Insulin-like effect of the phosphodiesterase type 5 inhibitor tadalafil onto male human skeletal muscle cells.

BACKGROUND: Phosphodiesterase type 5 inhibitors (PDE5i), widely used to treat male erectile dysfunction, seem to counteract insulin resistance (IR) in animals and humans. IR, primarily manifest in peripheral tissues and particularly in skeletal muscle, is due to impaired insulin signal transduction. Investigators have been focusing onto intracellular defects responsible for IR to identify suitable pharmacological tools targeted toward the specific defects. Albeit some effects of PDE5i have been reported onto animal muscular tissues or cells, whether and how they might affect metabolic processes directly in human skeletal muscle still remains unclear. AIM: We aimed to investigate in human fetal skeletal muscle cells (Hfsmc) the effect of tadalafil, one of PDE5i, onto some intracellular factors involved in response to insulin, such as ras-raf mitogen activated protein kinase (MAPK), phosphatidylinositol 3-kinase/protein kinase B (PKB/Akt), glycogen synthase kinase 3ß (GSK-3ß), and the transcriptional factor c-Myc; proliferation rate; lactate (lact) and free fatty acid (ffa) release; activity of citrate synthase (CS) and succinate dehydrogenase (SDH), both enzymes of Kreb's cycle; PDE5 gene expression. MATERIALS AND METHODS: Western blot analysis, enzyme-linked immunosorbent assay, enzymatic assays, cell count, MTT assay and Real Time PCR were performed in Hfsmc with and without tadalafil. RESULTS: In Hfsmc tadalafil affected the insulin-related intracellular cascade, by increasing MAPK, PKB/Akt, GSK-3ß phosphorylation and c-Myc expression. ffa release and CS activity also significantly increased, with no changes in SDH activity and lact release. CONCLUSIONS: Tadalafil, like insulin, targeted part of the machinery dedicated to energy management and metabolic control in human skeletal muscle cells.

Recolonization and recovery dynamics of the macrozoobenthos after sand extraction in relict sand bottoms of the Northern Adriatic Sea.

The long-term effects of sand extraction on macrozoobenthic communities were investigated in an offshore area in the Northern Adriatic Sea characterised by relict sands formed during the last Adriatic post-glacial transgression. Surveys were carried out before, during and 1, 6, 12, 18, 24 and 30 months after extraction at three impacted and seven reference stations. The operations did not influence the physical characteristics of the sediment, but they caused almost complete defaunation at dredged sites. Univariate and multivariate analyses highlighted that the macrozoobenthic community responses to the dredging operations were (1) a rapid initial recolonisation phase by the dominant taxa present before dredging, which took place 6-12 months after sand extraction; (2) a slower recovery phase, that ended 30 months after the operations, when the composition and structure of the communities were similar in the dredged and reference areas. This pattern of recolonisation-recovery fits well with the commonly encountered scenario where the substratum merely remains unchanged after marine aggregate extraction.

High glucose causes apoptosis in cultured human pancreatic islets of Langerhans: a potential role for regulation of specific Bcl family genes toward an apoptotic cell death program.

Type 2 diabetes is characterized by insulin resistance and inadequate insulin secretion. In the advanced stages of the disease, beta-cell dysfunction worsens and insulin therapy may be necessary to achieve satisfactory metabolic control. Studies in autopsies found decreased beta-cell mass in pancreas of people with type 2 diabetes. Apoptosis, a constitutive program of cell death modulated by the Bcl family genes, has been implicated in loss of beta-cells in animal models of type 2 diabetes. In this study, we compared the effect of 5 days' culture in high glucose concentration (16.7 mmol/l) versus normal glucose levels (5.5 mmol/l) or hyperosmolar control (mannitol 11 mmol/l plus glucose 5 mmol/l) on the survival of human pancreatic islets. Apoptosis, analyzed by flow cytometry and electron and immunofluorescence microscopy, was increased in islets cultured in high glucose (HG5) as compared with normal glucose (NG5) or hyperosmolar control (NG5+MAN5). We also analyzed by reverse transcriptase-polymerase chain reaction and Western blotting the expression of the Bcl family genes in human islets cultured in normal glucose or high glucose. The antiapoptotic gene Bcl-2 was unaffected by glucose change, whereas Bcl-xl was reduced upon treatment with HG5. On the other hand, proapoptotic genes Bad, Bid, and Bik were overexpressed in the islets maintained in HG5. To define the pancreatic localization of Bcl proteins, we performed confocal immunofluorescence analysis on human pancreas. Bad and Bid were specifically expressed in beta-cells, and Bid was also expressed, although at low levels, in the exocrine pancreas. Bik and Bcl-xl were expressed in other endocrine islet cells as well as in the exocrine pancreas. These data suggest that in human islets, high glucose may modulate the balance of proapoptotic and antiapoptotic Bcl proteins toward apoptosis, thus favoring beta-cell death.

