Genomic characterization and chromosomal mapping of 5 river buffalo skeletal muscle differentiation master genes.

River buffalo (Bubalus bubalis, 2n = 50, BBU) is a species of economic relevance in a number of countries. This species shows a very peculiar biology and a great capacity for environmental adaptation. There has been an increasing economic interest as well as a growing demand for a more detailed knowledge of molecular features in this species. From this perspective we report a genomic, transcriptional and cytogenetic analysis of 5 master genes involved in skeletal muscle development. Of these 5 genes, MYOD1, MYF5, MYF6 and MYOG belong to the basic helix-loop helix protein family while MSTN belongs to the TNF-B protein family. In mammals, these genes are involved in the early stages of skeletal muscle differentiation, development and regeneration. These pivotal biological functions are finely regulated in a tissue- and temporal-specific manner. We used a comparative genomic approach to obtain the buffalo specific sequences of MYOD1 and MYF6. The nucleotide sequence similarity and the protein domain conservation of the newly obtained sequences are analysed with respect to bovine and other mammalian species showing sequence similarity. The presence of a polymorphism in MYOD1 coding sequence is described and its possible effect discussed. Using a quantitative PCR approach, we compared the level of the 5 transcripts in adult and fetal muscle. These genes were physically localised on river buffalo R-banded chromosomes by FISH using bovine genomic BAC-clones. Here, we present a genomic and cytogenetic analysis which could offer a background to better characterise the buffalo genes involved in muscle function and which may be responsible for buffalo-specific meat features.

Beneficial effects of melatonin on nicotine-induced vasculopathy.

Tobacco smoking is responsible for death of many people each year and increases the risk of developing numerous disorders, particularly cardiovascular disease and cancer. Among the components of cigarette smoke, nicotine is known to excert proatherosclerotic, prothrombotic and proangiogenic effects on vascular endothelial cells. The current study was designed to investigate the mechanisms by which nicotine induces endothelial dysfunction and further to examine whether melatonin protects against nicotine-induced vasculopathy. Four groups of male rats (controls, melatonin-treated, nicotine treated [100 microg/mL in drinking water], and nicotine plus melatonin [5 mg/kg/day] treated) were used in this study. After 28 days all the animals were killed by decapitation and the aorta was removed. We evaluated the hydroxyproline content, and the different expression of proteins involved in several types of stress (ERK1/2), in fibrosis (TGF-beta1, NF-kappaB) and in recruitment of circulating leukocytes onto the vessel wall, including intercellular adhesion molecule-1 (ICAM-1) and vascular cellular adhesion molecule-1 (VCAM-1). These metabolic pathways are important in the development of nicotine-induced atherosclerosis and hypertension. Our results show that nicotine induces marked structural and functional alterations in the aorta. Nicotine receptor binding results in activation and phosphorylation of ERK 1/2. This enzyme, in turn, activates both TGF-beta1 and NF-kappaB; they stimulate respectively the synthesis of type I collagen, responsible of fibrosis, and moreover ICAM-1, VCAM-1 and reactive oxygen species. Based on these findings, melatonin is able to minimize the negative effects of nicotine by blocking the activation of ERK and the other signalling pathways in which this enzyme is involved.

Red wine polyphenols prevent cyclosporine-induced nephrotoxicity at the level of the intrinsic apoptotic pathway.

Flavonoids, polyphenol derivatives of plant origin, possess a broad range of pharmacological properties. A number of studies have found both pro/anti-apoptotic effects for many of these compounds. For these reasons we investigated whether Provinols flavonoids obtained from red wine, have anti-apoptotic properties. The investigations have been carried out in rats treated with Cyclosporine A (CsA). In particular, four groups of rats have been treated for 21 days with either olive oil (control group), with CsA, with Provinols, or with CsA and Provinols simultaneously. Oxidative stress, systolic blood pressure, body weight, biochemical parameters and different markers of pro/anti-apoptotic pathway were measured. CsA produced an increase of systolic blood pressure, a decrease in body weight, serum creatinine levels, urinary total protein concentration and creatinine clearance. Moreover, CsA induced renal alterations and the translocation of Bax and cytochrome c from cytoplasm to mitochondria and vice versa. These changes activated the caspase cascade pathway, that leads to morphological and biochemical features of apoptosis. Provinols restored morphological and biochemical alterations and prevented nephrotoxicity. In conclusion, this study may augment our current understanding of the controversial pro-/anti-apoptotic properties of flavonoids and their molecular mechanisms.

