Impaired regulation of the TNF-alpha converting enzyme/tissue inhibitor of metalloproteinase 3 proteolytic system in skeletal muscle of obese type 2 diabetic patients: a new mechanism of insulin resistance in humans.

AIMS/HYPOTHESIS: TNF-alpha levels are increased in obesity and type 2 diabetes. The regulation of TNF-alpha converting enzyme (TACE) and its inhibitor, tissue inhibitor of metalloproteinase 3 (TIMP3), in human type 2 diabetes is unknown. METHODS: We examined TACE/TIMP3 regulation: (1) in lean and obese normal glucose tolerant (NGT) individuals and in type 2 diabetes patients; (2) following 6 h of lipid/saline infusion in NGT individuals; and (3) in cultured human myotubes from lean NGT individuals incubated with palmitate. Insulin sensitivity was assessed by a euglycaemic clamp and TACE/TIMP3 was evaluated by confocal microscopy, RT-PCR, western blotting and an in vitro activity assay. Circulating TNF-alpha, TNF-alpha-receptor 1 (TNFR1), TNF-alpha-receptor 2 (TNFR2), IL-6 receptor (IL-6R), vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) levels were evaluated. RESULTS: TIMP3 levels were reduced and TACE enzymatic activity was increased in type 2 diabetes skeletal muscle. TACE expression, and TACE, TNF-alpha, TNFR1 and IL-6R levels were increased in type 2 diabetes, and positively correlated with insulin resistance. A 6 h lipid infusion into NGT individuals decreased insulin-stimulated glucose metabolism by 25% with increased TACE, decreased expression of the gene encoding TIMP3 and increased IL-6R release. Palmitate induced a dramatic reduction of TIMP3 and increased the TACE/TIMP3 ratio in cultured myotubes. CONCLUSIONS/INTERPRETATION: TACE activity was increased in skeletal muscle of obese type 2 diabetes patients and in lipid-induced insulin resistance. We propose that dysregulation of membrane proteolysis by TACE/TIMP3 of TNF-alpha and IL-6R is an important factor for the development of skeletal muscle insulin resistance in obese type 2 diabetes patients by a novel autocrine/paracrine mechanism.

Microtubule nucleation and release from the neuronal centrosome.

We have proposed that microtubules (MTs) destined for axons and dendrites are nucleated at the centrosome within the cell body of the neuron, and are then released for translocation into these neurites (Baas, P. W., and H. C. Joshi. 1992. J. Cell Biol. 119:171-178). In the present study, we have tested the capacity of the neuronal centrosome to act as a generator of MTs for relocation into other regions of the neuron. In cultured sympathetic neurons undergoing active axonal outgrowth, MTs are present throughout the cell body including the region around the centrosome, but very few (< 10) are directly attached to the centrosome. These results indicate either that the neuronal centrosome is relatively inactive with regard to MT nucleation, or that most of the MTs nucleated at the centrosome are rapidly released. Treatment for 6 h with 10 micrograms/ml nocodazole results in the depolymerization of greater than 97% of the MT polymer in the cell body. Within 5 min after removal of the drug, hundreds of MTs have assembled in the region of the centrosome, and most of these MTs are clearly attached to the centrosome. A portion of the MTs are not attached to the centrosome, but are aligned side-by-side with the attached MTs, suggesting that the unattached MTs were released from the centrosome after nucleation. In addition, unattached MTs are present in the cell body at decreasing levels with increasing distance from the centrosome. By 30 min, the MT array of the cell body is indistinguishable from that of controls. The number of MTs attached to the centrosome is once again diminished to fewer than 10, suggesting that the hundreds of MTs nucleated from the centrosome after 5 min were subsequently released and translocated away from the centrosome. These results indicate that the neuronal centrosome is a highly potent MT-nucleating structure, and provide strong indirect evidence that MTs nucleated from the centrosome are released for translocation into other regions of the neuron.

Inhibition of microtubule nucleation at the neuronal centrosome compromises axon growth.

