N-terminal region of human chemokine receptor CXCR3: Structural analysis of CXCR3(1-48) by experimental and computational studies.

Our study on the highly charged N-terminal peptide of the human chemokine receptor CXCR3 by spectroscopic methods in solution and by means of molecular dynamics simulations showed that the charge content modulates the intrinsic structural preference of its flexible backbone. Collectively, our findings suggest that the structural organization of a protein should be seen as a part of a continuum in which the ratio between electrostatic and hydrophobic interactions and the intrinsic flexibility are important properties used to optimize the folding. When this ratio changes and the structure is intrinsically flexible, the structural organization of the system moves along the continuum of the possible conformational states. By all this combined information, one can describe the structure of CXCR3(1-48) as an ensemble of conformations. In fact, the peptide shows stretches of negative charges embedded in a flexible sequence which can be used to maximize promiscuous interactions relevant to molecular recognition but globally the peptide appears as a poly-structured globule-like ensemble that is dynamically stabilized by H-bonds. We have approached the study of the most populated ensembles with subset selection to explain our experimental data also by evidencing that the changes into the fraction of charged residues discriminate between dynamically poly-structured states, conceivably because of small free energy barriers existing between the different conformations of CXCR3(1-48). Therefore, the overlap of a highly flexible backbone, negatively charged residues and sites which can be modified by post-translational modifications represent the structural organization that controls the molecular mechanisms underlying the biological functions carried out by CXCR3(1-48).

The role of E-cadherin down-regulation in oral cancer: CDH1 gene expression and epigenetic blockage.

BACKGROUND: The prognosis of the oral squamous cell carcinoma (OSCC) patients remains very poor, mainly due to their high propensity to invade and metastasize. E-cadherin reduced expression occurs in the primary step of oral tumour progression and gene methylation is a mode by which the expression of this protein is regulated in cancers. In this perspective, we investigated E-cadherin gene (CDH1) promoter methylation status in OSCC and its correlation with Ecadherin protein expression, clinicopathological characteristics and patient outcome. METHODS: Histologically proven OSCC and paired normal mucosa were analyzed for CDH1 promoter methylation status and E-cadherin protein expression by methylation-specific polymerase chain reaction and immunohistochemistry. Colocalization of E-cadherin with epidermal growth factor (EGF) receptor (EGFR) was evidenced by confocal microscopy and by immunoprecipitation analyses. RESULTS: This study indicated E-cadherin protein down-regulation in OSCC associated with protein delocalization from membrane to cytoplasm. Low E-cadherin expression correlated to aggressive, poorly differentiated, high grade carcinomas and low patient survival. Moreover, protein down-regulation appeared to be due to E-cadherin mRNA downregulation and CDH1 promoter hypermethylation. In an in vitro model of OSCC the treatment with EGF caused internalization and co-localization of E-cadherin with EGFR and the addition of demethylating agents increased E-cadherin expression. CONCLUSION: Low E-Cadherin expression is a negative prognostic factor of OSCC and is likely due to the hypermethylation of CDH1 promoter. The delocalization of E-cadherin from membrane to cytoplasm could be also due to the increased expression of EGFR in OSCC and the consequent increase of E-cadherin co-internalization with EGFR.

Poor glycaemic control in type 2 diabetes patients reduces endothelial progenitor cell number by influencing SIRT1 signalling via platelet-activating factor receptor activation.

