Isolation and complete genome characterization of novel reassortant orthoreovirus from common vole (<Emphasis Type="Italic">Microtus arvalis</Emphasis>)

A novel mammalian orthoreovirus (MRV) strain was isolated from the lung tissue of a common vole (Microtus arvalis) with Tula hantavirus infection. Seven segments (L1–L3, M2–M3, S2, and S4) of the Hungarian MRV isolate MORV/47Ma/06 revealed a high similarity with an MRV strain detected in bank vole (Myodes glareolus) in Germany. The M1 and S3 segment of the Hungarian isolate showed the closest relationship with the sequence of a Slovenian human and a French murine isolate, respectively. The highest nucleotide and amino acid identity values were above 90 and 95% in all of the comparisons to the reference sequences in GenBank, except for the S1 with a maximum of 69.6% nucleotide and 75.4% amino acid identity. As wild rodents are among the main sources of zoonotic infections, the reservoir role of these animals and zoonotic potential of rodent origin MRVs need to be further investigated.     

Sequence Analysis of Porcine Adenovirus Serotype 5 Fibre Gene: Evidence for Recombination

The nucleic acid and deduced amino acid sequence of the fibre gene of the HNF-61 strain of porcine adenovirus serotype 5 (PAdV-5) was determined and compared to that of the HNF-70 strain of the same serotype (Nagy et al., J Gen Virol 82, 525–529, 2001) and also to adenovirus fibre genes from the genera Mastadenovirus and Atadenovirus. The putative HNF-61 and HNF-70 proteins were similar to each other, with 90% amino acid identity. Conserved amino acid sequences described for mastadenovirus fibre shafts were identified in the shaft regions of both PAdV-5 fibres, except for the so-called TLWT motif. The head regions of the PAdV-5 fibre did not resemble any of the known mastadenovirus fibre heads, but they showed characteristics of the fibre head protein sequences of viruses grouped in the proposed genus Atadenovirus (Benkö et al., Virus Taxonomy, Seventh Report of the International Committee on the Taxonomy of Viruses, Academic Press, New York, San Diego, 2000, pp. 227–238). The findings suggested recombination between viruses of different adenovirus genera.     

Chemical stabilization of tetrahydrobiopterin by L-ascorbic acid: contribution to placental endothelial nitric oxide synthase activity

The aim of this study was to characterize the mechanism of the chemical interaction between L-ascorbic acid (ASC) and tetrahydrobiopterin (BH(4)) in vitro and to examine its effect on the activity of endothelial nitric oxide synthase (eNOS) in first trimester human placentae. At room temperature, in Tris-HCl buffer (pH 7.4), both ASC and BH(4) were readily oxidized by dissolved O(2) or H(2)O(2). BH(4) was more sensitive to auto-oxidation, while ASC was more susceptible to oxidation by H(2)O(2). Addition of 36 micromol/l BH(4) to 143 micromol/l ASC increased the initial rate of ASC oxidation 3.2-fold in a catalase-sensitive manner, indicating that enhanced ASC oxidation is partly due to the formation of H(2)O(2). In the presence of catalase, BH(4) still stimulated 1.9-fold the initial rate of ASC oxidation, suggesting that another auto-oxidation product of BH(4), most probably quininoid-BH(2) (qBH(2)), could also stimulate ASC oxidation while itself being reduced back to BH(4). ASC prevented the auto-oxidation of BH(4) in a concentration-dependent fashion, with 3 mmol/l ASC providing an almost complete stabilization of 25 micromol/l BH(4). Importantly, basal eNOS activity in placental microsomes was stimulated 2.5-fold by 0.5 micromol/l BH(4), and 0.5 mmol/l ASC enhanced the BH(4)-stimulation 1.4-fold, with a smaller effect on basal eNOS activity. Taken together, the findings support the notion that the stabilizing action of ASC on BH(4) is related to the ASC-mediated reductive reversal of the auto-oxidation process of BH(4). Moreover, we demonstrated that concentrations of ASC present in the placenta as a common vitamin C supply are sufficient to protect cellular free BH(4) and may contribute to the stimulation of placental eNOS activity.     

