Nitric oxide inhibition of Drp1-mediated mitochondrial fission is critical for myogenic differentiation.

During myogenic differentiation the short mitochondria of myoblasts change into the extensively elongated network observed in myotubes. The functional relevance and the molecular mechanisms driving the formation of this mitochondrial network are unknown. We now show that mitochondrial elongation is required for myogenesis to occur and that this event depends on the cellular generation of nitric oxide (NO). Inhibition of NO synthesis in myogenic precursor cells leads to inhibition of mitochondrial elongation and of myogenic differentiation. This is due to the enhanced activity, translocation and docking of the pro-fission GTPase dynamin-related protein-1 (Drp1) to mitochondria, leading also to a latent mitochondrial dysfunction that increased sensitivity to apoptotic stimuli. These effects of NO inhibition were not observed in myogenic precursor cells containing a dominant-negative form of Drp1. Both NO-dependent repression of Drp1 action and maintenance of mitochondrial integrity and function were mediated through the soluble guanylate cyclase. These data uncover a novel level of regulation of differentiation linking mitochondrial morphology and function to myogenic differentiation.

A novel mutation in KCNQ2 associated with BFNC, drug resistant epilepsy, and mental retardation.

BACKGROUND: Benign familial neonatal convulsion (BFNC) is a rare autosomal dominant disorder caused by mutations in two genes, KCNQ2 and KCNQ3, encoding for potassium channel subunits underlying the M-current. This current limits neuronal hyperexcitability by causing spike-frequency adaptation. METHODS: The authors describe a BFNC family with four affected members: two of them exhibit BFNC only while the other two, in addition to BFNC, present either with a severe epileptic encephalopathy or with focal seizures and mental retardation. RESULTS: All affected members of this family carry a novel missense mutation in the KCNQ2 gene (K526N), disrupting the tri-dimensional conformation of a C-terminal region of the channel subunit involved in accessory protein binding. When heterologously expressed in CHO cells, potassium channels containing mutant subunits in homomeric or heteromeric configuration with wild-type KCNQ2 and KCNQ3 subunits exhibit an altered voltage-dependence of activation, without changes in intracellular trafficking and plasma membrane expression. CONCLUSION: The KCNQ2 K526N mutation may affect M-channel function by disrupting the complex biochemical signaling involving KCNQ2 C-terminus. Genetic rather than acquired factors may be involved in the pathophysiology of the phenotypic variability of the neurologic symptoms associated with BFNC in the described family.

Autoantibodies to amphiphysin I and amphiphysin II in a patient with sensory-motor neuropathy.

A proportion of patients with peripheral neuropathies has circulating autoantibodies directed against neural antigens. In some cases, autoantibodies may play a pathogenic role. We studied a patient with a progressive sensory-motor axonal neuropathy of unknown etiology, looking for circulating autoantibodies against neural antigens and we showed that the patient's serum contained anti-amphiphysin I (AMP I) and amphiphysin II (AMP II) autoantibodies. A sural nerve biopsy revealed an axonal neuropathy. Indirect immunofluorescence experiments with the patient's serum showed a staining of rat axons due to alpha-AMP I autoantibodies and a specific labelling of cytoplasm and Schmidt-Lanterman incisures of Schwann cells due to alpha-AMP II autoantibodies. In conclusion we identified a patient affected by a sensory-motor neuropathy with autoantibodies against both AMP I and AMP II.

The role of somatostatin and octreotide in bowel obstruction: pre-clinical and clinical results.

Malignant bowel obstruction is a common complication in patients with advanced abdominal or pelvic cancer. Whereas surgery should be considered in all cases of malignant bowel obstruction, many advanced and terminal cancer patients are considered unfit for surgery. In such patients with a short life expectancy, gastrointestinal symptoms such as nausea, vomiting, continuous and/or colicky pain, can be controlled by using a pharmacologic approach made up of analgesics, antiemetics and antisecretory drugs, without the use of a venting nasogastric tube. Among the antisecretory drugs, octreotide has been shown to reduce nausea and vomiting in bowel-obstructed patients owing to a reduction of gastrointestinal secretions, thus allowing in most patients removal of the nasogastric tube and the associated distress. Preclinical and clinical studies that demonstrated the role of somatostatin and octreotide in bowel obstruction are reviewed.

The role of disodium pamidronate in the management of bone pain due to malignancy.