AIMS/HYPOTHESIS: Downregulation of levels of endothelial progenitor cells (EPCs) during in-vitro short-term exposure to high glucose concentrations relates to reduced activity of silent information regulator 1 (SIRT1) and increased synthesis of platelet-activating factor (PAF). We investigated the possible relationship between PAF and SIRT1 pathways in EPCs during altered glucose homeostasis. METHODS: SIRT1 and PAF receptor (PAF-R) levels were determined by western blot, RT-PCR and confocal laser-scanning microscopy. In-vivo experiments were performed on 48 type 2 diabetic patients (25 with poor glycaemic control and 23 with good glycaemic control) and 20 control individuals. In-vitro experiments with the PAF-R antagonist CV3988 were performed on EPCs isolated from leucocyte-rich buffy coat of healthy human donors. RESULTS: Decreased SIRT1 protein levels were observed in EPCs from type 2 diabetic patients compared with control individuals (p < 0.01). Notably, the SIRT1 level was consistently lower in patients with poor glycaemic control than in those with good glycaemic control (p < 0.01). Diabetic patients also showed an upregulation of PAF-Rs; this response occurred to a greater extent in individuals with poor glycaemic control than in those with good glycaemic control. In-vitro experiments confirmed that EPCs respond to PAF stimulation with decreased SIRT1 protein and SIRT1 mRNA levels. Moreover, reduction of SIRT1 levels and activity were abolished by CV3988. CONCLUSIONS/INTERPRETATION: These findings unveil a link between PAF and SIRT1 pathways in EPCs that contributes to the deleterious effect of hyperglycaemia on the functional properties of EPCs, crucial in diabetes and peripheral vascular complications.

Ion, protein, phospholipid and energy substrate content of oviduct fluid during the oestrous cycle of buffalo (Bubalus bubalis).

The aim of this research was to analyse the composition of oviduct fluid (ODF) in buffalo cows at different oestrous cycle phases to fulfil the requirements of buffalo embryos in vitro. ODF was collected by chronic cannulation from three cows that were synchronized by administering a synthetic prostaglandin. Based on hormonal profiles, the pre-ovulatory, ovulatory, post-ovulatory and luteal phases of the oestrous cycle were defined. The volume of ODF produced (ml/24 h) was influenced by the oestrous cycle, with values (mean ± SE) around ovulation (1.0 ± 0.2) greater (p < 0.05) than in both the luteal (0.4 ± 0.1) and the post-ovulatory phases (0.5 ± 0.1), but not different from the intermediate values in the pre-ovulatory phase (0.8 ± 0.2). Among cycle phases, no differences were found in sodium, potassium, calcium and magnesium concentrations (130.0 ± 1.1, 5.1 ± 0.3, 2.8 ± 0.1 and 0.59 ± 0.04 mmol/l respectively). Interestingly, the chloride secretion (µm/24 h) was higher (p < 0.05) at ovulation (150.2 ± 16.5) than during both the luteal (73.7 ± 22.0) and the post-ovulatory phases (63.7 ± 11.2), with intermediate values in the pre-ovulatory phase (113.4 ± 23.5). Glucose concentration (mmol/l) was higher (p = 0.056) in the pre-ovulatory phase (0.06 ± 0.02) than in the luteal (0.02 ± 0.01) and post-ovulatory (0.02 ± 0.01) phases but not different from values in the ovulatory phase (0.04 ± 0.02). Concentrations of pyruvate and lactate among oestrous cycle phases were similar (0.08 ± 0.01 and 1.0 ± 0.1 mmol/l respectively). The total quantity of phospholipids (µmol/24 h) was greater (p < 0.05) at ovulation (0.21 ± 0.02) compared with the luteal, pre-ovulatory and post-ovulatory phases of the cycle (0.09 ± 0.02, 0.13 ± 0.02 and 0.09 ± 0.01 respectively). No differences were found in either the protein concentration (1.8 ± 0.3 mg/ml) or the quantity of proteins secreted in 24 h (1.8 ± 0.4 mg) among oestrous cycle phases. In conclusion, this study provides the first characterization of buffalo ODF during the oestrous cycle, showing species-specific differences that may be useful for developing suitable media for buffalo in vitro embryo production.

Laparoscopic treatment of incarcerated hernia through right broad ligament in patients with bilateral parametrium defects.

We present the first case reported in the literature of small bowel obstruction due to internal incarcerated hernia through a diagnosed bilateral broad ligament defect, and treated by laparoscopy. A 36-year-old white woman, gravida 0, para 0, was admitted to our hospital with intestinal obstruction symptoms. A laparoscopic approach was performed with 3 trocars and internal incarcerated hernia due to a defect in the right broad ligament was found. There was a similar defect in the left broad ligament. The small bowel, once reduced, appeared viable. Closure of both defects was carried out by laparoscopy with 2-0 monofilament absorbable running suture. The patient's postoperative course was unremarkable and she was discharged from the hospital 4 days after the surgical procedure. The classification of defect was a bilateral fenestrae type I defect. Congenital ethiology is plausible because of the presence of bilateral defects and the absence of surgical trauma, pregnancy, pelvic inflammatory disease, endometriosis in the clinical history.