Longins: a new evolutionary conserved VAMP family sharing a novel SNARE domain.

This article describes the discovery of a novel SNARE domain that might be involved in the regulation of membrane fusion. This domain is shared by a novel family of VAMPs called long VAMPs or longins. Members of this family are more conserved among eukaryotes than are classical VAMPs, possibly because of their underlying basic SNARE function.

Mutation analysis of the MECP2 gene in British and Italian Rett syndrome females.

Rett syndrome is an X-linked dominant neurological disorder, which appears to be the commonest genetic cause of profound combined intellectual and physical disability in Caucasian females. Recently, this syndrome has been associated with mutations of the MECP2 gene, a transcriptional repressor of still unknown target genes. Here we report a detailed mutational analysis of 62 patients from UK and Italian archives, representing the first comparative study among different populations and one of the largest number of cases so far analyzed. We review the literature on MECP2 mutations in Rett syndrome. This analysis has permitted us to produce a map of the recurrent mutations identified in this and previous studies. Bioinformatic analysis of the mutations, taking advantage of structural and evolutionary data, leads us to postulate the existence of a new functional domain in the MeCP2 protein, which is conserved among brain-specific regulatory factors.

Identification of novel RP2 mutations in a subset of X-linked retinitis pigmentosa families and prediction of new domains.

X-linked Retinitis Pigmentosa (XLRP) shows a huge genetic heterogeneity with almost five distinct loci on the X chromosome. So far, only two XLRP genes have been identified, RPGR (or RP3) and RP2, being mutated in approximately 70% and 10% of the XLRP patients. Clinically there is no clearly significative difference between RP3 and RP2 phenotypes. In the attempt to assess the degree of involvement of the RP2 gene, we performed a complete mutation analysis in a cohort of patients and we identified five novel mutations in five different XLRP families. These mutations include three missense mutations, a splice site mutation, and a single base insertion, which, because of frameshift, anticipates a stop codon. Four mutations fall in RP2 exon 2 and one in exon 3. Evidence that such mutations are different from the 21 RP2 mutations described thus far suggests that a high mutation rate occurs at the RP2 locus, and that most mutations arise independently, without a founder effect. Our mutation analysis confirms the percentage of RP2 mutations detected so far in populations of different ethnic origin. In addition to novel mutations, we report here that a deeper sequence analysis of the RP2 product predicts, in addition to cofactor C homology domain, further putative functional domains, and that some novel mutations identify RP2 amino acid residues which are evolutionary conserved, hence possibly crucial to the RP2 function.

Evidence suggesting protein tyrosine phosphorylation in plants depends on the developmental conditions.

Protein tyrosine phosphorylation plays a central role in a variety of signal transduction pathways regulating animal cell growth and differentiation, but its relevance and role in plants are controversial and still largely unknown. We report here that a large number of proteins from all plant subcellular fractions are recognized by recombinant, highly specific, anti-phosphotyrosine antibodies. Protein tyrosine phosphorylation patterns vary among different adult plant tissues or somatic embryo stages and somatic embryogenesis is blocked in vivo by a cell-permeable tyrosyl-phosphorylation inhibitor, demonstrating the involvement of protein tyrosine phosphorylation in control of specific steps in plant development.

Cytotoxicity, blood compatibility and antimicrobial activity of two cyanoacrylate glues for surgical use.

The biocompatibility of two cyanoacrylate surgical glues (Glubran and Glubran 2), supplied by General Enterprise Marketing, Viareggio, Lucca, Italy, was tested through cytotoxicity and blood compatibility tests and the evaluation of antimicrobial activity. Cytotoxicity and blood compatibility tests were performed on the polymerized glues. Using the neutral red uptake test, the extracts from Glubran and Glubran 2 after polymerization were non-toxic to L929 cells only when diluted 1: 10 with culture medium. Glubran and Glubran 2 induced a significant decrease of activated partial thromboplastin time (APTT), which is favourable with regard to the desired haemostasis. The APTT shortening determines a haemostatic effect and therefore contribute to the tissue adhesion induced by the glues. Otherwise, no significant variation of prothrombin activity, fibrinogen, platelet number, total and differential leukocyte count was induced by the glues, which, in addition, did not show haemolytic effect. There was no difference between Glubran and Glubran 2 regarding haemocompatibility. The antimicrobial ability of the unpolymerized glues was tested onto Bacillus subtilis var. niger for 3 weeks: neither Glubran nor Glubran 2 were found effective in this respect. In conclusion, we can assume that cytotoxicity was severe with the undiluted glues, but was acceptable when glues were diluted. On the contrary, blood compatibility was acceptable for the intended use of the glues. No difference was found between Glubran and Glubran 2 after polymerization.

