Long-term results with growth hormone therapy in idiopathic hypopituitarism

More than 30 years after its introduction, growth hormone (GH) treatment is well established in children with GH deficiency. Nevertheless, the long-term results of this therapy, expressed as height, are generally considered unsatisfactory. We report on results obtained in a group of GH-deficient children who were treated with daily injections of recombinant GH within the first 5 years of life and who reached an adult height very close to their target height. The full catch-up growth to the target height demonstrated in these patients suggests that replacement therapy should be started early and continued until adulthood. Height at onset of puberty is an important variable which might significantly influence the adult height. The significant and prolonged influence of birth weight on growth response to GH therapy underlines the important role of fetal growth in planning early treatment of GH-deficient children.     

Tricyclic Imipramine Modification of the Circadian Rhythms of Hypothalamic Serotonin, its Precursors and Acid Catabolite in Individually Housed Rats

Circadian rhythms of serotonin (5HT), its precursors tryptophan (TP) and 5-hydroxy-tryptophan (5HTP) and its acid catabolite 5-hydroxy-indoleacetic acid (5HIAA), were determined in the hypothalamus of control rats and rats which had been treated continuously with subcutaneous imipramine (10 mg/kg/day) for 2 weeks.

Rats were individually housed and entrained to LD12:12. Controls showed the 5HT and TP peaks in the light and dark periods respectively, as reported in the literature, but no inverted correlation (antiphase) between SHT and 5HIAA rhythms.

Imipramine significantly modified circadian rhythm characteristics: the 5HT acrophase was advanced, that of TP and 5HIAA was delayed. Imipramine also significantly increased hypothalamic SHT and TP concentrations.     

Peroxisomes as novel players in cell calcium homeostasis

Ca2+ concentration in peroxisomal matrix ([Ca2+](perox)) has been monitored dynamically in mammalian cells expressing variants of Ca2+-sensitive aequorin specifically targeted to peroxisomes. Upon stimulation with agonists that induce Ca2+ release from intracellular stores, peroxisomes transiently take up Ca2+ reaching peak values in the lumen as high as 50-100 microm, depending on cell types. Also in resting cells, peroxisomes sustain a Ca2+ gradient, [Ca2+](perox) being approximately 20-fold higher than [Ca2+] in the cytosol ([Ca2+](cyt)). The properties of Ca2+ traffic across the peroxisomal membrane are different from those reported for other subcellular organelles. The sensitivity of peroxisomal Ca2+ uptake to agents dissipating H+ and Na+ gradients unravels the existence of a complex bioenergetic framework including V-ATPase, Ca2+/H+, and Ca2+/Na+ activities whose components are yet to be identified at a molecular level. The different [Ca2+](perox) of resting and stimulated cells suggest that Ca2+ could play an important role in the regulation of peroxisomal metabolism.     

Protein phosphatase 2A subunit PR70 interacts with pRb and mediates its dephosphorylation

The retinoblastoma tumor suppressor protein (pRb) regulates cell proliferation and differentiation via phosphorylation-sensitive interactions with specific targets. While the role of cyclin/cyclin-dependent kinase complexes in the modulation of pRb phosphorylation has been extensively studied, relatively little is known about the molecular mechanisms regulating phosphate removal by phosphatases. Protein phosphatase 2A (PP2A) is constituted by a core dimer bearing catalytic activity and one variable B regulatory subunit conferring target specificity and subcellular localization. We previously demonstrated that PP2A core dimer binds pRb and dephosphorylates pRb upon oxidative stress. In the present study, we identified a specific PP2A-B subunit, PR70, that was associated with pRb both in vitro and in vivo. PR70 overexpression caused pRb dephosphorylation; conversely, PR70 knockdown prevented both pRb dephosphorylation and DNA synthesis inhibition induced by oxidative stress. Moreover, we found that intracellular Ca²⁺ mobilization was necessary and sufficient to trigger pRb dephosphorylation and PP2A phosphatase activity of PR70 was Ca²⁺ induced. These data underline the importance of PR70-Ca²⁺ interaction in the signal transduction mechanisms triggered by redox imbalance and leading to pRb dephosphorylation.     

