Application of response surface methodology (RSM) for the optimization of supercritical CO2 extraction of oil from patè olive cake: Yield, content of bioactive molecules and biological effects in vivo.

The two-phase technology for olive oil extraction generates large amounts of patè olive cake (POC), a by-product that is rich in bioactive health-promoting compounds. Here, response surface methodology (RSM) was used to maximize supercritical-CO2 oil extraction from POC, while minimizing operative temperature, pressure and time. Under the optimal parameters (40.2 °C, 43.8 MPa and time 30 min), the oil yield was 14.5 g·100 g-1 dw (~65% of the total oil content of the freeze-dried POC matrix), as predicted by RSM. Compared with freeze-dried POC, the oil contained more phytosterols (13-fold), tocopherols (6-fold) and squalene (8-fold) and was a good source of pentacyclic triterpenes. When the biological effects of POC oil intake (20-40 µL·die-1) were evaluated in the livers of BALB/c mice, no significant influence on redox homeostasis was observed. Notably, a decline in liver triglycerides alongside increased activities of NAD(P)H:Quinone Oxidoreductase 1, Carnitine Palmitoyl-CoA Transferase and mitochondrial respiratory complexes suggested a potential beneficial effect on liver fatty acid oxidation.

Dietary fatty acids influence sperm quality and function.

Recently, obesity has been linked to male infertility. In animal models the administration of a high-fat diet caused a reduction in sperm quality, by impairing gamete energy metabolism. The aim of this study was to investigate a possible effect of dietary fatty acids supplementation in the modulation of sperm energy metabolism and, in turn, in the improvement of sperm quality in rats fed a high-fat diet. Sexually mature male Sprague-Dawley rats were divided into four groups and fed for 4 weeks a standard diet (control group), a high-fat diet (enriched in 35% of fat and 15% sucrose), a high-fat diet supplemented with 2.5% olive oil (a source of monounsaturated fatty acids) or a high-fat diet supplemented with 2.5% krill oil (a source of n-3 polyunsaturated fatty acids). Liver and adipose tissue weight, plasma glucose, insulin and lipid concentrations were determined. Activities of enzymes involved in sperm energetic metabolism were evaluated by spectrophotometric assays. Sperm mitochondrial respiratory efficiency was also assayed. The obtained results suggest that olive oil partially counteracts the negative effects of a high-fat diet on sperm quality, by increasing gamete motility, by reducing oxidative stress and slightly improving mitochondrial respiration efficiency. On the other hand, krill oil determines an increase in sperm concentration and motility, an increase in the activities of lactate dehydrogenase, Krebs cycle enzymes and respiratory chain complexes; a parallel increase in the cellular levels of ATP and a reduction in oxidative damage were also observed. These results suggest that dietary fatty acids are able to positively influence sperm quality and function.

Characterization of endocrine features and genotype-phenotypes correlations in blepharophimosis-ptosis-epicanthus inversus syndrome type 1.

OBJECTIVE: Blepharophimosis syndrome (BPES) is an autosomal dominant genetic condition resulting from heterozygous mutations in the FOXL2 gene and clinically characterized by an eyelid malformation associated (type I) or not (type II) with premature ovarian failure. The distinction between the two forms is critical for female patients, as it may allow to predict fertility and to plan an appropriate therapy. Identifying an underlying causative mutation is not always predictive of the clinical type of BPES since genotype-phenotype correlations are not yet fully delineated. Here, we describe the clinical and hormonal phenotypes of three female patients with BPES type 1 from two novel families, correlate their phenotypes with identified mutations, and investigate the effects of hormone replacement therapy (HRT). METHODS: Clinical, biochemical, and genetic evaluation were undertaken in all the patients and genotype-phenotype correlation was analyzed. The effects of substitutive hormonal therapy on secondary sexual characteristics development and induction of menarche were evaluated. RESULTS: All patients presented with primary amenorrhea or other signs of ovarian dysfunction. Two distinct mutations, a missense p.H104R change and an in-frame p.A222_A231dup10 duplication in the FOXL2 gene were identified. Observed phenotypes were not in accordance with the prediction based on the current genotype-phenotype correlations. HRT significantly improved secondary sexual characteristics development, as well as the induction of menarche. CONCLUSIONS: This study highlights the importance of early recognition of BPES and emphasizes the need of personalized therapy and follow-up in female patients carrying distinct FOXL2 mutations.