Preconditioning-specific reduction of c-fos expression in hippocampal granule and pyramidal but not other forebrain neurons of ischemic brain: a quantitative immunohistochemical study

To specify targets for an ischemic preconditioning paradigm (ischemic tolerance), c-fos expressions in ischemic (induced by 10 min bilateral carotid-occlusions subsequent to coagulation of vertebral arteries) and preconditioned rats (treated for 4 min carotid-occlusions 72 h before ischemia) were compared in 12 forebrain areas/nuclei. Fos immunostaining was applied to serial sections of the forebrain and the density (cell number/area measured) of Fos-immunopositive (Fos+) neurons, as well as their percentile changes were determined in five hippocampal and seven extrahippocampal areas/nuclei of ischemic and preconditioned rats. The ratio of counts found in ischemic over control animals showed several fold increase of Fos+ cells in the three layers (granule cell, molecular and polymorphic) of the dentate gyrus, CA3 and CA1 pyramidal neurons, as well as in thalamic and hypothalamic nuclei and limbic cortical areas. In contrast, preconditioning did not alter c-fos expressions significantly in the extrahippocampal brain areas investigated. These results strengthen the hypothesis that the hippocampal and dentate neurons are more susceptible to ischemic tolerance than cells in other brain regions. In fact stress-response and induction of ischemic tolerance in different forebrain areas can be distinguished.     

Usage patterns and attitudes towards emergency contraception: the International Emergency Contraception Research Initiative

Objectives:?The aim of the survey was to gain understanding of women’s usage patterns and attitudes towards emergency contraception (i.e., the ‘morning after pill’) and to gain insight into the role and attitudes of pharmacists as providers of emergency contraception.

Methods:?As part of the International Emergency Contraception Research Initiative, approximately 6500 women (15–49 years) and nearly 500 pharmacists from 14 countries in Western, Central and Eastern Europe, and Central Asia completed questionnaires via web-based interrogation or computer-assisted/paper-assisted personal interviews.

Results:?Common to almost all countries and cultures was that, while awareness of emergency contraception was high (≥84% of respondents, except in Kazakhstan), usage was generally low (4–18%). In Austria, the Czech Republic, Spain, and the UK, better underlying protection with hormonal contraceptives or male condoms would have meant less need for emergency contraception. In Bulgaria, Lithuania, Romania, and Russia, greater dependence on less reliable contraceptive methods such as calendar?+?withdrawal was associated with higher use of the emergency contraceptive pill (11–18%) but also with higher abortion rates (19–21%). Overt rejection of emergency contraception in the event of an accident was low, except in countries (e.g., Austria, Poland) where the misperception that it acts as an abortifacient was common. Except for Bulgaria, pharmacists elsewhere tended to have limited knowledge and moralistic attitudes towards emergency contraception.

Conclusions:?Improved educational efforts, probably country-specific, are required to increase the use of highly effective methods of regular contraception and overcome barriers to acceptance of emergency contraception as a suitable postcoital solution to avoid unwanted pregnancy or abortion.     

Immunohistochemical verification of ductal differentiation in prostate cancer

Recent studies have shown that patients with prostate carcinomas exhibiting ductal differentiation have an unfavourable prognosis compared with those with purely acinar adenocarcinomas. We studied the expression of nine immunohistochemical markers to evaluate their value in delineating carcinomas with and without ductal differentiation. Thirteen tumours showing cellular characteristics and growth patterns typical of ductal differentiation were identified among 110 analysed prostatectomy specimens. The levels of cytoplasmic expression of chromogranine A (69% vs 19%, p = 0.0003) and nuclear expression of p53 (76% vs 12%, p < 0.0001) as well as nuclear expression of Ki-67 (69% vs 26%, p = 0.0047) in the tumour cells, were found to be statistically significantly different in the two tumour categories. Assessment of chromogranine A, p53 and Ki-67 in prostate carcinoma may serve as useful adjunctive diagnostic tools for delineating more aggressive prostate cancer cases exhibiting ductal differentiation.     

Molecular comparison of the fibre proteins of bovine adenovirus subtype A and subtype B

The fibre gene of the bovine adenovirus type 2 (BAdV2) subtype B was prepared for sequencing by using cloning, sub-cloning and PCR amplification techniques. The nucleotide sequence of the total fibre gene was determined, and it was found to consist of 1,647 nucleotides, coding for a polypeptide of 549 amino acids. The fibre gene regions of BAdV2 A and B subtypes were aligned. The nucleotide identity of the total fibre gene was found to be 60.5%; however, the homology showed great differences in the different subregions coding for the shaft and knob part of the fibre, and the two subtypes were almost identical in the tail subregion. Remarkable changes indicating deletion, insertion and point mutations were found in the shaft subregion when BAdV2/A and B subtypes were compared. We concluded that the differences found in the haemagglutinating activity of the two subtypes of BAdV2 can mostly be explained by the changes in the polypeptide structure of the fibre shaft.     

