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.

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.

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.

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.

[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.

NGF-withdrawal induces apoptosis in pancreatic beta cells in vitro.

AIMS/HYPOTHESIS: Using primary cultures of human pancreatic islets, purified human pancreatic beta cells and the mouse beta TC6-F7 cell line, we analysed the expression of nerve growth factor, (NGF/NGF) receptors in beta cells. To investigate whether NGF could sub-serve an autocrine antiapoptotic role in beta cells, we studied the effects of NGF withdrawal using a neutralizing monoclonal anti-NGF antibody. METHODS: The expression of NGF and NGF receptors (gp140(Trk-A) and p75(NTR)) were analysed by RT-PCR and immunofluorescence. Pulse-chase experiments and beta cell/PC12 co-cultures were used to investigate NGF production and secretion from beta cells. Possible apoptosis induced by NGF withdrawal was monitored by phosphatidylserine translocation, nucleosomal formation, DNA laddering and FACS analysis. Involvement of transcription/translation mechanisms were investigated as well as the gp140(Trk-A) required. Finally, signal transduction pathways typically involved in apoptotic mechanisms were analysed by western blot analysis. RESULTS: We show that NGF and both NGF receptors, gp140(Trk-A) and p75(NTR) are expressed in beta cells where NGF is produced and secreted in a biologically active form. NGF-withdrawal induces beta-cell transcription/translation independent apoptosis but mediated by gp140(Trk-A). Analysis of signal transduction pathways revealed that NGF withdrawal inhibits the PI3-K, protein kinase B (AKT), Bad survival pathway and activates c-Jun kinase (JNK) whereas ERKs and p38 mitogen-activated protein kinase (MAPK) are not affected. Moreover, Bcl-XL, but not Bcl-2 protein expression are reduced. CONCLUSION/INTERPRETATION: We suggest that the integrity of the NGF/NGF receptor system and NGF bioavailability participate in controlling beta-cell survival in culture which represents a key issue for improving possibilities for transplantations in the treatment of diabetes.

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.

Nerve growth factor inhibits apoptosis in memory B lymphocytes via inactivation of p38 MAPK, prevention of Bcl-2 phosphorylation, and cytochrome c release.

Survival of memory B lymphocytes is tightly linked to the integrity of the Bcl-2 protein and is regulated by a nerve growth factor (NGF) autocrine circuit. In factor-starved memory B cells, the addition of exogenous NGF promptly induced p38 mitogen-activated protein kinase (MAPK), but not c-Jun N-terminal kinase (JNK), dephosphorylation. Conversely, withdrawal of endogenous NGF was followed by p38 MAPK activation and translocation onto mitochondria, whereby it combined with and phosphorylated Bcl-2, as assessed by co-immunoprecipitation and kinase assays in vivo and in vitro. Mitochondria isolated from human memory B cells, then exposed to recombinant p38 MAPK, released cytochrome c, as did mitochondria from Bcl-2-negative MDCK cells loaded with recombinant Bcl-2. Apoptosis induced by NGF neutralization could be blocked by the specific p38 MAPK inhibitor SB203580 or by Bcl-2 mutations in Ser-87 or Thr-56. These data demonstrate that the molecular mechanisms underlying the survival factor function of NGF critically rely upon the continuous inactivation of p38 MAPK, a Bcl-2-modifying enzyme.

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.

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.

Multicentre evaluation of the Boehringer Mannheim/Hitachi 917 analysis system.

The new selective access analysis system BM/Hitachi 917 was evaluated in an international multicentre study, mainly according to the ECCLS protocol for the evaluation of analysers in clinical chemistry. Forty-three different analytes, covering 56 different methods--enzymes, substrates, electrolytes, specific proteins, drugs and urine applications--were tested in seven European clinical chemistry laboratories. Additionally, the practicability of the BM/ Hitachi 917 was tested according to a standardized questionnaire. Within-run CVs (median of 3 days) for enzymes, substrates and electrolytes were <2% except for creatine-kinase MB isoform and lipase at low concentration. For proteins, drugs and urine analytes the within-run CVs were < 4% except for digoxin and albumin in urine. Between-day median CVs were generally < 3% for enzymes, substrates and electrolytes, and < 6% for proteins, drugs and urine analytes, except for lipase, creatine kinase and MB isoform, D-dimer, glycosylated haemoglobin, rheumatoid factors, digoxin, digitoxin, theophylline and albumin in urine in some materials. Linearity was found according to the test specifications or better and there were no relevant effects seen in drift and carry-over testing. The interference results clearly show that also for the BM/Hitachi 917 interference exists sometimes, as could be expected because of the chemistries applied. It is a situation that can be found in equivalent analysers as well. The accuracy is acceptable regarding a 95-105% recovery in standard reference material, with the exception of the creatinine Jaffé method. Most of the 160 method comparisons showed acceptable agreement according to our criteria: enzymes, substrates, urine analytes deviation of slope +/- 5%, electrolytes +/- 3%, and proteins and drugs +/- 10%. The assessment of practicability for 14 groups of attributes resulted in a grading of one-three scores better for the BM/Hitachi 917 than the present laboratory situation. In conclusion, the results of the study showed good analytical performance and confirmed the usefulness of the system as a consolidated workstation in medium-sized to large clinical chemistry laboratories.

[The Verona Medical Interview Classification System/Patient (VR-MICS/P): the tool and its reliability]. / Il Verona-Medical Interview Classification System/Patient (VR-MICS/P). Presentazione dello strumento e studio di attendibilità.