Hemorrhagic corpus luteum cysts: an unusual problem for pediatric surgeons.

STUDY OBJECTIVE: Hemorrhagic corpus luteum cysts (HCLC) constitute a common disorder in pediatric subjects undergoing surgical intervention. HCLCs especially develop in the early period after menarche, and they are commonly associated with dysfunctional ovulation. DESIGN: Retrospective analysis of surgery outcome of HCLC patients. SETTING: Pediatric Surgery Unit, S. Chiara University Hospital. PARTICIPANT: 13 girls with HCLC diagnosis. INTERVENTIONS: Surgical treatment of HCLCs. MAIN OUTCOME MEASURES: We reviewed the clinical presentation and outcome of 13 post-menarcheal girls surgically treated for HCLCs in the Pediatric Surgical Unit from 2002 to 2006. RESULTS: Primary presentation was persistent abdominal pain in 84.6% and acute abdominal pain in 15.4% of patients, respectively. Ultrasound examination showed complex ovarian masses in 77.23% cases and simple ovarian masses in 33.7% cases, respectively. Although laparoscopic excision of HCLC was performed in more than 45% cases, laparotomic approach was commonly required. After conservative surgery, ovarian size and viability were normal, as assessed by 6-month ultrasound scan. No recurrences of disease and regular menses were reported at 2 years follow-up. CONCLUSIONS: In pediatric subjects with HCLC that required surgical intervention, no complications or disorder recurrence were reported. In order to preserve ovarian function, conservative surgery has to be performed whenever feasible.

Functional ovarian lesions in children and adolescents: when to remove them.

STUDY OBJECTIVE: Functional ovarian lesions represent 45% of all pediatric adnexal abnormalities. Their surgical management, even if frequent, is not clear, especially in pediatric age. MATERIALS AND METHODS: We retrospectively reviewed 22 pediatric patients surgically treated for functional ovarian lesions from 2000 to 2006. The following characteristics were analysed: age, size of the lesion, ultrasound (US) aspect and clinical presentation. RESULTS: The average age was 16.1 years of age (range: 6 months-18 years). Of the 22 functional lesions, 12 (55%) were follicular cysts and 10 (45%) corpus luteum ones. The average size was 6.7 cm (range: 5.1-33 cm). US scan showed simple lesions in 10 cases (45%) and complex ones in 12 cases (55%). In 16 girls (72.8%) the presenting symptom was abdominal pain while 2 patients (9%) presented abdominal distention. In the other 4 patients (16.2%) the lesion was found accidentally during US examination. CONCLUSION: In pediatric subjects, functional ovarian cysts rarely required surgical intervention, though no complications or disorder recurrence were reported. To preserve ovarian function, conservative surgery has to be performed whenever feasible.

Two novel mutations and a new STK11/LKB1 gene isoform in Peutz-Jeghers patients.

Peutz-Jeghers syndrome (PJS) is a rare autosomal dominantly inherited disorder with variable expression and incomplete penetrance characterized by mucocutaneous pigmentation, predisposition to hamartomatous intestinal polyposis, and various other neoplasms. It occurs in approximately 1 in 8,300 to 29,000 live births. In nearly 50% of patients PJS is caused by germ line mutations in the STK11/LKB1 serine/threonine kinase gene, the only kinase gene currently known to act as a tumor suppressor. We have performed a mutation search in the STK11/LKB1 gene in 8 sporadic cases and 3 PJS families using a combination of different screening techniques. We have identified four mutations, two of which I177N and the IVS2+1A->G, were previously unreported. We have also evaluated the presence of cDNA alterations by means of RT-PCR analysis and direct cDNA sequencing and have found two aberrant transcripts in a single PJS case despite the lack of any apparent genomic alteration. Finally, we report the presence of a novel STK11/LKB1 cDNA isoform observed in all the normal subjects studied as well as in the majority of the PJS patients.

