Down-regulation of neuronal form of Nitric oxide synthase in the Nurse cell of Trichinella spiralis.

The free radical nitric oxide (NO) and Ca2+ are critical regulators of skeletal muscle exercise performance and fatigue. The major source of NO in skeletal muscle cells is the neuronal form of the enzyme Nitric oxide synthase (nNOS). One of the most peculiar characteristics of the Nurse cell of Trichinella spiralis (T. spiralis) is the complete loss of the contractile capabilities of its derivative striated muscle fiber. The aim of the present study was to clarify the expression of nNOS protein and mRNA in striated muscles during the muscle phase of T. spiralis infection in mice. Muscle tissue samples were collected from mice at days 0, 14, 24, and 35 post infection (d.p.i.). The expression of nNOS was investigated by immunohistochemistry, and the expression levels of mRNA of mouse Nitric oxide synthase 1 (Nos1) by real-time PCR. The presence of nNOS protein was still well observable in the disintegrated sarcoplasm at the early stage of infection. The cytoplasm of the developing and mature Nurse cell showed the absence of this protein. At least at the beginning of the Nurse cell development, Trichinella uses the same repairing process of skeletal muscle cell, induced after any trauma and this corroborates very well our results concerning the nNOS expression on day 14 p.i. At a later stage, however, we could suggest that the down-regulation of nNOS in the Nurse cell of T. spiralis either serves a protective function or is an outcome of the genetic identity of the Nurse cell.

The Synthesis of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine Kinase (GNE), α-dystroglycan, and ß-galactoside α-2,3-sialyltransferase 6 (ST3Gal6) By Skeletal Muscle Cell As a Response To Infection with Trichinella Spiralis.

The Nurse cell of the parasitic nematode Trichinella spiralis is a unique structure established after genetic, morphological and functional modification of a small portion of invaded skeletal muscle fiber. Even if the newly developed cytoplasm of the Nurse cell is no longer contractile, this structure remains well integrated within the surrounding healthy tissue. Our previous reports suggested that this process is accompanied by an increased local biosynthesis of sialylated glycoproteins. In this work we examined the expressions of three proteins, functionally associated with the process of sialylation. The enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is a key initiator of the sialic acid biosynthetic pathway. The α-dystroglycan was the only identified sialylated glycoprotein in skeletal muscles by now, bearing sialyl-α-2,3-Gal-ß-1,4-Gl-cNAc-ß-1,2-Man-α-1-O-Ser/Thr glycan. The third protein of interest for this study was the enzyme ß-galactoside α-2,3-sialyltransferase 6 (ST3Gal6), which transfers sialic acid preferably onto Gal-ß-1,4-GlcNAc as an acceptor, and thus it was considered as a suitable candidate for the sialylation of the α-dystroglycan. The expressions of the three proteins were analyzed by real time-PCR and immunohistochemistry on modified methacarn fixed paraffin tissue sections of mouse skeletal muscle samples collected at days 0, 14 and 35 post infection. According to our findings, the up-regulation of GNE was a characteristic of the early and the late stage of the Nurse cell development. Additional features of this process were the elevated expressions of α-dystroglycan and the enzyme ST3Gal6. We provided strong evidence that an increased local synthesis of sialic acids is a trait of the Nurse cell of T. spiralis, and at least in part due to an overexpression of α-dystroglycan. In addition, circumstantially we suggest that the enzyme ST3Gal6 is engaged in the process of sialylation of the major oligosaccharide component of α-dystroglycan.

Identification of a novel HLA-B null allele, HLA-B*41:45N, by HLA typing of two related Caucasoid individuals.

The new HLA-B*41:45N allele differs from B*41:02 by insertion of 21 nucleotides in exon 2.

Identification of the novel HLA-A*11:192 allele by HLA typing of a Caucasian individual.

We report the new HLA-A*11:192 allele differing from A*11:01 by one nucleotide in exon 2.

Increased number of multi-oocyte follicles (MOFs) in juvenile p27Kip1 mutant mice: potential role of granulosa cells.