Expression of the CD69 activation antigen on lymphocytes of patients with hip prosthesis.

The aim of the study was to evaluate the sensitization to metals in patients with Co-Cr hip prosthesis. Peripheral blood mononuclear cells (PBMC) were collected from 14 healthy donors and three groups of patients: 10 candidates for primary total joint replacements, 11 patients with well-fixed implant and 13 patients with aseptic loosening of the hip prosthesis. PBMCs were cultured with the metal ions employed for implant manufacturing and the expression of CD69 activation antigen on CD3/T lymphocytes was detected by flow cytometry. Chromium extract increased significantly the expression of CD3/CD69 phenotype in patients with loosening of hip prosthesis. The chromium-induced 'activation index' was higher in patients with loosening of hip prosthesis than in healthy donors and in pre-implant patients. The cobalt-stimulated PBMC of patients with either well-fixed or loosened prosthesis had an 'activation index' significantly higher than healthy donors. The activation index values were used to graduate the PBMC-response as 'normal' (> or = 0.9 and < 2), 'low' (< 0.9) and 'high' (> or = 2): an high-activation index was observed only in chromium-exposed PBMC of patients with prosthesis. Our data show that chromium released from orthopedic implants could be responsible for the lymphocyte sensitization and flow cytometry is an easy and reliable method for monitoring the hypersensitivity state in patients with metal prostheses. Activated lymphocytes in the peri-implant tissue are likely to elicit a localized immune response and contribute to maintain the inflammatory process evolving in the implant failure.

In vitro cytokine production by mononuclear cells exposed to bone cement extracts.

The authors evaluated the ability of bone cement to modify the profile of pro-inflammatory cytokines secreted by the immune cells. Peripheral blood mononuclear cells (PBMC) collected from healthy individuals were cultured with cement extracts and tested to assess the release of IL-1beta, TNFalpha, GM-CSF and IL-6 in both unstimulated and PHA-stimulated PBMC. The cytokine release of unstimulated PBMC was very poor, and in particular the IL-1beta was undetectable: the addition of cement extract increased both TNFalpha and GM-CSF release and decreased IL-6, sometimes significantly. The most recurrent observation in PHA-stimulated PBMCs exposed to bone cement extract was the increase in both IL-1beta and IL-6 release, while both the mean concentration and the index of release of TNFalpha and GM-CSF were changeable. In conclusion our results showed that leachable components of some bone cements can induce in vitro the release of pro-inflammatory cytokines which are known to be involved in the bone resorption associated with aseptic loosening of hip prostheses. These findings allowed us to identify materials endowed with the highest inflammatory power.

Cytokine release in mononuclear cells of patients with Co-Cr hip prosthesis.

The aim of this study was the evaluation of the release of bone-resorbing cytokines in peripheral blood mononuclear cells (PBMC) of patients with aseptic loosening of Co-Cr hip prostheses. TNF-alpha, IL-6, and GM-CSF were measured in both unstimulated and PHA-stimulated PBMC, and in PBMC cultured in the presence of chromium and cobalt extracts. Blood samples from healthy donors were used as controls. Serum samples of both patients and healthy donors were tested to determine the concentration of metal ions. The proportion of lymphocyte, monocyte and lympocyte subpopulation in cultured PBMC did not differ in patients and the control group. In unstimulated PBMC the release of TNF was significantly higher in patients than in the control group, while IL-6 was significantly decreased and no change was observed for GM-CSF. When the PBMC were challenged with chromium extract, all the 'index of cytokine release' resulted higher in patients than in the control group; cobalt extract increased both the TNF and GM-CSF index, but not the index of IL-6 release. Metal concentrations in serum from patients were significantly increased and correlated with the TNF release in PBMC stimulated with both metal extract. Our results suggest that a CoCr-implant releases a large amount of metal ions which could mediate the priming or the renewal of a cell-mediated hypersensitivity reaction. The prevalence of circulating lymphocytes responsible for the delayed hypersensitivity, namely Thl, would justify both the significant increase of TNF and the significant decrease of IL6 in unstimulated PBMC of patients, as well as the significant increase of the 'index of cytokine release' after the challenge with metal ions.

Results of long-term experimental studies on the carcinogenicity of methyl tert-butyl ether.