Micrometastasis in colorectal cancer.

Colorectal cancer is one of the most common malignancies in the word and can be usually cured if diagnosed at an early stage. In the European area the estimated number of new cases of cancer in 1995 were approximately 334.000 and 189.000 died of cancer in that year. Despite recent progress in early detection and surgical therapy, the mortality remained unchanged over the past decades. The major reason for this disturbing discrepancy is that occult dissemination of viable cancer cells can occur at any stage of tumorigenesis. Occult dissemination of the tumor cells in patients with operable cancer may be considered a determinant of subsequent metastasis formation. Several groups have therefore designed immunocytochemical and molecular assays to identify such minimal amounts of residual tumor cells that have successfully invaded secondary organs. The question whether circulating tumor cells represent metastatic dissemination or are merely cancer cells without metastatic potential that have detached from the primary tumor, has been debated for over half a century.

Control of mouse cumulus cell-oocyte complex integrity before and after ovulation: plasminogen activator synthesis and matrix degradation.

During the preovulatory period, cumulus cells (CCs) form a hyaluronan-protein extracellular matrix (cumulus expansion) that positively influences oocyte fertilization. Degradation of this matrix and CC-oocyte complex (COC) dissociation occurs within a few hours of ovulation and parallels the aging of oocytes. Modulation of CC proteolytic activity by gonadotropins and oocyte soluble factors has been hypothesized to determine such cumulus matrix changes. In the present study, we investigated plasminogen activator (PA) synthesis by COCs during the expansion and disassembly processes. Our results show that the secretion of tissue type PA and urokinase type PA (uPA) by oocytes and CCs, respectively, does not change significantly during expansion but dramatically increases thereafter. Compact COCs were isolated from immature mice, primed 48 h earlier with 5 IU PMSGs, and were induced to expand in vitro with 100 ng/ml FSH in the presence of 1% FCS. Full expansion was achieved at 16 h, when hyaluronan synthesis ceased. Release of hyaluronan and CCs from the COC matrix began between 18 and 20 h of culture, which indicates that matrix degradation started at this time. PA activities in culture media were determined by SDS-PAGE, followed by a zymography at various time intervals between 4 and 32 h of culture. Secreted tissue type PA and uPA activity abruptly increased between 16 and 20 h after FSH stimulation. Slot blot hybridization of CC messenger RNA showed that uPA messenger RNA levels correlated with the increase in uPA activity. Similar temporal patterns of PA synthesis and matrix degradation were found in COCs induced to expand in vivo by injection of 5 IU human CG into PMSG-primed mice. Cultures of CCs, both in the presence and absence of oocytes, revealed that uPA synthesis is repressed in FSH-stimulated CCs by an oocyte-soluble factor for the first 16 h of culture, whereas CC responsiveness to this factor is lost thereafter. In conclusion, the data show that a sophisticated interplay between oocyte and CCs causes the two cell types to simultaneously secrete PA activity after ovulation. The fact that matrix degradation parallels PA production strongly supports the hypothesis that these enzymes may destabilize the expanded COC matrix.

Hyaluronan and proteoglycans in ovarian follicles.