STUDY QUESTION: Why are female mice that lack a functional p27 protein infertile? SUMMARY ANSWER: The absence of a functional p27 leads to a dramatic increase in the number of multi-oocyte follicles (MOFs) in juvenile female mice; p27 would promote the individualization of follicles favoring the development of fertile eggs. WHAT IS KNOWN ALREADY: p27-/- female mice are infertile. p27 suppresses excessive follicular endowment and activation and promotes follicular atresia in mice. MATERIALS AND METHODS: Ovaries from wild type (WT) and p27Kip1 mutant mice aged 2, 4 and 12 weeks were subjected to immunohistochemistry/immunofluorescence. The slides with whole organs serially sectioned were scanned and examined by image analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Compared with WT, p27Kip1 mutant pre-pubertal mice had a greater number of oocytes, a greater number of growing follicles and a greater number of MOFs. These differences were statistically significant (P < 0.05), particularly in the case of MOFs (P > 0.001). The unusually large number of MOFs in juvenile p27-deficient mice is a novel observation. In WT mice p27 protein remains present in the oocyte nucleus but gradually decreases in the ooplasm during follicular growth, while granulosa cells show dynamic, follicle stage-related changes. LIMITATIONS, REASONS FOR CAUTION: These results have been obtained in mice and they cannot be directly extrapolated to humans. WIDER IMPLICATIONS OF THE FINDINGS: The dramatic increase in the numbers of MOFs in juvenile p27 mutants has not been previously reported. The number of MOFs declines sharply as the mice become sexually mature, pointing to their negative selection. These findings open a new approach to the study of sterility. STUDY FUNDING/COMPETING INTERESTS: This study has been funded by the Basque Government, Dept. of Health grant 2007111063 and Dept. of Industry (Saiotek) grant S-PC11UN008. Jairo Perez-Sanz was the recipient of a grant from Fundación Jesús de Gangoiti Barrera. The authors have no conflicts of interest to declare.

Detection of a novel HLA-DQB1 allele, designated DQB1*06:49.

We report a novel allele HLA-DQB1*06:49 with a G→T transversion, most closely resembling HLA-DQB1*06:02:01.

Detection of a novel HLA-A allele, designated A*02:334.

We report a novel allele HLA-A*02:334 with the transition G→A at nucleotide position 282.

Survival role of superoxide dismutase 1 on human granulosa luteinized cells in vitro.

OBJECTIVE: Deleterious effects of free radicals do not only result from the amount of free radicals produced but also are related to the efficiency and to the activities of enzymatic antioxidant systems. We investigated the effect of exogenous superoxide dismutase (SOD1) or Cu-chelating agent diethyldithiocarbamate (DDC) on the apoptosis (caspase-3 activity) of human granulosa luteinized cells (hGLC) in vitro. METHODS: The effects of SOD1 and DDC were studied using in vitro culture system, caspase-3 and the total SOD activity in hGLCs were measured using AcDEVD-PNA substrate and Beaushap and Fridovich methods, respectively, after 48 h of the culture period. RESULTS: The activity of SOD1 was the lowest in GLCs treated with 100 µM DDC as compared to control cells and to the cells supplemented with Cu, Zn-SOD or DDC (10 µM). The effect of DDC was associated with elevated caspase-3 activity as compared to control cells. CONCLUSIONS: It was demonstrated for the first time that the supplementation of cultured hGLCs with Cu2+, Zn2+-SOD (200 U/ml) maintains the viability of hGLCs via caspase-3 suppression.

A new HLA-B*52 allele, B*52:23, detected in a patient before bone marrow transplantation.

The new allele HLA-B*52:23 differs from B*52:01:01 at position 208 in exon 2.

A new HLA-C*07 variant allele, C*07:108, identified by sequence-based typing.

The new HLA-C*07:108 differs from C*07:01:01 by two transversions in exon 3.

A novel HLA-DQB1*03:02 variant designated DQB1*03:02:05.

The new DQB1*03:02:05 differs from DQB1*03:02:01 by two nucleotide exchanges in exon 2.

Identification of a new HLA-DRB1* 14 allele, DRB1*14:99, by HLA typing of an Asian individual.

We report the new HLA-DRB1*14:99 allele differing from DRB1*14:01:01 by one nucleotide in exon 2.

Identification of a novel HLA-C*02 allele, HLA-C*02:02:09.