[Short-term results in colorectal surgery. Statistical analysis about mortality, morbidity and hospital stay]. / Risultati a breve temine della chirurgia colo-rettale. Studio statistico su mortalità, morbilità e tempi di degenza.

AIM: The surgical approach on the colon and rectum represents a wide slice of the surgical procedures carry out in election or emergency in a general surgery unit. The literature reports prospective and retrospective studies evidencing emergency surgery, advanced age, comorbidity and other factors can determinate a worsening of short-term outcome (postoperative mortality, morbidity and hospital stay). The aim of the study was to verify, through a statistical analysis on a group of patients operated on the colon and the rectum, which are the factors weighting on the short-term outcome. METHODS: Our retrospective study is carried out on 150 patients consecutively operated on the colon and rectum from January 2002 to September 2004 in elective or emergency surgery in the Unity of General Surgery of the Hospital S. Maria Nuova Azienda Sanitaria of Florence. The variables for the statistical analysis were: sex, age, comorbidity, nature of pathology, timing of surgery, type of emergency, lesion location, surgical intervention, presence of social factors delaying the discharge, blood transfusion, Physiological and Operative Severity Score for the enUmeration of Mortality and Morbidity (POSSUM-score). RESULTS: The mortality study found the advanced age (>70 years) as risk factor in the univariate analysis, not confirmed in the multivariate one. The morbidity study found advanced age, presence of comorbidity and blood transfusion as risk factors in the univariate analysis, not confirmed in the multivariate one. The POSSUM-score represents in both multivariate analyses the only statistically meaningful parameter correlated with mortality (P<0.005) and morbidity (P<0.05). The multivariate analysis in the study on the hospital stay found that more staged surgery carry to a lengthening of hospital stay (P<0.0001); in minor such measure blood transfusion (P=0.0005), emergency surgery (P=0.002) and presence of social factors (P=0.008); comorbidity (P=0.02) and advanced age (P=0.03) had less statistical weight. CONCLUSIONS: Despite of the literature, this study found none of the analyzed variables related on postoperative mortality and morbidity in statistically meaningful way. The POSSUM-score demonstrated once again validity in estimating the probability of dead and of postoperative complications. The variables that influenced in lengthening of hospital stay were: more staged surgery, blood transfusion, emergency surgery, presence of social factors conditioning the discharge, comorbidity and advanced age of the patients. The good results about mortality and morbidity can be explained by the fact we prefer in emergency more staged surgery that protect the patients from complications related to the anastomosis, the presence of sub-intensive surgical beds with a constant monitoring of high risk patients and the close collaboration between surgeons and intensive care medical doctors.

Apoptosis induced by interferon-alpha and antagonized by EGF is regulated by caspase-3-mediated cleavage of gelsolin in human epidermoid cancer cells.

We have previously reported that interferon-alpha (IFNalpha) induces apoptosis and EGF can antagonize this effect in human epidermoid cancer KB cells. Since apoptosis occurs together with cytoskeleton reorganization we have evaluated if IFNalpha and EGF could modulate cell remodeling in our experimental conditions. We have found that 48 h 1,000 IU/ml IFNalpha induced structural reorganization of stress fibers and membrane delocalization and partial capping of the actin severing protein gelsolin. The transfection of KB cells with both a wild type (WT) or a C-terminal truncated form of gelsolin caused overexpression of the protein and an increase of both the spontaneous and IFNalpha-induced apoptosis and cell cytoskeletal modifications. In fact, after 48 h of treatment IFNalpha induced 45% of apoptotic cell death in parental cells while an approximately 80% of cell population was apoptotic in transfected cells. These effects occurred together with an increase of the expression and consequent degradation of gelsolin. Again the addition of EGF to IFNalpha-treated transfected cells caused a recovery of the apoptosis. Notably, IFNalpha and EGF did not modify the expression of other molecules associated to cytoskeleton such as focal adhesion kinase and vinculin. In the same experimental conditions IFNalpha induced also gelsolin cleavage that occurred together with caspase-3 activation and release of cytochrome c. All these effects were antagonized by the exposure of IFNalpha-treated KB to 10 nM EGF for the last 12 h. Moreover, the specific inhibition of caspase-3 with 20 microM DEVD completely abrogated apoptosis and gelsolin cleavage induced by IFNalpha. In conclusion, our data are the first demonstration that IFNalpha can induce morphological cell changes that are peculiar of apoptosis onset through the caspase-3-mediated cleavage of gelsolin. Furthermore, we have demonstrated that EGF is able to antagonize these effects through the inhibition of caspase-3 activation.