A crude extract of Artocarpus integrifolia contains two lectins with distinct biological activities.

The crude extract derived from seeds of Artocarpus integrifolia (jack fruit) contains two fractions with different biological activities for lymphocytes. One fraction is the D-galactose-binding lectin, jacalin, obtained by affinity purification on a D-galactose agarose column. The other, which is a component of the flow-through fraction (FT), is responsible for the mitogenic activity observed with human PBMC and murine spleen cells. In contrast, jacalin inhibits FT- and ConA-induced proliferative activity of human PMBC and murine spleen cells. This inhibition is not due to toxicity, because: (1) jacalin induces significant levels of IL-3/GM-CSF but not of IL-2 and/or IL-4 in murine spleen cells; (2) jacalin does not affect the capacity of these cells to secrete IL-2 or IL-4 as supernatants obtained from spleen cells sequentially stimulated with jacalin and ConA contain IL-2 and/or IL-4 as well as IL-3/GM-CSF. The ligand for the mitogen contained in the FT fraction is D-mannose as determined by sugar inhibition studies.

Effect of clot-detection methods and reagents on activated partial thromboplastin time (APTT). Implications in heparin monitoring by APTT.

Two automatic coagulometers, ACL 810 (Instrumentation Laboratory), a laser-nephelometric centrifugal analyzer, and KoaguLab 40 A (Ortho Diagnostics), an optical automatic coagulometer, were compared with the manual tilt-tube method for the performance of activated partial thromboplastin time (APTT). Seven commercial APTT reagents were used for duplicate determinations in 30 normal controls, 26 patients with liver disease, and 33 patients on full-dose heparin treatment. Clotting times were longer with the manual method than with ACL 810 and, to a lesser extent, with KoaguLab 40 A. Average imprecision of duplicate determinations (coefficient of variation [CV]) was less with ACL 810 (less than 1.5%) than with KoaguLab 40 A (2.9%) and the manual method (2.4%). Differences in slope of the regression curves of clotting times obtained with the coagulometers over the tilt-tube method were observed with all the reagents tested (P less than 0.01). Transformation of clotting times of controls, patients with liver disease, and patients on heparin therapy to APTT ratios did not eliminate the bias resulting from the different reagents (P less than 0.001) and clot-detection methods (P less than 0.001); in controls, significant (P less than 0.001) reagent-method interaction was also observed. The in vitro heparin sensitivity differed with the APTT reagents evaluated and was influenced by the clot-detection method used. Transformation of APTT ratios of anticoagulated patients to apparent plasma heparin levels--as derived from in vitro dose-response curves--effectively eliminated the bias resulting from the different clot-detection methods but had no effect on the bias resulting from the different APTT reagents. In vitro heparin activity curves thus have little, if any, relevance for the ex vivo monitoring of heparin treatment.

Comparison of two automated coagulometers and the manual tilt-tube method for the determination of prothrombin time.

