Se level was significantly (p<0.01) higher than in both healthy controls and benign prostate hyperplasia. Thus, the Se level in human healthy controls is lower than in kidney and liver but higher compared with other tissues.     

Antioxidant enzymes and lipid peroxides in children with cerebral palsy

Impaired antioxidant mechanisms are unable to inactivate free radicals that may induce a number of pathophysiological processes and result in cell injury. Thus, any abnormality in antioxidant defense systems could affect neurodevelopmental processes and could have an important role in the etiology of cerebral palsy (CP). The plasma levels of lipid peroxidation as plasma levels of malondialdehyde (MDA), activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) in plasma and erythrocytes were investigated in 34 CP children and compared with 61 normal controls. SOD, GPx and GR activities were spectrophotometrically assayed. Activities of SOD, GPx and GR in plasma did not differ significantly between CP children and the control group. Activities of erythrocyte GR in the CP patients were significantly lower compared with controls. MDA concentration did not differ statistically between the CP children and healthy subjects. In conclusion our results suggest that increased activities of erythrocyte GPx and decreased erythrocyte GR activities might be due to lesser physical activity of children with CP.     

Mycobacterium avium 104 deleted of the methyltransferase D gene by allelic replacement lacks serotype-specific glycopeptidolipids and shows attenuated virulence in mice

Mycobacterium avium is a major opportunistic pathogen of AIDS patients in the United States. The understanding of M. avium pathogenesis has been hampered by the inability to create gene knockouts by homologous recombination, an important mechanism for defining and characterizing virulence factors. In this study a functional methyltransferase D (mtfD) gene was deleted by allelic replacement in the M. avium strain 104. Methyltransferase D is involved in the methylation of glycopeptidolipids (GPLs); highly antigenic glycolipids found in copious amounts on the M. avium cell surface. Interestingly, the loss of mtfD resulted in M. avium 104 containing only the non-serotype specific GPL. Results also suggest that the mtfD encodes for a 3-O-methyltransferase. The absence of significant amounts of any serotype-specific GPLs as a consequence of mtfD deletion indicates that the synthesis of the core 3,4-di-O-methyl rhamnose is a prerequisite for synthesis of the serotype-specific GPLs. Macrophages infected with the mtfD mutant show elevated production of tumour necrosis factor-alpha (TNF-alpha) and RANTES compared to control infections. In addition, the M. avium 104 mtfD mutant exhibits decreased ability to survive/proliferate in mouse liver and lung compared to wild-type 104, as assessed by bacterial counts. Importantly, the mtfD mutant complemented with a wild-type mtfD gene maintained an infection level similar to wild-type. These experiments demonstrate that the loss of mtfD results in a M. avium 104 strain, which preferentially activates macrophages in vitro and shows attenuated virulence in mice. Together our data support a role for GPLs in M. avium pathogenesis.     

The imbalance between metalloproteinases and their tissue inhibitors is involved in the pathogenesis of dermatitis herpetiformis

Dermatitis herpetiformis (DH) is a subepidermal autoimmune disease characterized by skin and intestinal lesions consistent with coeliac disease. There are also some data that metalloproteinases (MMPs) are involved in the development of skin lesions in DH, however their exact role in this process is not fully understood. The aim of the study was to investigate whether MMPs and their inhibitors are involved in pathogenesis of DH. Skin biopsies were taken from 13 patients with active DH and from 10 healthy subjects. The localization and expression of MMPs and TIMPs were examined by immunohistochemistry. MMPs expression was detected in basal keratinocytes and in the whole epidermis in all of the DH subjects. Neutrophils in microabscesses and in blister fluid were also positive for MMPs. Expression of TIMPs was moderate or weak in all examined biopsies. Our results allow us to conclude that imbalance between these enzymes takes an important role in the pathogenesis of DH.