We tested the dependence of axon growth on microtubule (MT) nucleation from the neuronal centrosome. Nocodazole diminished MTs in freshly plated neurons by > 99%. Within 5 min of drug removal, MTs reassembled at the centrosome. This response was inhibited in cells microinjected with gamma-tubulin antibody. Within 2 hr of drug removal, uninjected neurons grew > 500 microns of axon. In roughly half of the antibody-injected cells, axon growth was abolished and MT levels were reduced by approximately 87% compared with uninjected cells. In the other antibody-injected cells, axon growth was compromised but not abolished, and MT levels were reduced by approximately 38%. Thus inhibition of MT nucleation at the centrosome hindered MT reassembly, and depending on the severity of this response, axon growth was either compromised or abolished.

Analysis of benzo[a]pyrene partitioning and cellular homeostasis in a rat liver cell line.

The uptake and subcellular partitioning of benzo[a]pyrene (BaP) were examined in a rat-liver cell line (Clone 9) using confocal and multiphoton microscopy. Following a 16-h treatment, intracellular accumulation of BaP increased with increasing concentration, and cytoplasmic BaP fluorescence reached saturation at 10 microM. Analysis of the kinetics of BaP uptake at this concentration indicated that BaP is rapidly partitioned into all cytoplasmic membranes within several min, although saturation was not reached until 4 h. Based upon the rapid uptake of BaP into membranes, the chronology of changes in gap junction-mediated intercellular communication (GJIC), plasma membrane potential (PMP), and steady state levels of intracellular Ca2+ in relation to the time-course for induction of microsomal ethoxyresorufin-0-deethylase (EROD) activity were examined. EROD activity in Clone 9 cells treated for 16 h increased with increasing concentrations of BaP and reached the highest levels at 40 microM BaP. In addition, kinetic analysis of EROD activity in Clone 9 cells treated with 10 microM BaP indicated that significant induction of EROD activity was not detected before 3 h, and it reached maximal levels by 16 h of treatment at this concentration. Both GJIC and PMP were directly affected by the partitioning of BaP into cellular membranes. The most sensitive index of BaP-induced changes in membrane function was GJIC which revealed a 25% suppression in cells exposed to 0.4 microM BaP for 16 h. Kinetic analysis revealed that suppression of GJIC occurred within 15 min of exposure of cells to 10 microM BaP, whereas significant suppression of PMP was not detected prior to 30-min exposure at this concentration. Elevation of basal Ca2+ level was also detected simultaneously with PMP at this dose. These data suggest that early changes in cellular membrane functions occur prior to detectable induction of EROD activity, although basal metabolic activation of BaP may contribute to these changes.

Short-term culture of peritoneum explants confirms attachment of endometrium to intact peritoneal mesothelium.

OBJECTIVE: To evaluate the initial adhesion of endometrium to the peritoneum. DESIGN: Descriptive study using light and confocal laser-scanning microscopy, immunohistochemistry, and transmission electron microscopy. SETTING: University-based laboratory. PATIENT(S): Women without endometriosis undergoing surgery for benign conditions. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Explants of peritoneum (n = 20), prepared from four patients, were cultured for 1 hour with mechanically dispersed proliferative or secretory endometrium. Peritoneum was cultured with endometrium from the same patient. Specimens were fixed and serially sectioned for hematoxylin and eosin stain, immunohistochemistry using an anti-cytokeratin monoclonal antibody, and transmission electron microscopy. RESULT(S): In 17 of 20 explants, endometrium was adherent to intact mesothelium. There was no evidence of transmesothelial invasion at any sites of attachment. Although in most cases endometrium was adherent to mesothelium via endometrial stroma, there were many sites of endometrial epithelium-mesothelium attachment. Confocal laser scanning microscopy demonstrated an intact monolayer of cytokeratin-positive cells below the sites of endometrial implantation. Transmission electron microscopy demonstrated intact, viable, mesothelial cells below sites of attachment. CONCLUSION(S): This study demonstrates that endometrium rapidly adheres to intact peritoneal mesothelium. In addition, this study demonstrates that endometrial epithelial cells, as well as stroma, can attach to mesothelium. Further studies are needed that characterize the mechanism of endometrial-mesothelial cell adhesion.