Proteoglycans are macromolecules formed by a protein backbone to which one or more glycosaminoglycan side chains are co-valently attached. They can be secreted by the cells, retained at the cell surface, or stored in intracellular vacuoles. Hyaluronan is an extremely long glycosaminoglycan which, at variance with other glycosaminoglycans, is released into the extracellular matrix as a free polysaccharide not co-valently linked to a core protein. Both proteoglycans and hyaluronan influence many aspects of cell behaviour by multiple interactions with other molecules. They are involved in matrix formation, cell-cell and cell-matrix adhesion, cell proliferation and migration, and show co-receptor activity for growth factors. Both proteoglycan and hyaluranon synthesis change significantly during ovarian follicle development and atresia. This review describes the structure of these molecules and their possible function in ovarian physiology.

Cytochemical localization of adenylate cyclase in the limb buds of Bufo bufo.

The importance of cyclic nucleotides in the regulation of the processes of differentiation and embryonic development is known. The possible role that cyclic adenosine monophosphate (cAMP) plays during the development of the posterior limb of Bufo bufo is studied by the cytochemical localization of adenylate cyclase (AC), an enzyme that catalyzes the synthesis of the cyclic nucleotide. The method is based on the reaction between the enzyme AC and its specific substrate AMP-PNP (5'-adenylylimidodiphosphate) in the presence of lead. The lead precipitates that form as secondary reaction products are evidence of enzymatic activity. Reaction products are present only at the epithelial level in the limb bud; initially, such products are visible only at the base of the bud, particularly on the epithelial fascia located at the boundary with the body. During successive elongation and toe formation, AC activity is only present on the cells of the proximal portion of each new segment. Enzymatic activity is never present in correspondence to the ectodermal apical crest. cAMP is probably not involved in the processes of cellular proliferation but, rather, in the processes of inducing differentiation of the internal mesenchymal cells.

Duodenal obstruction following elective abdominal aortic aneurysm repair.

A case where duodenal obstruction complicated elective repair of abdominal aortic aneurysm is reported. A review of the literature shows that the incidence of this rare complication has probably been underestimated. Patients should be treated expectantly, regardless of the pathogenesis of the obstruction.

Benign fibrous mesothelioma: report of a case.

A case of fibrous benign mesothelioma is reported, together with some considerations on the diagnosis and the treatment of this rare tumour. Preoperative diagnosis is often impossible and so surgery is of great value both for treatment and diagnosis.

Synthesis of a 11-deoxyprostanoid in the area of preclavulones: (+-)-8,12-trans-(5Z-14Z)-9-oxo-prosta-5,14-dienoic acid from 2-allyl-2-cyclopenten-1-one.

Following our research on the arachidonic acid metabolites and their derivatives with potential biological activity, we describe the synthesis of the (+-)-8,12-trans-(5Z, 14Z)-9-oxo-prosta-5,14-dienoic acid, a 11-deoxyprostanoid correlated to the class of Preclavulones, one of the unusual families of marine eicosanoids from the coral Clavularia Viridis with considerable biological interest.

Hydrophobic interaction chromatography of autoproteolysis products of human seminal plasma.

Hydrophobic interaction chromatography was employed for the fractionation of the glycopeptide mixture obtained after autoproteolysis and delipidization treatment of normal human seminal plasma samples. This sequence of procedures led to the elimination of highly hydrophobic components and to the concentration of the carbohydrate-rich fractions. A partial separation between the O- and N-glycosidic-linked oligopeptides was also achieved after the chromatographic step.

Conversion of longstanding atrial flutter to sinus rhythm and transient complete A-V block following oral administration of verapamil. Report of a case.

In a patient with ischaemic heart disease chronic atrial flutter reverted to sinus rhythm during treatment with oral Verapamil, given at dosage of 240 mg once a day in order to prevent spontaneous angina. Such an unexpected event was accompanied by a remarkable sinus node depression and by a transient complete a-v block, that gradually subsides leaving a slight permanent a-v conduction delay. Either a higher bioavailability of the drug or an extremely increased sensitivity of the receptors can explain such a marked electrophysiological effect at a moderate drug dosage.