The patella and tibial condyle position after combined and after closing wedge high tibial osteotomy

High tibial osteotomy changes the patella and tibial condyle position, which makes the subsequent total knee replacement technically demanding. From 1 January 1993 to 31 December 2000, combined osteotomy [After the first osteotomy made 2 cm distally to the joint line, a bone wedge is removed based laterally. Its tip ends at the center of the tibial condyle (half bone wedge). The distal part of the tibia is placed into the valgus position and the half bone wedge is placed into the gap opened medially.] was performed on 103 knees and closing wedge osteotomy was performed on 47 consecutive knees. Eighty combined (group A) and 41 closing wedge (group B) osteotomy were studied. All knees were assessed radiologically before surgery, in the 10th postoperative week, in the 12th postoperative month and at the time of the final follow-up (in group A—66.15 months, in group B—66.61 months). We examined the change of the femorotibial angle, of the patellar height according to the method of Insall and Salvati, of the tibial slope angle according to the method of Bonnin, of the tibial condylar offset according to the method of Yoshida and of the distance between the lateral tibial plateau and the top of the fibular head. In group A and B, the recurrence of the varus deformity was not noted and valgus alignment did not increase in any case. In group-A, the Insall–Salvati ratio remained unchanged in 65% of knees. The tibial slope angle decreased in both groups. There was correlation between the change of the tibial condylar offset and the angle of the correction in both groups. There was correlation between the change of the distance between the lateral tibial plateau and the top of the fibular head. After combined osteotomy, the transposition of the tibial condyle and the decrease of the distance between the lateral tibial plateau and the top of the fibular head was less than after closing wedge osteotomy, although the average angle of correction was more after combined osteotomy (11.835°), than after closing wedge osteotomy (9.465°). Theoretically, the recurrence of the varus deformity, the increase of the valgus alignment and (in majority of cases) the shortening of the patellar tendon do not compromise the likelihood of successful conversion to the subsequent total knee replacement, either after combined or after closing wedge osteotomy. The combined osteotomy does not lead to considerable transposition of the tibial condyle and to considerable lateral tibial bone loss; therefore, theoretically, the combined osteotomy does not impair the subsequent total knee replacement.     

The effects of CRF and the urocortins on [3H]GABA release from the rat amygdala--an in vitro superfusion study

Corticotropin-releasing factor (CRF) is the major neuromodulator of the hypothalamic-pituitary-adrenal axis, regulating the behavioural, endocrine, autonomic and immune responses to stress. Together with the recently discovered members of the CRF peptide family, urocortin 1, urocortin 2 and urocortin 3, it also has neurotransmitter actions. Previous publication has demonstrated that stress induces CRF release in the paraventricular nucleus of the hypothalamus and the release of both CRF and GABA in the amygdala. Accordingly, the aim of the present study was to determine the effects of the members of the CRF peptide family on GABA release from the amygdala by using an in vitro superfusion system. In order to study the participation of different CRF receptors (CRF1 and CRF2) in this process, rat amygdalar slices were pretreated with selective CRF1 and CRF2 antagonists. CRF and urocortin 1 significantly increased the release of [(3)H]GABA from the slices following electrical stimulation, whereas urocortin 2 and urocortin 3 were ineffective. The actions of CRF and urocortin 1 were blocked by the selective CRF1 receptor antagonist antalarmin, but were not inhibited by the selective CRF2 receptor antagonist astressin 2B, both administered in equimolar doses. Our results demonstrate that the release of GABA from the amygdala is mediated by CRF and urocortin 1 through the activation of CRF1 receptors.     

β‐catenin expression and claudin expression pattern as prognostic factors of prostatic cancer progression

OBJECTIVE

To investigate the patterns of expression of the junctional proteins β‐catenin and claudins in different prognostic groups of patients with prostatic cancer, to determine their value as prognostic markers.

PATIENTS AND METHODS

We evaluated the samples of 30 patients who had a radical prostatectomy for organ‐confined cancer (pT2N0M0), men with clinically advanced cancer, and a control group with benign prostatic hyperplasia. Using immunohistochemistry applied to tissue microarrays, each group was evaluated for claudin‐1, ‐2, ‐3, ‐4, ‐5, ‐7, ‐8 and ‐10, and β‐catenin expression.

RESULTS

There were differences among the three groups in the expression of claudin‐1 (P = 0.001), ‐2 (P = 0.014), ‐3 (P = 0.027), ‐4 (P = 0.001), ‐8 (P = 0.001) and β‐catenin (P = 0.002), regardless of Gleason score. By contrast, claudin‐5, ‐7 and ‐10 patterns were not significantly different among the groups. Furthermore, claudin‐1 (P = 0.014) and ‐4 (P = 0.004) could be used to distinguish between those patients who had metastases and those who did not.

CONCLUSIONS

The pattern of claudin expression could be a novel diagnostic marker in re‐classifying adenocarcinomas, and an additional sensitive predictive factor for a clinically poor prognosis. Our results suggest that patients with organ‐confined and advanced cancer are subsets with distinct claudin expression profiles, and that claudin‐4 is related to cellular differentiation in prostate cancer, which is not only the receptor molecule for the Clostridium perfringens enterotoxin, and thus a theoretical future therapeutic target for prostate cancer, but also a marker of progression.