OBJECTIVE: To assess the reliability and to describe the categories and the procedure to apply the VR-MICS/P (Verona-Medical Interview Classification System/Patient). SETTING: The interviews used for the reliability study were audiotaped. Five general practitioners (GPs) working in two general practices in South-Verona recorded their consultations. SAMPLE: 50 interviews selected randomly from 120, 10 for each GP. The selection criterion for the participating patients was a GHQ-12 score of 3 and the consultation for a new illness episode. MAIN OUTCOME MEASURES: The VR-MICS/P classifies patients' verbal behaviours into 21 categories, 15 of them are defined by form (cue or statement) and content. PROCEDURE: Two trained raters classified 50 interviews. Before applying the classification system each interview is divided into units which are numbered to define doctor's and patient's sequence of speech. RESULTS: The reliability of VR-MICS/P was satisfactory (Kappa 0.85). Similarity Index (Dice, 1945) for categories varied between 0.71 and 0.94. Reliability for form and content classification was satisfactory too (Similarity Index between 0.81 and 0.89 and between 0.84 and 0.94, respectively). CONCLUSIONS: The VR-MICS/P is a reliable measure for describing patients' verbal behaviours during medical interviews. It can be used together with the VR-MICS/D (Verona-Medical Interview Classification System/Doctor; Saltini et al., 1998) to describe the medical interview, the quality of doctor-patient interview and can be used as a measure of patient centredness.

Interferon-alpha-induced inhibition of B16 melanoma cell proliferation: interference with the bFGF autocrine growth circuit.

The molecular mechanisms underlying the growth inhibition induced by interferon-alpha (IFN-alpha) in B16 murine melanoma cells were investigated. IFN-alpha did not induce cell apoptosis, but strongly interfered with the synthesis of basic fibroblast growth factor (bFGF), which acts as an autocrine growth factor in this system. Inhibition of bFGF synthesis was observed at the same concentrations (50-500 pM, 10-100 U/ml) of IFN-alpha able to induce growth arrest of B16 melanoma cells. Although the synthesis of acidic (a)FGF was only slightly affected by IFN-alpha, the cytokine induced release of an aFGF-related low-molecular-weight peptide, which was able to interfere with bFGF binding to surface receptors. Thus, the molecular mechanisms of IFN-alpha activity on melanoma cells include a specific modulation of the bFGF autocrine circuit.

Multicentre evaluation of KONE Optima analysis system.

Optima from KONE Instruments Corporation is a new selective laboratory analyzer for turbidimetric, colorimetric and ion selective electrode measurements. Overall analytical performance of Optima and reagents provided by KONE was evaluated according to ECCLS guidelines, in a multicentre study involving four different laboratories, including substrates (cholesterol, high-density lipoprotein-cholesterol, creatinine), specific proteins (transferrin, IgG), enzyme activities (gamma-glutamyltransferase, alanine aminotransferase) and electrolytes (sodium, potassium, chloride). The results obtained attest good precision of the system for all the analytes tested: the range of within-run CV is 0.5 %-4.3 %, and range of between-day CV % is 0.8 %-7.9 % (median of four laboratories). Except for total cholesterol (5 % overestimation compared to the reference method) accuracy of measurement is adequate. Creatinine and uric acid assays were subjects of interference (defined as deviation > 10 % from target value) by bilirubin and haemoglobin respectively.

Differential effects of selective beta-adrenergic blockade on insulin sensitivity and release in control subjects and in patients with angina and normal coronary arteries (syndrome X).

AIMS: Betablockers are very effective in patients with angina and angiographically smooth coronary arteries (syndrome X), but may exacerbate the state of insulin resistance that is known to be present in such patients. The aim of the study was to evaluate the effects of short-term treatment with atenolol on carbohydrate metabolism in syndrome X patients, as compared to normal subjects. METHODS AND RESULTS: Seventeen patients (15 females, 55 +/- 8 years, BMI 23.4 +/- 2.7 kg/m2) and 11 controls (5 females, 50 +/- 7 years, BMI 23.1 +/- 2.0 kg/m2) were studied twice by an intravenous glucose tolerance test (IVGTT, 0.5 g/kg) after ten days of both placebo and atenolol (100 mg o.d.), given in random order. Metabolic indices measuring glucose effectiveness and insulin sensitivity were derived from minimal model analysis of the glucose and insulin profiles measured during the IVGTT. Indices of first- and second-phase insulin release were also calculated from the IVGTT insulin response. Atenolol had different metabolic effects on normal subjects and syndrome X patients. Despite the fact that the drug was found to be effective in relieving symptoms of chest pain, it induced a significant (p < 0.05) worsening of insulin resistance in syndrome X patients. No such effect was observed in control subjects. On the other hand, atenolol produced a marked reduction (40%, p < 0.05) of first-phase insulin release in control subjects, but no significant change of the same index in syndrome X patients. CONCLUSION: These results show that betablockers are very effective for controlling symptoms and improving quality of life in syndrome X patients. However, they appear to further impair the ability to dispose glucose. Long-term studies on the net effects of beta-blockade administration for the treatment of such patients are warranted.

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.

Automation of human sperm cell analysis by flow cytometry.

Semen sample analysis is routinely performed by microscopical evaluation and manual techniques by laboratory operators; the analysis is affected by a wide imprecision related to variability among observers, influencing its clinical validity. Our aim was to automate sperm analysis with the use of flow cytometry for evaluation of cell counts and typing and with the use of a new membrane-permeant nucleic acid stain for evaluation of sperm viability. Statistical analysis of the comparison between manual and automated methods for sperm counts was performed by the Bland and Altman method; the mean difference was 0.243 x 10(6) sperms/ mL. The precision of the flow cytometric analysis was evaluated with whole sperm; the between-run CV was 7.5% and the within-run CV was 2.5%. Data observed suggest that flow cytometric sperm analysis, with high precision and accuracy and low costs, can be proposed for routine use in clinical laboratories.