Quantitative imaging of protein-protein interactions by multiphoton fluorescence lifetime imaging microscopy using a streak camera.

Fluorescence lifetime imaging microscopy (FLIM) using multiphoton excitation techniques is now finding an important place in quantitative imaging of protein-protein interactions and intracellular physiology. Recent developments in multiphoton FLIM methods are reviewed and a novel multiphoton FLIM system using a streak camera is described. An example of a typical application of the system is provided in which the fluorescence resonance energy transfer between a donor-acceptor pair of fluorescent proteins within a cellular specimen is measured.

Composition of the extracellular matrix of the peritoneum.

OBJECTIVE: To localize the extracellular matrix proteins collagen I, collagen IV, fibronectin, and laminin in the peritoneal membrane. STUDY DESIGN: Peritoneal biopsies (n = 13) from the anterior abdominal wall and the uterine serosa (n = 3) were incubated with antibodies to collagen IV, laminin, collagen I, and fibronectin. Specimens were examined using light and confocal laser scanning microscopy. RESULTS: All of the extracellular matrix (ECM) proteins were present immediately under the mesothelium. Collagen (Col) IV and laminin (LM) were seen in the smooth muscle of microvascular structures, in the subendothelial basement membrane, and were present in a fascicular pattern in the peritoneal stroma. Collagen I was distributed diffusely in the peritoneal stroma. Fibronectin was also present in the subendothelial basement membrane. CONCLUSIONS: The resolution of the confocal microscope allowed for localization of extracellular matrix proteins in relation to the mesothelium. The presence of collagen IV, laminin, collagen I, and fibronectin under the mesothelium suggests that cells invading the peritoneum must have the ability to degrade and remodel this matrix.

Vascular injuries in ergotamine abuse: a case report.

The case reported here concerns a 40-year-old woman who has suffered from severe migraine without aura since she was 23. The patient has been taking 1-3 suppositories of Virdex (2 mg of ergotamine tartrate, 250 mg of aminophenazone, caffeine) every day for the past three years. In addition to headache, the onset of short, sporadic cramps in the limbs together with paresthesia, hyposthenia, hypothermia and skin pallor with a slight increase in diastolic pressure, made hospital treatment necessary. Instrumental investigation through a stress test on a moving carpet, doppler, echo-doppler and digitalized angiography of the arterial vessels of the lower limbs shows the presence of bilateral impairment attributable to the chronic intake of ergotamine.

Sumatriptan overuse in episodic cluster headache: lack of adverse events, rebound syndromes, drug dependence and tachyphylaxis.

This observational study was designed to examine the pattern of sumatriptan use in patients with cluster headache using more than the recommended daily dose of subcutaneously injected (s.c.) sumatriptan. Thirteen patients suffering from episodic cluster headache were asked to record the characteristics of their attacks and drug intake for 1 year. All reported a high daily frequency of attacks (more than 3 per day) and the related overuse of s.c. sumatriptan. The results show that the overall incidence of adverse events among patients receiving sumatriptan injections for the treatment of cluster headache is low. The extended administration of this drug in episodic cluster headache did not result in tolerance problems or tachyphylaxis. Only 4 patients experienced minor adverse events and recovered more slowly than the others. They suffered from migraine without aura and cluster headache, and showed a family history of migraine. Even though they must be viewed with caution, due to the observational nature of the study and the low number of patients included, these results suggest that the profile of sumatriptan may differ in cluster headache compared with migraine.

Patient education and migraine: a pilot study.

Our study examines the effectiveness of an educational approach to migraine patients. A course in migraine education was set up for 30 patients suffering from this disease; meetings were structured taking into consideration specific educational aims, with parameters evaluated before the course, at the end of the course and at a 3-month follow-up. The results, particularly the increase in the migraineurs' knowledge of their disease and the decrease in the use of symptomatic drugs, suggest the effectiveness of the course. Furthermore, our study suggests that there is a need to build educational processes into therapeutic protocols, as they enable patients to manage their chronic diseases more correctly.

