Circadian pacemaking in cells and circuits of the suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal circadian pacemaker of the brain. It co-ordinates the daily rhythms of sleep and wakefulness, as well as physiology and behaviour, that set the tempo to our lives. Disturbance of this daily pattern, most acutely with jet-lag but more insidiously with rotational shift-work, can have severely deleterious effects for mental function and long-term health. The present review considers recent developments in our understanding of the properties of the SCN that make it a robust circadian time-keeper. It first focuses on the intracellular transcriptional/ translational feedback loops (TTFL) that constitute the cellular clockwork of the SCN neurone. Daily timing by these loops pivots around the negative regulation of the Period (Per) and Cryptochrome (Cry) genes by their protein products. The period of the circadian cycle is set by the relative stability of Per and Cry proteins, and this can be controlled by both genetic and pharmacological interventions. It then considers the function of these feedback loops in the context of cytosolic signalling by cAMP and intracellular calcium ([Ca(2+) ]i ), which are both outputs from, and inputs to, the TTFL, as well as the critical role of vasoactive intestinal peptide (VIP) signalling in synchronising cellular clocks across the SCN. Synchronisation by VIP in the SCN is paracrine, operating over an unconventionally long time frame (i.e. 24 h) and wide spatial domain, mediated via the cytosolic pathways upstream of the TTFL. Finally, we show how intersectional pharmacogenetics can be used to control G-protein-coupled signalling in individual SCN neurones, and how manipulation of Gq/[Ca(2+) ]i -signalling in VIP neurones can re-programme the circuit-level encoding of circadian time. Circadian pacemaking in the SCN therefore provides an unrivalled context in which to understand how a complex, adaptive behaviour can be organised by the dynamic activity of a relatively few gene products, operating in a clearly defined neuronal circuit, with both cell-autonomous and emergent, circuit-level properties.

Activated ROCK II by-passes the requirement of the CDK2 activity for centrosome duplication and amplification.

Initiation of centrosome duplication and DNA replication is coupled, which is primarily achieved by the late G1 phase-specific activation of cyclin-dependent kinase 2 (CDK2)-cyclin E, which triggers both centrosome duplication and DNA replication. Uncoupling of these two events contributes to overduplication of centrosomes, resulting in the presence of more than two centrosomes (centrosome amplification). Centrosome amplification, which is frequently observed in cancers, contributes to tumor development through destabilizing genomes. Nucleophosmin (NPM/B23) is one of the phosphorylation targets of CDK2-cyclin E for the initiation of centrosome duplication. It has been found that NPM/B23 phosphorylated on Thr199 by CDK2-cyclin E acquires a high binding affinity to ROCK II kinase. The Thr199-phosphorylated NPM/B23 physically interacts with and super-activates the centrosomally localized ROCK II, which is a critical event for centrosomes to initiate duplication. Here, we provide direct evidence for the activation of ROCK II as a primary and sufficient downstream event of CDK2-cyclin E for the initiation of centrosome duplication and for the induction of centrosome amplification.

Degree of concordance between double-balloon enteroscopy and capsule endoscopy in obscure gastrointestinal bleeding: a multicenter study.

BACKGROUND AND STUDY AIMS: Capsule endoscopy is considered the diagnostic procedure of choice in patients with obscure gastrointestinal bleeding (OGIB). Double-balloon endoscopy (DBE) offers both diagnostic and therapeutic potential, but is invasive, complex, and time-consuming. The aim was to evaluate diagnostic agreement between capsule endoscopy and DBE in patients with OGIB, and secondarily the diagnostic gain of DBE when capsule endoscopy detected only blood or clots in the small-bowel lumen. METHODS: Multicenter prospective study carried out at six institutions in Italy. RESULTS: 193 patients (119 men, mean age 61.6 +/- 16.2) first underwent capsule endoscopy and then DBE. The most frequent positive findings at capsule endoscopy were vascular lesions (74 patients, 38.3 %), blood or clot in the lumen (34, 17.6 %), and tumor mass (20, 10.4 %). The most frequent findings at DBE were vascular lesions (72 patients, 37.3 %), neoplasia (30, 15.5 %) and ulcers/inflammatory lesions (12, 6.2 %). Overall kappa coefficient was 0.46 (95 %CI 0.38 - 0.54), with maximum concordance for vascular (0.72 [95 %CI 0.59 - 0.84]) and inflammatory (0.78 [0.58 - 0.99]) lesions and minimum for polyps (0.46 [0.16 - 0.80]). Blood in the lumen was the only positive finding at capsule endoscopy in 34 cases; of these, 12 had negative DBE findings whereas 10 had vascular lesions, 6 neoplasia, 1 ulcer, and 5 diverticula. CONCLUSION: Capsule endoscopy and DBE have good agreement for vascular and inflammatory lesions but not for polyps or neoplasia. DBE provides valuable adjunctive information, particularly in patients with neoplasia or polyp at capsule endoscopy. DBE clarified the origin of bleeding in two-thirds of patients with capsule endoscopy showing only blood in the lumen.