Two automatic coagulometers--ACL 810 (Instrumentation Laboratory), a laser-nephelometric centrifugal analyzer, and KoaguLab 40 A (Ortho Diagnostics), an optical automatic coagulometer--were compared with the manual tilt-tube method for the performance of prothrombin time (PT). Seven ISI- (International Sensitivity Index) calibrated commercial thromboplastin reagents were used for duplicate determinations in 30 normal subjects, 30 patients with liver disease, and 30 patients receiving stabilized oral anticoagulation. Clotting times were longer with the manual method than with ACL 810 and, to a lesser extent, with KoaguLab 40 A. Average imprecision of duplicate determinations (CV) was less than 1% with ACL 810; KoaguLab 40 A and the manual method had similarly higher imprecisions (2.8% and 2.7%). Differences in origin and slope of the regression curves of clotting times obtained with the coagulometers over the tilt-tube method were observed with all the reagents tested. Transformation of clotting times to PT ratios did not eliminate the bias resulting from the different clot-detection methods. A higher percentage of patients with liver disease had abnormal PT ratios when their plasma was tested with the coagulometers than with the manual method. Transformation of PT ratios to International Normalized Ratios effectively eliminated the bias resulting from the different thromboplastin reagents but had no effect on the bias resulting from the different clot-detection methods. A significant proportion of patients appeared excessively anticoagulated (INR greater than 4.5) with the coagulometers but not with the manual method. These results highlight the need for standardization of both instrumentations and reagents to improve monitoring of oral anticoagulant treatment.

Role of leucocytes in free radical production during myocardial revascularisation.

OBJECTIVE: To evaluate the role of leucocytes in free radical production in patients with depressed or normal ejection fraction undergoing coronary bypass. DESIGN: Two randomised control trials. SETTING: Tertiary care centre. PATIENTS AND INTERVENTIONS: In the first study, 22 patients with ejection fractions of < or = 40% received blood cardioplegic reperfusion with (n = 11) or without (n = 11) leucocyte depletion. In the second study, 22 patients with ejection fractions > or = 45% received either leucocyte depleted (n = 11) or blood cardioplegia (n = 11). MAIN OUTCOME MEASURES: Glutathione, hypoxanthine, and lipid peroxidation products were measured in coronary sinus blood and plasma before aortic cross clamping and at 0, 15, and 30 minutes after unclamping. Haemodynamic variables and creatine kinase MB isoenzymes were monitored on the first postoperative day. Comparison between treatments was performed on difference (delta) between measurements at time 0 and at baseline, and on slopes obtained by fitting measurements after unclamping with a linear regression model. RESULTS: At unclamping no difference in delta for plasma glutathione redox ratio (oxidised/total glutathione, %) was observed between treated and control groups with low ejection fraction (delta = 16 (SD 8.39) and 24 (7.0) redox ratio %, respectively). Baseline value recovery rate (redox ratio %/min) was significantly faster in treated v control patients (slope -0.912 (0.380) v -0.158 (0.200), P < 0.005, respectively). Cardiac index showed a trend to greater improvement in the treated group (slope 0.04 (0.03) v 0.003 (0.002) 1/min/m2/h, P < 0.02, treated v controls, respectively). In patients with normal ejection fraction, leucocyte depletion did not result in significant improvement v controls. CONCLUSIONS: Leucocyte depletion seems to provide benefit only in patients with left ventricular dysfunction.

Relation between left ventricular function and oxidative stress in patients undergoing bypass surgery.

OBJECTIVE: To determine whether preoperative left ventricular ejection fraction (LVEF) is related to the degree of myocardial oxidative stress during bypass surgery in man. DESIGN: Observational study. SETTING: Tertiary care centre. PATIENTS AND INTERVENTIONS: 31 patients (LVEF range was 20% to 68%) undergoing elective coronary bypass surgery with blood cardioplegic reperfusion were studied. Arterial and coronary sinus blood was collected before aortic cross clamping (T0) and at 0 (T1), 15 (T2), and 30 (T3) minutes after unclamping. Transmural left ventricular biopsies were also obtained from 15 patients at T0 and at T1. MAIN OUTCOME MEASURES: Glutathione and adenine nucleotides were measured in myocardial biopsies, while coronary sinus-artery differences for glutathione, nucleotides, and products of lipid peroxidation were calculated from blood specimens. Creatine kinase (myocardial band; CK-MB) was measured in plasma at four and 12 hours after operation. RESULTS: Myocardial glutathione and adenine nucleotides were correlated (p < 0.02) with preoperative LVEF both at T0 (r = 0.909 and 0.672) and T1 (r = 0.603 and 0.605). Oxidised glutathione released from the heart during reperfusion was inversely correlated with LVEF (r = -0.448, -0.466, and -0461 at T1, T2, and T3, p < 0.01), while reduced glutathione (r = 0.519 and 0.640 at T1 and T2) and glutathione redox ratio (r = 0.647, 0.714, 0.645, and 0.702 at T0, T1, T2, and T3) showed a direct correlation (p < 0.01). Lipid peroxidation at T1 was negatively related to LVEF (r = -0.492). CK-MB was also negatively related to LVEF (r = -0.440 at 4 h and -0.462 at 12 h). CONCLUSIONS: The capacity to counterbalance oxidative burst following ischaemia and reperfusion appears to be related to the functional ability of the heart.