[Symptomatic headaches in internal medicine: the classification, physiopathological and diagnostic aspects]. / Le cefalee sintomatiche in medicina interna: aspetti classificativi, fisiopatologici e diagnostici.

Symptomatic or secondary headache occurs when pain itself is a symptom of disease. It is well known that within the general population the percent frequency of secondary headache is lower than that of primary headache. Moreover, some forms do not seem to evidence particular clinical, diagnostic or physiopathological importance. The Authors investigate here a number of clinical aspects of secondary headache, in particular headache in vascular disease (stroke, hypertension, Horton's arteritis). Particular attention is paid to headache in brain neoplasia due to the interest brought about by the diagnostic problems of this disease. Lastly postural headache and its prevalence in the general population is examined. Various physiopathological aspects of this form (stress, psychosocial events) are evaluated.

[Raynaud's phenomenon and calcium blocking agents. A preliminary open study with flunarizine]. / Fenomeno di Raynaud e farmaci calcio-antagonisti. Studio preliminare in aperto con flunarizina.

Raynaud's phenomenon (Raynaud's disease), an accessual vascular acrosyndrome characterised by an important constriction of distal arterioles, has still no specific pharmacological therapy. In the last years, the use of calcium-entry-blockers (nifedipine, diltiazem, verapamil, nicardipine), drugs able to control the contractility of the vessels, showed some positive results. Considering this data, we appraised the efficacy of flunarizine, another calcium-entry-blocker, in a preliminary study of 28 patients (23 females, 5 males, aged between 15 and 48 years) suffering from Raynaud's disease. Apart from a statistically insignificant improvement of subjective symptoms (i.e. acroparesthesias, cold extremities) flunarizine (10 mg/day for 1 month) did not have positive results. Finally, this drug caused some side-effects: drowsiness, increase of weight and appetite, but without a real necessity for withdrawal of therapy.

[Brain aging and calcium antagonist drugs. A preliminary open study with nimodipine]. / Invecchiamento cerebrale e farmaci calcio-antagonisti. Studio preliminare in aperto con la nimodipina.

Forty patients aged 65-80 with minor-medium signs of brain aging were treated for 6 months with daily 90 mg doses of nimodipine. Treatment was well tolerated in that the minor side effects reported by a few of the patients never required withdrawal of the drug. Treatment proved useful, as shown by a variety of mental tests, in 69.5% of cases. In 20% conditions remained unchanged and in 9.5% further deterioration was observed. The authors conclude that nimodipine may be considered a useful therapeutic resource for phenomena of brain aging.

[Evaluation of the efficacy of oral sumatriptan in the management of migraine attacks. Clinical results]. / Valutazione dell'efficacia di sumatriptan nella terapia orale dell'attacco di emicrania. Risultati clinic.

This italian multicentre, double blind, parallel groups study compared the efficacy, safety and tolerability of oral sumatriptan, given as new film-coated tablet, with placebo in the acute treatment of migraine. 88 Patients received placebo and 162 patients received sumatriptan 100 mg (plus an optional dose 2 h later if the headache persisted plus a further optional dose for recurrence within 24 h). Sumatriptan was significantly more effective than placebo at releiving headache at 2 h (51% versus 31%, P = 0.003) and 4 h (71% versus 35%, P < 0.001). Fewer sumatriptan-treated patients required a second dose compared with placebo-treated patients (49% versus 74%, P < 0.001). Sumatriptan was more effective than placebo at relieving nausea, vomiting and photophobia/phonophobia. Few patients were evaluable for treatment of headache recurrence, and statistical analysis was not possible. More sumatriptan-treated patients than placebo-treated patients reported adverse events (29% versus 16%) but the difference was not statistically significant. More of these events were mild to moderate in severity, of short duration and resolved without treatment. Sumatriptan had no clinically significant effect on blood pressure, heart rate, electrocardiogram or laboratory test results. It is concluded that oral sumatriptan 100 mg, given as a film-coated, tablet, provides an effective and well-tolerated acute treatment for migraine.