Capsule endoscopy for the detection of bleeding Meckel's diverticulum. A case report.

The case of a 59-year-old man with a history of recurrent severe gastrointestinal bleeding due to Meckel's diverticulum is described. The diagnosis was achieved by means of capsule endoscopy. The histological examination revealed the presence of Meckel's diverticulum with ectopic gastric mucosa. The use of capsule endoscopy for the detection of Meckel's diverticulum is discussed.

Distribution and localization of vinculin-talin-integrin system and dystrophin-glycoprotein complex in human skeletal muscle. Immunohistochemical study using confocal laser scanning microscopy.

The vinculin-talin-integrin system and the dystrophin-glycoprotein complex (DGC) are two protein systems with structural and signaling functions, allowing interaction between muscle fibers and extracellular matrix. Although numerous studies have been conducted on these systems, their localization and distribution patterns along the nonjunctional sarcolemma are not clear. On this basis, we carried out an indirect immunofluorescence study on the vastus lateralis muscle of human adults not affected by neuromuscular diseases to better define these patterns. Our results showed that all tested proteins of the two systems have a costameric distribution; all tested proteins of the two systems colocalize with each other (about 90-95% of the cases); only alpha-sarcoglycan in a few cases (about 6%) does not colocalize with other proteins; in about 9-10% of the cases, dystrophin and beta-dystroglycan colocalize partially with other proteins; all tested proteins can be localized in different fibers, both in the region of the sarcolemma over I or A bands. The colocalization between the vinculin-talin-integrin and DGC systems may imply their functional interaction involving the structural aspect, by providing a stronger adhesion between sarcolemma and extracellular matrix in well-defined regions of the muscle fiber. Besides, their colocalization may suggest the existence of a mechanism of mutual modulation of the transmitted signals. This reciprocal control may determine, in different conditions, the prevalence of one system over another with a consequent transmission of different messages to the sarcolemma-associated cytoskeleton.

The New Stories/New Cultures after-school enrichment program: a direct cultural intervention.

This article describes the organization, curriculum, and outcomes for New Stories/New Cultures, an activity-based program for after-school enrichment in five schools in the low-income neighborhood near a major American university. The program encourages students (70% Hispanic-American, 30% African-American) to experience themselves as producers of culture, not just as consumers. Its methods include (a) creative team use of video equipment and other expressive media and (b) lessons about media literacy (i.e., making critical choices about images and activities depicted in popular culture and commercials). Outcome measures with the cohort of fifth and sixth graders support the programs occupation-based philosophy. They show that students are more likely to experience themselves as building skills when engaged in activities that are both challenging and enjoyable. The students reported greatest engagement and enjoyment in activities that were creative, team-based, and involving media production. These same activities were correlated with increased self-esteem. The term direct cultural intervention is used to describe the application of occupational principles and critical perspectives to provide a population with conceptual tools and skills for interpreting and successfully navigating the social world.

Integrin function and regulation in development.