Pectin methylesterase inhibitor.

Pectin methylesterase (PME) is the first enzyme acting on pectin, a major component of plant cell wall. PME action produces pectin with different structural and functional properties, having an important role in plant physiology. Regulation of plant PME activity is obtained by the differential expression of several isoforms in different tissues and developmental stages and by subtle modifications of cell wall local pH. Inhibitory activities from various plant sources have also been reported. A proteinaceous inhibitor of PME (PMEI) has been purified from kiwi fruit. The kiwi PMEI is active against plant PMEs, forming a 1:1 non-covalent complex. The polypeptide chain comprises 152 amino acid residues and contains five Cys residues, four of which are connected by disulfide bridges, first to second and third to fourth. The sequence shows significant similarity with the N-terminal pro-peptides of plant PME, and with plant invertase inhibitors. In particular, the four Cys residues involved in disulfide bridges are conserved. On the basis of amino acid sequence similarity and Cys residues conservation, a large protein family including PMEI, invertase inhibitors and related proteins of unknown function has been identified. The presence of at least two sequences in the Arabidopsis genome having high similarity with kiwi PMEI suggests the ubiquitous presence of this inhibitor. PMEI has an interest in food industry as inhibitor of endogenous PME, responsible for phase separation and cloud loss in fruit juice manufacturing. Affinity chromatography on resin-bound PMEI can also be used to concentrate and detect residual PME activity in fruit and vegetable products.

Two Arabidopsis thaliana genes encode functional pectin methylesterase inhibitors.

We have identified, expressed and characterized two genes from Arabidopsis thaliana (AtPMEI-1 and AtPMEI-2) encoding functional inhibitors of pectin methylesterases. AtPMEI-1 and AtPMEI-2 are cell wall proteins sharing many features with the only pectin methylesterase inhibitor (PMEI) characterized so far from kiwi fruit. Both Arabidopsis proteins interact with and inhibit plant-derived pectin methylesterases (PMEs) but not microbial enzymes. The occurrence of functional PMEIs in Arabidopsis indicates that a mechanism of controlling pectin esterification by inhibition of endogenous PMEs is present in different plant species.

[Diagnostic laparoscopy in non traumatic abdominal emergencies]. / La laparoscopia diagnostica nelle urgenze addominali di tipo non traumatico.

The development of video-laparoscopic techniques and surgical experience in this field have led to a resurgence of laparoscopy diagnostic purposes and have made it often the first action of surgical therapy with totally mini-invasive procedure. The purpose of this paper is to underline the clinical pictures of non traumatic acute abdomen that can profit from laparoscopy diagnostic ally and in related problems. The authors, in their initial experience of management of the urgency with laparoscopic approach, hold that laparoscopy can be not only the key to clarify a preoperative diagnostic doubt, but can reveal especially in some situations, such as pelvic pathologies in women of childbearing age the most correct surgical approach.

Kiwi protein inhibitor of pectin methylesterase amino-acid sequence and structural importance of two disulfide bridges.