Methyl-tert-butyl ether (MTBE) was submitted to long-term carcinogenicity bioassays on Sprague-Dawley rats. The test compound was delivered in olive oil by stomach tube (gavage), at the doses of 1000, 250, and 0 mg/kg b.w. to groups of 60 males and 60 females, once daily, 4 times weekly, for 104 weeks. All animals were kept under control until spontaneous death. MTBE was found to cause in males an increased incidence of Leydig cell testicular tumors in the group treated with the higher dose, and in females a dose-related increase of leukemias, an increase of dysplastic proliferations of lymphoreticular tissues, and also an increase of uterine sarcomas at the lower tested dose. On the basis of the presented data, MTBE must be considered a potential carcinogen.

MECP2 gene mutation analysis in the British and Italian Rett Syndrome patients: hot spot map of the most recurrent mutations and bioinformatic analysis of a new MECP2 conserved region.

Rett syndrome (RTT) is an X-linked dominant neurological disorder, which appears to be the most common genetic cause of profound combined intellectual and physical disability in Caucasian females. This syndrome has been associated with mutations of the MECP2 gene, a transcriptional repressor of unknown target genes. We report a detailed mutational analysis of a large cohort of RTT patients from the UK and Italy. This study has permitted us to produce a hot spot map of the mutations identified. Bioinformatic analysis of the mutations, taking advantage of structural and evolutionary data, leads us to postulate the existence of a new functional domain in the MeCP2 protein, conserved among brain-specific regulatory factors.

The long styloid process syndrome or Eagle's syndrome.

Eleven cases of Eagle's syndrome or long styloid process syndrome are presented. It is a rare entity, which is not commonly suspected in clinical practice. Symptoms were dull and persistent pharyngeal pain, dysphagia, and facial pain. In addition to careful clinical examination, the use of plain radiographs, orthopantomograms or CT scan has allowed accurate preoperative diagnosis of this syndrome. Surgical treatment has positively resolved the symptoms in these cases. The International literature is discussed regarding anatomy, symptoms and treatment of the long styloid process syndrome.

Phenotypic relationships between longevity, type traits, and production in Chianina beef cattle.

Longevity is an increasingly important trait in beef cattle. Increased longevity decreases costs for the farmer and increases revenue. The objective of this research was to investigate the phenotypic relationship between type traits and longevity in Chianina beef cattle, and the relationship between production and longevity, to analyze the effect of voluntary culling. Data included records on reproductive, productive, and type traits provided by the National Association of Italian Beef Cattle Breeders from 6,395 Chianina cows. The average length of productive life was 1,829 d. The herd-year had a strong effect on the risk of culling. The effects of 22 type traits were analyzed. All the muscularity traits analyzed were highly significant (P < 0.01) and as a group had the largest effect on longevity, followed by dimension, refinement, and leg traits. Cows that calved before 35 mo of age had a lower probability of being culled than cows calving after 35 mo of age. Variation in herd size had a strong effect on risk ratio, with lower risk for intermediate classes. Cows with approximately one calf per year remained in the herd longer than did cows with fewer calves. Straight-legged animals had a 59% greater probability of being replaced than cows with a moderate angle to the hock, whereas sickle-legged animals had only a 3% higher probability of being culled than average cows. Udder conformation had no effect on longevity. In summary, results of this study indicate that herd-year effects and muscularity traits were the most important factors for longevity for Chianina cows among the factors studied.

[Isolated splenic metastasis of ovarian endometrioid carcinoma. A clinical case]. / Metastasi splenica isolata di un carcinoma endometrioide dell'ovaio. Caso clinico.

The authors describe a case of solitary spleen metastasis of an endometrioid ovarian cancer exceptionally reported in the literature. The follow-up of the patient is 12 months without recurrence.

Effectiveness of magnesium pidolate in the prophylactic treatment of primary dysmenorrhea.

To evaluate Magnesium (Mg) effectiveness in the treatment of primary dysmenorrhea, 30 volunteer dysmenorrheic women of mean age 22.6 years were selected from the out-patients of the Dept. of Obstetrics and Gynaecology of the University of Parma during the period January-December 1989. Patients affected by secondary dysmenorrhea were excluded from the trial. The women considered were asked to self-evaluate their menstrual pain for 6 subsequent cycles using the VAS (Visual Analogue Scale). In the first cycle, as control, no drug was administered; in the following ones, every woman was given 4.5 mg oral Mg Pidolate in 3 administrations daily, from the 7th day preceding the onset of menses till the 3rd day of menstruation. Data were statistically analyzed. In Mg-treated cycles compared with the control one, first day dysmenorrhea progressively decreased, with a significant drop (p < 0.05) from the 1st to the 6th cycle. A similar trend, but not statistically significant, was seen for the 2nd and 3rd day of cycle. No side effect was remarked. These data suggest Mg administration to be a reliable therapy of primary dysmenorrhea.