We report HLA-C*02:02:09 as a novel allele with a transition C->T at position 354.

Dysregulation of upstream binding factor-1 acetylation at K352 is linked to impaired ribosomal DNA transcription in Huntington's disease.

Huntington's disease (HD) is an autosomal-dominant neurological disorder caused by expanded CAG repeats in the Huntingtin (Htt) gene, but it is not known how this mutation causes neurodegeneration. Herein, we found that dysfunction of upstream binding factor-1 (UBF-1) is linked to reduced ribosomal DNA (rDNA) transcription in HD. We identified that UBF1 acetylation at Lys (K) 352 by CREB binding protein (CBP) is crucial for the transcriptional activity of rDNA. UBF1 mutation (K352A, K352Q, and K352R) decreased rDNA transcriptional activity. Moreover, both CBP-dHAT mutant and knockdown of CBP by siRNA reduced acetylation of UBF1 and resulted in the decreased transcription of rDNA into rRNA. ChIP analysis showed a significant reduction of UBF1 occupancy in the promoter of rDNA in STHdh(Q111) cell line model of HD. These results demonstrate that abnormal activity of UBF1 and its acetylation by CBP are linked to impaired rDNA transcription in HD. This novel mechanism suggests that modulation of UBF-mediated rDNA synthesis by CBP may be a therapeutic target for improving neuronal rDNA transcription in HD.

[Screening for gestational diabetes in Bulgaria--preliminary results].

Gestational diabetes mellitus (GDM) is defined as carbohydrate intolerance with onset or first recognition during pregnancy. There is no data regarding the prevalence of GDM in Bulgaria. The aim of this study is to present the preliminary results from a pilot study for GDM in Bulgaria, including the prevalence and the epidemiological differences between women with or without this disorder. A 2 h oral glucose tolerance test (OGTT) with 75g glucose was performed on 330 pregnant women between 24 and 28 weeks of gestation, using ADA criteria to define pregnancies complicated by GDM. Blood glucose levels, insulin levels, HbA1c, hsCRP and lipid parameters were evaluated. GDM was found in 11.3% of the patients. Women with GDM were older than the ones without the disorder (p < 0.006), had higher fasting glucose levels (p < 0.0001), higher fasting insulin levels (p < 0.0001), higher HOMA index (p < 0.0001) and higher HbA1c (p < 0.001). There was a significant difference in body mass index (BMI) before pregnancy (p < 0.003) and at the time of screening (p < 0.001) between GDM and control groups (p < 0.001). GDM women more frequently had thyroid disease, developed early preeclampsia, had family history of type 2 diabetes mellitus and history of PCOS. In conclusion the prevalence of GDM in high risk Bulgarian population is relatively high--11.3%. This confirms the necessity of establishing of a national screening program. If financial recourses are limited selective screening in high risk patients should be considered.

[Our and foreign experience in surgical treatment of CIN III-cold knife conisation versus LLETZ].

AIM: Our aim was to assess the treatment results of patients with CIN III- surgically treated with cold knife cone biopsy compared with the results of treatment with LLETZ and laser conisation. MATERIALS AND METHODS: 600 patients were assessed and researched for 10 years period. Some of them were from the Specialized Hospital for Active Treatment in Oncology-Sofia, some from RHW and some from the Medical University of Varna-Cathedra of Obstetrics and Gynaecology. 350 patients were operated by the classical method- cold knife conisation, 200 by LLETZ and 50 by laser cone biopsy. RESULTS: We didn't find any difference in patients age, their cytological results and the results of their treatment. The LLETZ showed good results according to resected margins. The same good results were observed with laser conisation. Invasive disease and recurrence were observed in 150 patients--50 of them having adenocarcinoma in situ. CONCLUSIONS: Our research showed that there is no difference in the results of both groups- treated by the classical way--cold knife conisation or by LLETZ and laser conisation.

[Frequency of miscarriages among pregnant women with autoimmune thyroid disorders].