A protein acting as a powerful inhibitor of plant pectin methylesterase was isolated from kiwi (Actinidia chinensis) fruit. The complete amino-acid sequence of the pectin methylesterase inhibitor (PMEI) was determined by direct protein analysis. The sequence comprises 152 amino-acid residues, accounting for a molecular mass of 16 277 Da. The far-UV CD spectrum indicated a predominant alpha-helix conformation in the secondary structure. The protein has five cysteine residues but neither tryptophan nor methionine. Analysis of fragments obtained after digestion of the protein alkylated without previous reduction identified two disulfide bridges connecting Cys9 with Cys18, and Cys74 with Cys114; Cys140 bears a free thiol group. A database search pointed out a similarity between PMEI and plant invertase inhibitors. In particular, the four Cys residues, which in PMEI are involved in the disulfide bridges, are conserved. This allows us to infer that also in the homologous proteins, whose primary structure was deduced only by cDNA sequencing, those cysteine residues are engaged in two disulfide bridges, and constitute a common structural motif. The comparison of the sequence of these inhibitors confirms the existence of a novel class of proteins with moderate but significant sequence conservation, comprising plant proteins acting as inhibitors of sugar metabolism enzymes, and probably involved in various steps of plant development.

Analysis of free and esterified ergosterol in tomato products.

A new extraction and chromatographic procedure to quantify free and esterified ergosterol in tomato products was devised. The extraction solution was composed of a dichloromethane/methanol mixture in a 2:1 (v/v) ratio. This extraction solvent allowed for higher ergosterol recovery from tomato products (an average of 25% more) compared to hexane, which is frequently employed for ergosterol extraction. Both free and esterified ergosterol were determined by HPLC reverse-phase chromatography employing a Nova-Pak C-18 column (300 x 3.9 mm), filled with 4 mm average particle size and a guard column of the same material. The elution was performed at a flow rate of 1 mL. min(-1) with a linear gradient of solvent A (methanol/water, 80:20, v/v) and solvent B (dichloromethane). The gradient, starting at sample injection, was from 0 to 50% B for 20 min for the free ergosterol analysis and additional 15 min at 50% B to analyze the ergosterol esters. This technique has proven to be more sensitive for ergosterol determination than other reported chromatographic procedures. Moreover, ergosterol esters, extracted from various fungal sources, separated well and were easily quantified.

Cellulose-based hydrogels as body water retainers.

In this work the possibility of using hydrogels as body water retainers for a therapeutic aid in pathologies such as oedemas of various origins was explored. For such a purpose, the material requires a good compatibility and a controlled swelling capacity without altering the body electrolyte homeostasis. The hydrogel was designed to meet the swelling requirements with the physiological constraints and its biocompatibility was assessed either in vitro or in vivo. Absorption tests were performed in order to define the swelling behavior by varying the pH and ion content of the external solution. The hydrogel swelling capacity was assessed in the presence of various solvents, in order to evaluate its absorption capacity in solutions similar to biological fluids. In addition, the capacity of the gel to modify electrolyte homeostasis by adsorbing ions such as calcium, potassium and sodium was tested. In order to assess the gel biocompatibility after contact of the hydrogel with intestinal cells, arachidonic acid relase was determined. No significant intracellular increase of free arachidonic acid was found in the cells after up to 2 h of contact with the gel. The results suggest that, as far as brief periods are concerned, the gel does not cause an inflammatory response in intestinal cells.

2-aminoanthracene as an analytical tool with the acetylation reaction catalyzed by arylamine N-acetyltransferase.

The polynuclear aromatic amine, 2-aminoanthracene, was found to be acetylated with high efficiency in the presence of acetyl-CoA by pigeon liver arylamine N-acetyltransferase (EC 2.3.1.5). As a consequence of acetylation the fluorescence properties of the compound dramatically change and the reaction time course can be easily followed fluorometrically at the emission wavelength of 425 nm upon excitation at 360 nm. When 2-aminoanthracene is employed with pigeon arylamine N-acetyltransferase, as the ultimate acceptor of the acetyl group in coupled fluorometric assays, it is possible to measure enzymatic activities, such as pyruvate dehydrogenase or carnitine acetyltransferase, in continuous assays rapidly and with high sensitivity or to determine with as much sensitivity important metabolites such as acetylcarnitine or acetyl-CoA.

[Ileal duplication in adults. A very rare nosological entity]. / La duplicazione ileale dell'adulto. Una rarissima entità nosologica.