Integrins are a large family of membrane receptors, consisting of alpha and beta subunits, that play a pivotal role in the interaction of cells with the extracellular matrix. Such interaction regulates the organization of cells in organs and tissues during development as well as cell differentiation and proliferation. We have shown that unfertilized oocytes express integrins that might be important during fertilization. We also analyzed nervous system and muscle tissue development showing that integrin expression is precisely regulated during organization of these tissues. The results indicate that two distinct integrin alpha subunits mediate the outgrowth of processes in nerve and glial cells. Alpha1 integrin, a laminin receptor, is up-regulated by nerve growth factor and other differentiation stimuli and is involved in neurite extension by nerve cells. In contrast, process extension by glial cells is likely to involve the alphaV integrin. Moreover, the latter integrin subunit is also transiently expressed in muscle of the embryo body where it localizes predominantly at developing myotendinous junctions. After birth this integrin disappears and is substituted by the alpha7 subunit. At the same time, important changes also occur in the expression of the associated beta subunit. In fact, the beta1A isoform which is expressed in fetal muscles, is substituted by beta1D. These isoforms are generated by alternative splicing and differ in only a few amino acid residues at the COOH terminus of the protein. This region of the molecule is exposed at the cytoplasmic face of the plasma membrane and is connected to the actin filaments. Our results show that beta1D, which is expressed only in striated muscle tissues, binds to both cytoskeletal and extracellular matrix proteins with an affinity higher than beta1A. Thus, beta1D provides a stronger link between the cytoskeleton and extracellular matrix necessary to support mechanical tension during muscle contraction. These results indicate that cells can regulate their interactions with the extracellular matrix by changing their expression of alpha integrin subunits and thus ligand specificity, or by more subtle changes involving alternative usage of different cytoplasmic domains. The important role of both alpha and beta integrin subunit cytoplasmic domains during development is further illustrated by the analysis of targeted mutations which we have generated by homologous recombination in mice.

Distinct roles of the adaptor protein Shc and focal adhesion kinase in integrin signaling to ERK.

It has been proposed that integrins activate ERK through the adaptor protein Shc independently of focal adhesion kinase (FAK) or through FAK acting on multiple target effectors, including Shc. We show that disruption of the actin cytoskeleton by cytochalasin D causes a complete inhibition of FAK but does not inhibit Shc signaling and activation of ERK. We have then generated primary fibroblasts carrying a targeted deletion of the segment of beta(1) subunit cytoplasmic domain required for activation of FAK. Analysis of these cells indicates that FAK is not necessary for efficient tyrosine phosphorylation of Shc, association of Shc with Grb2, and activation of ERK in response to matrix adhesion. In addition, integrin-mediated activation of FAK does not appear to be required for signaling to ERK following growth factor stimulation. To examine if FAK could contribute to the activation of ERK in a cell type-specific manner through the Rap1/B-Raf pathway, we have used Swiss-3T3 cells, which in contrast to primary fibroblasts express B-Raf. Dominant negative studies indicate that Shc mediates the early phase and peak, whereas FAK, p130(CAS), Crk, and Rap1 contribute to the late phase of integrin-dependent activation of ERK in these cells. In addition, introduction of B-Raf enhances and sustains integrin-mediated activation of ERK in wild-type primary fibroblasts but not in those carrying the targeted deletion of the beta(1) cytoplasmic domain. Thus, the Shc and FAK pathways are activated independently and function in a parallel fashion. Although not necessary for signaling to ERK in primary fibroblasts, FAK may enhance and prolong integrin-mediated activation of ERK through p130(CAS), Crk, and Rap1 in cells expressing B-Raf.

Melusin is a new muscle-specific interactor for beta(1) integrin cytoplasmic domain.