Are diabetic metabolic compensation and CA19.9 really correlated?

Diabetes has been claimed to be a risk factor for pancreatic carcinoma, but it is probably a consequence of gland invasion from the neoplastic tissue. A link between diabetes and pancreatic carcinoma was suggested by means of biochemical markers of the diseases, namely glycated hemoglobin and CA19.9. Moreover, CA19.9 was proposed as a sensitive and useful marker of the severity of exocrine damage in diabetes, since the mucin decreased when metabolic compensation improved. We examined 64 diabetic patients (36 insulin dependent, 16 non insulin dependent, 12 treated with diet) by measuring CA19.9 using two different immunometric methods and glycemia and glycated hemoglobin. We observed that a correlation between CA19.9 and biochemical markers of metabolic compensation of diabetes was inexistent and no differences between insulin dependent and non insulin dependent patients were found. A high concentration of CA19.9 in a diabetic patient should be interpreted and evaluated in the same manner as for a non diabetic patient.

Measurement of erythrocyte acetylcholinesterase and plasma cholinesterase activity by a differential pH technique.

We describe a new electrochemical method for the determination of erythrocyte acetylcholinesterase activity (EC 3.1.1.7) and plasma cholinesterase (EC 3.1.1.8) activity, based on the measurements of pH variation due to release of acetic acid from acetylcholine. The major advantages of the differential pH procedure are simplicity, high reproducibility, no need for pre-treatment of samples, automatic correction of sample blanks, and speed and direct measurement of enzymatic reaction. The proposed methods are linear up to 7400 U/L at 30 degrees C and correlate well with the manual spectrophotometric method of Ellman for plasma cholinesterase and for washed erythrocytes. We adapted the same technique for the determination of erythrocyte cholinesterase using whole blood as sample and quinidine sulphate as inhibitor of pseudocholinesterase.

Surgical treatment of tracheostomal stenosis.

HYPOTHESIS: Stenosis of the tracheostome is a frequent complication following total laryngectomy; the problems created by tracheostomal stenosis are the result of reduced airflow and consequent turbulence. Many authors have studied etiological factors for the onset of stomal stenosis, and a number of procedures have been recommended for the surgical correction of such stenosis. STUDY DESIGN: A prospective analysis of 12 patients who underwent surgical correction of stomal stenosis is presented. METHODS: At the Institute of Clinical Otolaryngology we have recently defined a surgical technique for the correction of stomal stenosis that combines radial incisions, V-shaped flaps, and interposing flaps. This technique enables us to correct all the types of stenosis, and we have treated 12 patients to date. RESULTS: To date, the average follow-up has been 17 months (range, 3-36 mo), and the results are encouraging. Early stenosis of the tracheostoma reappeared in one patient, who had successful repeat surgery with the same technique. CONCLUSIONS: Early results suggest the routine use of this surgical technique in the treatment of stomal stenosis.

Neurotrophins and neurotrophin receptors mRNAs expression in pancreatic islets and insulinoma cell lines.

CONTEXT: It is worth noting that islets and betaTC6-F7 cells share a common pattern of expression of neurotrophins and neurotrophin receptors. Recently, several studies have hypothesized a role for nerve growth factor in pancreatic development and maturation, suggesting that nerve growth factor may be a survival factor for pancreatic beta-cells. OBJECTIVE: The aim of the present study was to investigate the pattern of expression of neurotrophins and their relative receptors both in rat pancreatic islets and in a wide panel of insulinoma cell lines. MAIN OUTCOME MEASURES: A semi-quantitative reverse-transcription polymerase chain reaction analysis was performed on ribonucleic acids extracted from these cells. RESULTS: Reverse transcription-polymerase chain reaction analysis demonstrates that brain-derived neurotrophic factor, as well as neurotrophins 3 and 4, are expressed both in islets and in all insulinoma cells, while nerve growth factor is expressed only in islets, betaTC6-F7 cells and, at a low level, in RIN 1046-38 cells. Receptors protein tyrosine kinase A and C are ubiquitously expressed both in islets and insulinoma cells. Tyrosine kinase B is absent in HIT-T15 cells. CONCLUSIONS: These data indicate that betaTC6-F7 cells are a suitable model for studying the role of neurotrophins in the survival of beta-cells.