A number of controlled studies have recently demonstrated the role of disodium pamidronate in the prevention of skeletal complications in patients with metastatic bone disease due to breast cancer and multiple myeloma. They have also shown that it relieves pain and is well tolerated. The aim of this open prospective study was to evaluate the acceptability of a new schedule of pamidronate infusion and to assess pain, analgesic consumption and the Karnofsky Performance Status (KPS) in patients with metastatic bone pain treated with pamidronate in association or not with chemotherapy, radiotherapy, and hormone therapy. Patients with different types of cancer and at least one painful bone metastasis were treated with two cycles of 60 mg intravenous (iv) pamidronate weekly for three consecutive doses, with a 3-week interval between the two cycles (six infusions over 7 weeks), followed by one infusion every 3 weeks for a total of 24 infusions. Two hundred patients were enrolled in the study, of whom 94 received at least the first six infusions; 25 patients received all 24 infusions. Pamidronate was well tolerated in the majority of the patients both during the first six infusions and during the whole study period. In the patients under study, pain intensity decreased compared with T0 after the first two infusions (second week of treatment). The mean equivalent daily dose of oral morphine required ranged from 21.5 to 41.5 mg/day and was low and stable during the study. For the patients who remained in the study, the KPS remained around 70 during the whole treatment period and intrasubject analysis showed a substantial stability of the KPS within each subject. A first fracture occurred within 321 days in 25% of the whole population under study. Pamidronate represents a further valid therapy to add to an already consolidated list of therapies such as radiotherapy, chemotherapy, hormone therapy and orthopaedic intervention in the pain management of patients with bone metastases. Future studies are necessary to evaluate the role of pamidronate and the appropriate schedule in patients with advanced or terminal cancer who are no longer being treated with oncological therapies.

Pain experienced by patients hospitalized at the National Cancer Institute of Milan: research project "towards a pain-free hospital".

According to the data of the literature, the prevalence of pain in cancer patients at various stages of the disease and the settings of care range from 38 to 51%, with an increase of up to 74% in the advanced and terminal stages. Despite published World Health Organization (WHO) guidelines for pain management, 42 to 51% of cancer patients receive inadequate analgesia and 30% receive no analgesics at all. A 3-year Research Project "Towards a Pain-free Hospital", which began one year ago, is ongoing at the National Cancer Institute of Milan. The research is organized in three subsequent steps. In the 1st one, a series of patient- and staff-oriented evaluation tools are used to assess the level of appropriateness of pain communication, assessment, management and control of the in-patients. The 2nd step will implement a number of continuing educational interventions aimed at improving patient awareness and staff knowledge of the appropriate pain assessment and management in order to respond to the patient's pain problem. In the 3rd step, all the assessment tools used in step one will be applied again to establish the prevalence of pain, the causes and intensity and patient satisfaction with pain management and to evaluate the impact of the interventions performed during the 2nd step regarding the overall ability of our hospital to tackle pain emergency in the hospitalized cancer population. The results relative to the 1st step are herein reported, in particular as regards the study on prevalence, causes, severity of pain, the interference of pain with sleep, mood and concentration, the use of pain medications and the relief obtained, the structural validity and internal consistency of the assessment tool used. A total of 258 patients hospitalized for at least 24 h were interviewed by 9 physicians using a brief structured questionnaire prepared ad hoc: 51.5% of the patients presented pain during the previous 24 h caused by surgery (49.6%) or by the tumor mass itself (29.3%). Out of the 133 patients with pain, a high degree (much or very much) of pain at rest was present in 27.1% and pain on movement in 30.8%; 31.6% did not take any analgesic treatment, and 14.3% of the latter reported a high degree of pain at rest and 21.4% on movement. Pain interfered with sleep from much to very much in 28.8% and with irritability and nervousness in 15.9% of the patients. In the 91 patients taking analgesics, 57.2% reported a high degree of pain relief. A high degree of pain and interference, however, was associated with low relief levels. The assessment tool used was shown to have a good structural validity and internal consistency (Chrombach alpha index of interference scale = 0.73). Although the Milan Cancer Institute has the longest tradition in Italy of pain assessment by means of validated tools and pain management according to the WHO guidelines and educational efforts in this field, the results of the study clearly show that it is necessary to persevere with continuing educational and informative programs in order to reduce the frequency and severity of pain and thus improve the quality of life of in-patients.

Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration.