AIM OF THE STUDY: To analyse the influence of antithyroid antibodies (ATA) on the frequency of spontaneous abortions (SA) by pregnant women with a normal thyroid gland function. DESIGN OF THE STUDY: Prospective clinical study on 42 selected pregnant women with a normal thyroid gland function divided into two groups: I-st group--ATA positive pregnant [n = 28] and II-end group ATA negative pregnant [n = 14]. RESULTS: Increased ATA have been found by 30 (71.4%) out of the studied 42 pregnant women. There has been no significant difference found among the values of FT3, FT4 and TSH for women with positive and negative ATA. SA have been observed at 63.3% (19/30 women) from the ATA positive ones and at 25% (4/12 women) from the ATA negative ones (P = 0.001). By the ATA positive women with SA the average values of antithyroglobulin autoantibody (Tg-Ab) (Tg-Ab positive.-189.6 +/- 49.8 IU/ml vs. Tg-Ab -negative 118.2 +/- 58.3 IU/ml, P = 0.02) and antithyroid peroxidase autoantibody (TPO-Ab) (TPO-Ab positive-176.9 +/- 57.4 IU/ml vs. TPO - negative 118.2 +/- 81.3 IU/ml, P = 0.004) are both found to be significantly higher. CONCLUSION: There is a correlation found between ATA and the increased risk of SA, where the increased concentration of ATA is combined with an increased frequency of SA.

[Risk of thrombophilia in carriers of thrombophilic genetic factors in unsuccessful assisted reproduction].

The aim of this study was to evaluate an association of carrier status of common inherited thrombophilic genetic mutations and implantation failure after assisted reproduction (ART): IVF and ICSI. Sixty seven women with failure of embryo implantation and ninety six controls--women without obstetric complication were investigated for carriage of factor V Leiden (FVL), G20210A prothrombin gene mutation, genetic variant C677T in methylentetrahydrofolate reductase gene (MTHFR) and polymorphism A2 in platelet glycoprotein IIb/IIIa (GIPr IIb/IIIa). A significantly higher prevalence of polymorphism A2 in GIPr IIb/IIIa was found in women with implantation failure in ART compared to controls (respectively 26.1% and 12.5%; OR: 2.571, 95% CI: 1.066-6.258, p = 0.033). A higher but not significant prevalence of G20210A prothrombin gene mutation carriage was found inpatients compared to controls (respectively 5.8% and 3.13%, OR: 1.968, 95% CI 0.356-11.539). The carriage of FVL was a little but not significantly higher in controls. The carriage of genetic variant C677T in MTHFR was the same in both groups. These data suggest that polymorphism A2 in GIPr IIb/IIIa and G20210A prothrombin gene mutation could be play a role in the etiology of IVF failures and the carriers of GIPr IIb/IIIa A1/A2 and G20210A prothrombin gene mutation are at higher risk of implantation failure and not successful ART outcome. The carriage of these two genetic defects should be investigated in women undergoing IVF and the antithrombotic or anticoagulant prophylaxis should be indicated for carriers of these two factors.

[Is it possible to reduce the incidence of preeclampsia in pregnant women with type I diabetes mellitus].

AIM OF STUDY: To follow the meaning of diabetic metabolic control in the early pregnancy springing up the preeclampsia (PR) in pregnant women with type I diabetes mellitus (T1DM). DESIGN OF STUDY: A prospective study has been done among 105 pregnant women with T1DM. The diabetic pregnant have been divided into two groups: 1st group--43 women with preplanning pregnancy and 2nd group--62 women with non-planning pregnancy. RESULTS: There was no difference in the frequency of PR among women with planning and non-planning pregnancy (25.6% vs. 30.6%; P=0.36). The mild and moderate cases of PR among the women with planning pregnancy are prevailing, while moderate and severe cases of PR were observed in non-planning pregnancy. In pregnant women with planning pregnancy there is no difference between the initial level HbA1c of women without PR and PR (7%+/-0.7 vs. 7.1%+/-0.9%; P=0.13). Pregnant women with non-planning pregnancy have significantly higher initial level of HbA1c (9.7%+/-1.7 vs. 8.3%+/-1.3%; P=0.01). Pregnant women with PR have significantly higher body weight, creatinin, diurnal urine albumin excretion and diurnal insulin dose. CONCLUSION: The chronicle hyperglycemia in the early pregnancy in combination with the established factors are increasing the risk of preeclampsia in women with non-planning pregnancy and poor glycaemic control.