The report presents a rare case of intestinal duplication in a 43-year old female. Intestinal duplication is a rare congenital malformation and is extremely exceptional in adults. A lot of etiopathogenic theories have been advanced to explain this malformation that can occur anywhere along the alimentary tract, even if the ileum remains the most common. It may be cystic or tubular. An important aspect of mucosal histology is the possibility of gastric heterotopy, conditioning a particular treatment. The literature shows 14 cases with clinical very different presentations and instrumental exams were rarely helpful for correct diagnosis. Treatment of choice is surgical complete resection of the duplication. When contiguous structures are involved intestinal bypass or Roux-on-Y anastomosis may be necessary with mandatory stripping of the mucosa when heterotopic gastric mucosa is present in order to prevent the risk of gastrointestinal haemorrhage or malignant transformation, an event possible in about 25% of the cases reported in the literature.

Anaphylaxis in children: clinical and allergologic features.

BACKGROUND: Despite the importance of anaphylaxis, little information is available on its clinical features. OBJECTIVE: To evaluate the clinical and allergologic features of anaphylaxis in children referred to the allergology and immunology unit of A. Meyer Children's Hospital (Florence, Italy) from 1994 to 1996. RESULTS: Ninety-five episodes of anaphylaxis occurred in 76 children (50 boys and 26 girls). Sixty-six children (87%) had only one episode of anaphylaxis, while 10 (13%) had two or more episodes. Sixty-two (82%) of the 76 patients had a personal history of atopic symptoms, although 14 (18%) did not. Sixty (79%) of the 76 children studied had at least one positive skin prick test to one or more of the common inhalant and/or food allergens. Children with venom-induced anaphylaxis usually had negative skin tests to the allergens tested. A younger age and eczema were more frequent among children with food-dependent anaphylaxis, whereas an older age together with urticaria-angioedema were common among those with exercise-induced anaphylaxis. The mean latent period (+/-SD) of the anaphylaxis episodes was 15.4 +/- 27.5 minutes. Skin and respiratory manifestations had an earlier onset and were more common than the gastrointestinal and cardiovascular ones. The most frequent clinical manifestation in children with food anaphylaxis was gastrointestinal symptoms, whereas cardiovascular symptoms were rare. The most probable causative agents in the 95 episodes described were foods (57%), drugs (11%), hymenoptera venom (12%), exercise (9%), additives (1%), specific immunotherapy (1%), latex (1%), and vaccines (2%), but in 6 cases (6%) the agent was never determined. Among the foods, seafood and milk were the most frequently involved. As for location, 57% of the anaphylactic events occurred in the home (54/95), 12% outdoors (11/95); 5% in restaurants (5/95); 3% in the doctor's office (3/95); 3% in hospitals (3/95); 3% on football fields (3/95); 2% on the beach (2/95); 1% in the gym (1/95); 1% at school (1/95); and 1% in the operating room (1/95). In the remaining 12% of cases (11/95) the site remained unknown. Sixty-two percent of the patients (59/95) were treated in an emergency room or hospital, while 32% (30/95) were not (this information is lacking in 6% of the cases [6/95]). Patients were treated with corticosteroids in 72% of the cases (68/95), with antihistamines in 20% (19/95), with epinephrine in 18% (17/95), with beta2-agonists in 5% (5/95), and with oxygen in 4% (4/95). CONCLUSIONS: In our area, foods, particularly seafood and milk, seem to be the most important etiologic factors triggering anaphylaxis. Food-induced anaphylaxis often occurs in younger children with a severe food allergy, whereas exercise-induced anaphylaxis occurs more often in older children with a history of urticaria-angioedema. The venom-induced variant usually presents itself in nonatopic subjects. Given the fact that most of the children had only one anaphylactic reaction, prevention is almost impossible. Epinephrine, although it is the first-choice treatment of anaphylaxis, often goes unused, even in hospitals and doctors' offices.

[The solitary rectal ulcer today. A review of the literature]. / L'ulcera solitaria del retto oggi. Revisione della letteratura.