Tight junctions are the most apical components of endothelial and epithelial intercellular cleft. In the endothelium these structures play an important role in the control of paracellular permeability to circulating cells and solutes. The only known integral membrane protein localized at sites of membrane-membrane interaction of tight junctions is occludin, which is linked inside the cells to a complex network of cytoskeletal and signaling proteins. We report here the identification of a novel protein (junctional adhesion molecule [JAM]) that is selectively concentrated at intercellular junctions of endothelial and epithelial cells of different origins. Confocal and immunoelectron microscopy shows that JAM codistributes with tight junction components at the apical region of the intercellular cleft. A cDNA clone encoding JAM defines a novel immunoglobulin gene superfamily member that consists of two V-type Ig domains. An mAb directed to JAM (BV11) was found to inhibit spontaneous and chemokine-induced monocyte transmigration through an endothelial cell monolayer in vitro. Systemic treatment of mice with BV11 mAb blocked monocyte infiltration upon chemokine administration in subcutaneous air pouches. Thus, JAM is a new component of endothelial and epithelial junctions that play a role in regulating monocyte transmigration.

Identification of a novel cadherin (vascular endothelial cadherin-2) located at intercellular junctions in endothelial cells.

Endothelial cells express two major cadherins, VE- and N-cadherins, but only the former consistently participates in adherens junction organization. In heart microvascular endothelial cells, we identified a new member of the cadherin superfamily using polymerase chain reaction. The entire putative coding sequence was determined. Similarly to protocadherins, while the extracellular domain presented homology with other members of the cadherin superfamily, the intracellular region was unrelated either to cadherins or to any other known protein. We propose for this new protein the name of vascular endothelial cadherin-2. By Northern blot analysis, the mRNA was present only in cultured endothelial cell lines but not in other cell types such as NIH 3T3, Chinese hamster ovary, or L cells. In addition, mRNA was particularly abundant in highly vascularized organs such as lung or kidney. In endothelial cells and transfectants, this cadherin was unable to bind catenins and presented a weak association with the cytoskeleton. This new molecule shares some functional properties with VE-cadherin and other members of the cadherin family. In Chinese hamster ovary transfectants it promoted homotypic Ca2+ dependent aggregation and adhesion and clustered at intercellular junctions. However, in contrast to VE-cadherin, it did not modify paracellular permeability, cell migration, and density-dependent cell growth. These observations suggest that different cadherins may promote homophilic cell-to-cell adhesion but that the functional consequences of this interaction depend on their binding to specific intracellular signaling/cytoskeletal proteins.

P-selectin is expressed on the oolemma of human and hamster oocytes following sperm adhesion and is also detected on the equatorial region of acrosome-reacted human spermatozoa.

Selectins are a family of adhesive molecules, involved in the interactions between leukocytes and endothelium and in platelet adhesion. P-selectin, one of the members of this family, is stored in alpha-granules and dense granules of platelets as well as in Weibel-Palade bodies of endothelial cells, and it is rapidly redistributed to the cell surface after activation. It recognizes carbohydrate structures as ligands, in particular sialyl-Lewis(x), which is part of the CD15 antigen. In this work we studied P-selectin expression on gametes. While zona-free human and hamster oocytes did not react with a monoclonal antibody directed against P-selectin, oocytes from both species displayed a reactivity with this antibody following their contact with human spermatozoa, as demonstrated both by covasphere binding and indirect immunofluorescence. Artificial activation of zona-intact human oocytes by means of the calcium lonophore A23187 induced the expression on the oolemma of a moiety reacting with anti-P-selectin antibody as well. P-selectin also appeared to be expressed on the sperm surface following the acrosome reaction, as demonstrated by a flow cytometric study of reactivity of spermatozoa with the anti-P-selectin antibody, using the expression of CD46 as a marker of the acrosome reaction. The localization of the P-selectin moiety on the equatorial region of the plasma membrane of acrosome reacted spermatozoa was confirmed by transmission electron microscopy using immunogold labelling. We suggest that P-selectin might be involved in gamete interactions.

Importancia de las determinaciones seriadas de estriol total, progesterona y hormona lactogeno placentaria sérica: mujeres con gestosis / Importance of the serial determinations of total estriol, progesterone and placental serous lactogen hormone: women with gestosis

Se presenta un estudio prospectivo realizado en nueve mujeres embarazadas con hipertensión inducida por el embarazo, en las que se realizaron mediciones de estriol total, progesterona y HPL, por métodos de RIA, a las 32, 34 y semanas de gestación. Los valores obtenidos en las tres hormonas dosadas resultaron significativamente más bajas que los habituales para el mismo laboratorio utilizado, por lo que se concluye que resultan de valor en la prevención, pronóstico y manejo de la patología gestósica del embarazo