Potent stimulation of murine B cells to proliferate and to secrete immunoglobulins by a lipoteichoic acid-like molecule produced by Clostridium botulinum C and D.

Bacterial products have served as important immunological tools to study lymphocyte activation. The lipopolysaccharides of the Gram-negative bacteria are well known to be potent activators of B lymphocytes. Several Gram-positive bacteria produce exotoxins that are superantigens for T cells. In the present study, we demonstrate that the Gram-positive bacteria Clostridium botulinum C and D produce a high molecular weight mitogen (Cb mitogen) that is a potent activator of murine B lymphocytes. The Cb mitogen was discovered as a consequence of our attempt to investigate a possible superantigen activity present in the botulinum exotoxins. We observed initially that mouse spleen cells were strongly stimulated to proliferate by culture supernatants of C. botulinum C and D. However, the characterization of the responding cell ruled out superantigen because only the B lymphocytes were stimulated to proliferate and to secrete immunoglobulins, and they did so independent of T cell help. In addition, the molecular characterization of the Cb mitogen demonstrated that the purified botulinum toxin was devoid of mitogenic activity. In contrast, the fractionation of the culture supernatant of C. botulinum C in an FPLC Superose 12 column indicated that the Cb mitogen was present in the void volume of the column (MW > or = 300 kDa) which had no toxigenic activity. However, the fractions containing molecules of 150 kDa were highly toxic for mice and had no mitogenic activity. The possibility that LPS was present as a contaminant in the Cb mitogen preparations was excluded because spleen cells from the LPS non-responder C3H/HeJ mice responded well to the Cb mitogen, and the antibiotic polymyxin B, which is an inhibitor of LPS, had no effect on the Cb-mitogen activity. However, an anti-lipoteichoic acid monoclonal antibody (3-1 mAb) inhibited to a great extent the proliferation of spleen cells induced by the Cb mitogen but had no effect on the LPS or concanavalin A stimulation of these cells. Moreover, the Cb mitogen was specifically adsorbed and eluted from a protein G Sepharose column to which the anti-lipoteichoic acid 3-1 mAb had been conjugated. These results support the view that lipoteichoic acid is a selective B cell mitogen.

Tumor-associated trypsin inhibitor as a possible marker in male infertility.

The concentrations of tumor-associated trypsin inhibitor (TATI) in the seminal plasma of infertile males was studied. The TATI levels in seminal plasma were not correlated with either sperm count or ejaculate volume. High levels were observed in some men with unexplained infertility and high or normal sperm counts, whereas normal levels were observed in males with antisperm antibodies. The concentrations in seminal plasma were stable in the same subjects. These results suggest that TATI may be an important marker of reproductive pathology in men.

Tumor-associated trypsin inhibitor in induced and acquired immunodeficiency. Studies on transplanted and HIV-infected patients.

A new tumor marker, tumor-associated trypsin inhibitor (TATI), was studied in 5 patients who received successful kidney or pancreas grafts and in 30 subjects with antibodies against human immunodeficiency virus. Serum TATI concentrations were very high during the four first days after transplantation. Thereafter the serum levels decreased when the peptide was eliminated through the kidney. Consequently, the urine values were very high. The TATI concentrations of HIV positive subjects were compared with serum levels of HIV antigen and antibody, by Western blotting and determination of peripheral T-lymphocyte subpopulations. The occurrence of high concentrations of TATI in some HIV positive subjects and especially in AIDS patients, suggests that TATI could be useful in exploring physiopathological aspects of severe immunodeficiencies even if TATI levels were not correlated with the commonly used markers of the immune system status. The increased levels of TATI in immunological disorders suggests its possible use in assessing the immune response against cancer.

Determination of serum cholesterol on the Reflotron (BBR) dry chemistry analyser. Assessment of accuracy.