The solitary rectal ulcer (SRU) is a benign lesion of adults of either sex, which presents with chronic constipation, peculiar defecatory disorders, rectal prolapse and smaller psychological abnormalities. The characteristic appearance of this disease is a "neither being always ulcerate, nor always solitary" lesion, but often with polypoid or granular feature, typically localized in anterior rectal wall, a few inches from anal channel. Distinctive histopathological specimens are localized mucosal distortion, hypertrophic proliferation of muscularis mucosae and obliteration of lamina propria by fibroblasts and muscle fibres from the muscularis mucosae. Very few intermittent or recurrent symptoms are rectal bleeding and mucous discharge with defecations, difficulty of a complete ampullar evacuation and sometimes pelvic or rectoperineal pain. Clinical picture and endoscopic biopsies led to diagnosis. Barium enema, defecography, transrectal ultrasound, manometry and electromyography have an additional role. Medical treatment is performed by high-fiber diet, but biofeedback training is very helpful. Surgical management is as an excisional surgery, as a rectopexy if there is prolapse. Fecal diversion and rectocolic resection are considered only for patients with obstinate and severe symptoms. Even in patients who seem to advocate a surgical approach it is important to heal a dyskinetic puborectalis muscle.

Net-b, a Ras-insensitive factor that forms ternary complexes with serum response factor on the serum response element of the fos promoter.

The Ras signalling pathway targets transcription factors such as the ternary complex factors that are recruited by the serum response factor to form complexes on the serum response element (SRE) of the fos promoter. We have identified a new ternary complex factor, Net-b. We report the features of the net gene and show that it produces several splice variants, net-b and net-c. net-b RNA and protein are expressed in a variety of tissues and cell lines. net-c RNA is expressed at low levels, and the protein was not detected, raising the possibility that it is a cryptic splice variant. We have studied the composition of ternary complexes that form on the SRE of the fos promoter with extracts from fibroblasts (NIH 3T3) cultured under various conditions and pre-B cells (70Z/3) before and after differentiation with lipopolysaccharide (LPS). The fibroblast complexes contain mainly Net-b followed by Sap1 and Elk1. Net-b complexes, as well as Sap1 and Elk1, are induced by epidermal growth factor (EGF) stimulation of cells cultured in low serum. Pre-B-cell complexes contain mainly Sap1, with less of Net-b and little of Elk1. There is little change upon LPS-induced differentiation compared to the increase with EGF in fibroblasts. We have also found that Net-b is a nuclear protein that constitutively represses transcription. Net-b is not activated by Ras signalling, in contrast to Net, Sap1a, and Elk1. We have previously reported that down-regulation of Net proteins with antisense RNA increases SRE activity. The increase in SRE activity is observed at low serum levels and is even greater after serum stimulation, showing that the SRE is under negative regulation by Net proteins and the level of repression increases during induction. Net-b, the predominant factor in ternary complexes in fibroblasts, may both keep the activity of the SRE low in the absence of strong inducing conditions and rapidly shut the activity off after stimulation.

Simultaneous determination of lysophospholipids by high-performance liquid chromatography with fluorescence detection.

A high-performance liquid chromatography (HPLC) procedure for the separation of choline lysophospholipids including 1-acyl-lysophosphatidylcholines and 1-O-alkyl-lysophosphatidyl- cholines, like the lysoform of the platelet activating factor (2-lysoPAF), is described. The lysophospholipids are derivatized at the sn-2 position of the hydroxyl group by 7-diethylaminocoumarin-3-carbonylazide, which converts them into the corresponding carbamoyl derivatives. The derivatized compounds were well separated by reversed-phase HPLC and quantified by fluorimetric detection. This method shows a high sensitivity and allows the separation and quantification of mixtures of lysophospholipids at picomolar level. The method was applied to assay enzyme activities, like phospholipase A2 and PAF-acetylhydrolase, on single phospholipids or their mixtures.

Determination of residual pectin methylesterase activity in food products.

A method for the determination of a low level of pectin methylesterase activity from vegetable products is described. The method is based on an affinity chromatography technique that employs a resin-bound pectin methylesterase inhibitor, purified from kiwi fruit, which selectively binds the pectin methylesterase. The resin has the capacity to concentrate the enzyme, allowing measurement of enzyme activities too low to be determined by commonly employed techniques and commensurate with those found in pasteurized food products. The enzyme is eluted from the resin at alkaline pH (9.5) and assayed by a pH-stat method. Depulped orange juices containing different amounts of pectin methylesterase were prepared and used to determine enzyme recovery. The results show a recovery of 90% with a standard deviation of 6.8%.