Here we describe the isolation and partial characterization of a new muscle-specific protein (Melusin) which interacts with the integrin cytoplasmic domain. The cDNA encoding Melusin was isolated in a two-hybrid screening of a rat neonatal heart library using beta(1)A and beta(1)D integrin cytoplasmic regions as baits. Melusin is a cysteine-rich cytoplasmic protein of 38 kDa, with a stretch of acidic amino acid residues at the extreme carboxyl-terminal end. In addition, putative binding sites for SH3 and SH2 domains are present in the amino-terminal half of the molecule. Chromosomic analysis showed that melusin gene maps at Xq12.1/13 in man and in the synthenic region X band D in mouse. Melusin is expressed in skeletal and cardiac muscles but not in smooth muscles or other tissues. Immunofluorescence analysis showed that Melusin is present in a costamere-like pattern consisting of two rows flanking alpha-actinin at Z line. Its expression is up-regulated during in vitro differentiation of the C2C12 murine myogenic cell line, and it is regulated during in vivo skeletal muscle development. A fragment corresponding to the tail region of Melusin interacted strongly and specifically with beta(1) integrin cytoplasmic domain in a two-hybrid test, but the full-length protein did not. Because the tail region of Melusin contains an acidic amino acid stretch resembling high capacity and low affinity calcium binding domains, we tested the possibility that Ca(2+) regulates Melusin-integrin association. In vitro binding experiments demonstrated that interaction of full-length Melusin with detergent-solubilized integrin heterodimers occurred only in absence of cations, suggesting that it can be regulated by intracellular signals affecting Ca(2+) concentration.

Differential onset of expression of alpha 7 and beta 1D integrins during mouse heart and skeletal muscle development.

beta 1D is a recently identified isoform of the beta 1 integrin subunit selectively expressed in skeletal and cardiac muscles. In the present study we determined the temporal expression of beta 1D and its association with alpha subunits during mouse development. By immunohistochemistry and western blot analysis we demonstrated that beta 1D begins to be expressed in skeletal muscles of 17 days embryo (stage E17). Its level progressively increases reaching maximal values few days after birth and remaining high in adult mice. At earlier stages of development (E11-E17) the beta 1A isoform is expressed in skeletal muscle cells. After E17 beta 1A is downregulated and disappears from muscle fibers few days after birth. In cardiac muscle the regulation of the beta 1D expression is different: beta 1D and beta 1A are coexpressed in the heart of E11 embryo. Subsequently expression of beta 1A declines, while beta 1D increases until it becomes the unique beta 1 isoform in cardiomyocytes few days after birth. Previous studies (Belkin et al J. Cell Biol. 132: 211-226, 1996) demonstrated that beta 1D in adult mouse cardiomyocytes is exclusively associated with alpha 7B. Western blot analysis shows that alpha 7B starts to be expressed in the heart only at stage E17, while beta 1D is expressed already at E11 embryo, indicating that alpha subunits other than alpha 7 should associate with beta 1D in early developmental stages. To investigate this aspect, beta 1 associated alpha subunits were identified by western blotting from cardiomyocytes integrin complexes immunoprecipitated with alpha subunit specific antibodies. We found that, during cardiomyocyte development, beta 1D associates with several alpha subunits namely with alpha 5, alpha 6A and alpha 7B. In conclusion these data show that the expression of the beta 1D muscle specific integrin during development occurs much earlier in heart than in skeletal muscle and it can dimerize with different alpha subunits.

Megasphaera elsdenii endocarditis.

A case of endocarditis caused by Megasphaera elsdenii is reported. This anaerobic grim-negative coccus has rarely been associated with human infections and has not previously been described as a cause of endocarditis.

Intracellular mucin production by lobular breast carcinoma cells.

Slides from ten cases of infiltrating lobular and ten of in situ lobular breast cancer were studied with mucicarmine stain to determine the incidence of intracellular mucin in such cases. Nine of the ten infiltrating carcinomas and six of the ten in situ tumors contained such cells. None of the cases contained infiltrating duct or "colloid" carcinoma and none had in situ carcinoma of major ducts.

[Chronic bronchopneumopathies in chest x-ray findings at the Centro di Medicina Preventiva e Sociale]. / Le broncopneumopatie croniche nelle rilevazioni schermografiche del Centro di Medicina Preventiva e Sociale

Screening of secundary school children and the general opulation of the V. Monaldi Hospital Preventive and Social Medicine Centre,Naples showed a by no means negligible incidence of chronic bronchopneumopathy, particulary in infants. It is felt that, if untreated, these forms may be responsible for chronic respiratory insufficency in adulthood. Marker success in this respect has been obtained with anti-inflammatory and anti-bronchospastic substances, particularly a diethyl-aminoethoxyethylic ester of alpha-phenylbutyric acid. Preventive and social management is thus primarily directed at infants and the aged.