[Laparoscopy-assisted hysterectomy and laparoscopic preparation. Apropos of a series of 177 cases]. / L'hystérectomie coelio-assistée et coelio-préparée. A propos d'une série de 177 cas.

Our objective was to determine the limits of laparoscopic-assisted vaginal hysterectomy (LAVH) and the value of a preoperative scoring system to determine the operative approach to hysterectomy. Between January 1991 and December 1996, 152 out of 177 patients had LAVH and 25 had laparoconversion. The mean operating time was 163 min. The overall postoperative complication rate was 8.4%. The hospital stay was 4.8 days for LAVH versus 6.2 days for laparoconversion (p < 0.01). For each patient, a preoperative scoring system was established according to uterine size, previous laparotomy, uterine mobility, pelvic adhesions and endometriosis stage. The laparoconversion rate increased according to the score, as it was 7.8% for a score < or = 7 and 80% for a score > 7. LAVH offers a technique to convert some abdominal hysterectomies into vaginal hysterectomies. The use of the preoperative scoring system may help to determine patients who may benefit from the laparoscopic route and those with a high risk of laparoconversion.

[Distal tubal surgery: a critical review of 104 laparoscopic distal tuboplasties]. / Chirurgie de la trompe distale. Analyse critique d'une série de 104 plasties tubaires distales coeliochirurgicales.

MATERIALS AND METHODS: Between January 1988 and June 1993, laparoscopic distal tuboplasty was carried out on 104 patients. The mean age of the patients was 32 years: the duration of infertility was 3.2 years; infertility was primary for 44 patients (42.3%) and secondary for 60 patients (57.7%). The patients were classified according to the French cooperative tubal scoring system: stage 1: 21 patients, stage II: 49 patients, stage III: 22 patients, stage IV: 12 patients; 37 patients underwent a fimbrioplasty (35%) whereas 67 patients underwent a neosalpingostomy (65%). No complications were observed either during or after the operation. All patients were discharged two or three days after the operative procedure. Antibiotic and antiinflammatory treatments were systematically administered post-operatively. RESULTS: Thirty-four patients obtained intrauterine pregnancies (32.5%) and 5 patients obtained ectopic pregnancies (4.8%); 76% of the intrauterine pregnancies were obtained within the 1st postoperative year. The pregnancy outcome was directly correlated to the tubal scoring system. Intrauterine pregnancy rate decreased with the severity of the tubal damage: stage 1: 12/21 (57%), stage II: 19/49 (38.7%), stage III: 3/22 (13.6%), stage IV: 0/12. CONCLUSION: Laparoscopic surgery represents the first-choice treatment of distal tubal occlusion in stage I and II. In stage IV, IVF should be suggested immediately. In stage III, the choice of treatment is more difficult: the main prognostic factor might be the tubal mucosal appearance.

[Laparoscopy-assisted and -prepared hysterectomy. A series of 80 cases]. / Hystérectomie coelio-assistée et coelio-préparée. A propos d'une série de 80 cas.

OBJECTIVE: Our objective was to determine the interest of laparoscopic assisted vaginal hysterectomy. STUDY DESIGN: Between January 1991 to december 1994, 80 patients had laparoscopically assisted vaginal hysterectomy. We reviewed with particular emphasis characteristic indications, complications. RESULTS: Eighty were performed as laparoscopically assisted vaginal hysterectomy. 14 patients (17.5%) had laparotomy conversion; because of size of uterus in 3 cases, suspected ovarian tumor in 3 cases. Pelvic adherences in 4 cases, urinary tract injuries in 1 case, hypercapnia in 1 case, hemorrhage in 2 cases. 9 patients experienced febrile morbidity and 1 urinary infection. 1 patient received 2 units of packed red blood cells. The hospital stay was 5 days for laparoscopically assisted vaginal hysterectomy versus 5.9 for laparotomic hysterectomy. CONCLUSION: Laparoscopically assisted vaginal hysterectomy offers a technique to convert certain abdominal hysterectomies into vaginal hysterectomies with a 17.5% laparoconversion rate.