This investigation is aimed at assessing the accuracy of S-cholesterol determination by means of the Reflotron, an analytical system based upon the dry chemistry approach. Two sets of sera, one of them including specimens with elevated concentrations of urea, creatinine, glucose, urate or bilirubin, were assayed in parallel with the Refloton system, with a class-A "reference" method (including alkaline hydrolysis and extraction) and with a routine liquid chemistry enzymatic method. Results for both sets of sera indicated good agreement of the tested system with the routine method, whilst a small proportional negative bias was observed in comparison with the "reference" method. The use of the reference method as a basis for comparison allows more conclusive statements about accuracy to be made.

[Nasopharyngeal carcinoma]. / Il carcinoma nasofaringeo.

The authors report the case of a 62-year-old woman who underwent a course of radiotherapy for an undifferentiated nasopharyngeal carcinoma. One year later the patient developed liver metastasis and underwent liver resection. The authors review the various aspect of this tumour, which is particularly frequent in the countries of South-East Asia but is exceptional in Europe and North America, focusing on the possibility of the operative management of the liver metastasis.

Low molecular weight, non-peptidic agonists of TrkA receptor with NGF-mimetic activity.

Exploitation of the biologic activity of neurotrophins is desirable for medical purposes, but their protein nature intrinsically bears adverse pharmacokinetic properties. Here, we report synthesis and biologic characterization of a novel class of low molecular weight, non-peptidic compounds with NGF (nerve growth factor)-mimetic properties. MT2, a representative compound, bound to Trk (tropomyosin kinase receptor)A chain on NGF-sensitive cells, as well as in cell-free assays, at nanomolar concentrations and induced TrkA autophosphorylation and receptor-mediated internalization. MT2 binding involved at least two amino-acid residues within TrkA molecule. Like NGF, MT2 increased phosphorylation of extracellular signal-regulated kinase1/2 and Akt proteins and production of MKP-1 phosphatase (dual specificity phosphatase 1), modulated p38 mitogen-activated protein kinase activation, sustained survival of serum-starved PC12 or RDG cells, and promoted their differentiation. However, the intensity of such responses was heterogenous, as the ability of maintaining survival was equally possessed by NGF and MT2, whereas the induction of differentiation was expressed at definitely lower levels by the mimetic. Analysis of TrkA autophosphorylation patterns induced by MT2 revealed a strong tyrosine (Tyr)490 and a limited Tyr785 and Tyr674/675 activation, findings coherent with the observed functional divarication. Consistently, in an NGF-deprived rat hippocampal neuronal model of Alzheimer Disease, MT2 could correct the biochemical abnormalities and sustain cell survival. Thus, NGF mimetics may reveal interesting investigational tools in neurobiology, as well as promising drug candidates.

Low molecular weight, non-peptidic agonists of TrkA receptor with NGF-mimetic activity.

Exploitation of the biologic activity of neurotrophins is desirable for medical purposes, but their protein nature intrinsically bears adverse pharmacokinetic properties. Here, we report synthesis and biologic characterization of a novel class of low molecular weight, non-peptidic compounds with NGF (nerve growth factor)-mimetic properties. MT2, a representative compound, bound to Trk (tropomyosin kinase receptor)A chain on NGF-sensitive cells, as well as in cell-free assays, at nanomolar concentrations and induced TrkA autophosphorylation and receptor-mediated internalization. MT2 binding involved at least two amino-acid residues within TrkA molecule. Like NGF, MT2 increased phosphorylation of extracellular signal-regulated kinase 1/2 and Akt proteins and production of MKP-1 phosphatase (dual specificity phosphatase 1), modulated p38 mitogen-activated protein kinase activation,sustained survival of serum-starved PC12 or RDG cells, and promoted their differentiation. However, the intensity of such responses was heterogenous, as the ability of maintaining survival was equally possessed by NGF and MT2, whereas the induction of differentiation was expressed at definitely lower levels by the mimetic. Analysis of TrkA autophosphorylation patterns induced by MT2 revealed a strong tyrosine (Tyr)490 and a limited Tyr785 and Tyr674/675 activation, findings coherent with the observed functional divarication. Consistently, in an NGF-deprived rat hippocampal neuronal model of Alzheimer Disease, MT2 could correct the biochemical abnormalities and sustain cell survival. Thus, NGF mimetics may reveal interesting investigational tools in neurobiology, as well as promising drug candidates.