The effects of interleukin-8 on airway smooth muscle contraction in cystic fibrosis

Background

Clara cells are the epithelial progenitor cell of the small airways, a location known to be important in many lung disorders. Although migration of alveolar type II and bronchiolar ciliated epithelial cells has been examined, the migratory response of Clara cells has received little attention.

Methods

Using a modification of existing procedures for Clara cell isolation, we examined mouse Clara cells and a mouse Clara-like cell line (C22) for adhesion to and migration toward matrix substrate gradients, to establish the nature and integrin dependence of migration in Clara cells.

Results

We observed that Clara cells adhere preferentially to fibronectin (Fn) and type I collagen (Col I) similar to previous reports. Migration of Clara cells can be directed by a fixed gradient of matrix substrates (haptotaxis). Migration of the C22 cell line was similar to the Clara cells so integrin dependence of migration was evaluated with this cell line. As determined by competition with an RGD containing-peptide, migration of C22 cells toward Fn and laminin (Lm) 511 (formerly laminin 10) was significantly RGD integrin dependent, but migration toward Col I was RGD integrin independent, suggesting that Clara cells utilize different receptors for these different matrices.

Conclusion

Thus, Clara cells resemble alveolar type II and bronchiolar ciliated epithelial cells by showing integrin mediated pro-migratory changes to extracellular matrix components that are present in tissues after injury.     

AFM imaging and theoretical modeling studies of sequence-dependent nucleosome positioning

Telomeric chromatin has different features with respect to bulk chromatin, since nucleosomal repeat along the chain is unusually short. We studied the role of telomeric DNA sequences on nucleosomal spacing in a model system. Nucleosomal arrays, assembled on a 1500-bp-long human telomeric DNA and on a DNA fragment containing 8 copies of the 601 strong nucleosome positioning sequence, have been studied at the single molecule level, by atomic force microscopy imaging. Random nucleosome positioning was found in the case of human telomeric DNA. On the contrary, nucleosome positioning on 601 DNA is characterized by preferential positions of nucleosome dyad axis each 200 bp. The AFM-derived nucleosome organization is in satisfactory agreement with that predicted by theoretical modeling, based on sequence-dependent DNA curvature and flexibility. The reported results show that DNA sequence has a main role, not only in mononucleosome thermodynamic stability, but also in the organization of nucleosomal arrays.     

Proteomic analysis of membrane microdomains derived from both failing and non-failing human hearts

Eukaryotic cells plasma membranes are organized into microdomains of specialized function such as lipid rafts and caveolae, with a specific lipid composition highly enriched in cholesterol and glycosphingolipids. In addition to their role in regulating signal transduction, multiple functions have been proposed, such as anchorage of receptors, trafficking of cholesterol, and regulation of permeability. However, an extensive understanding of their protein composition in human heart, both in failing and non-failing conditions, is not yet available. Membrane microdomains were isolated from left ventricular tissue of both failing (n = 15) and non-failing (n = 15) human hearts. Protein composition and differential protein expression was explored by comparing series of 2-D maps and subsequent identification by LC-MS/MS analysis. Data indicated that heart membrane microdomains are enriched in chaperones, cytoskeletal-associated proteins, enzymes and protein involved in signal transduction pathway. In addition, differential protein expression profile revealed that 30 proteins were specifically up- or down-regulated in human heart failure membrane microdomains. This study resulted in the identification of human heart membrane microdomain protein composition, which was not previously available. Moreover, it allowed the identification of multiple proteins whose expression is altered in heart failure, thus opening new perspectives to determine which role they may play in this disease.     

Screening for mutations in the neurofibromatosis type 2 (NF2) gene in sporadic meningiomas

Meningiomas are benign tumors of the central nervous system. They are usually sporadic but can also occur associated with the neurofibromatosis type 2 (NF2) syndrome. The gene responsible for NF2, recently isolated from chromosome 22, encodes a membrane-organizing protein that shows high sequence homology to a protein family thought to link the cytoskeleton with membrane proteins. Mutations of the NF2 gene have been described in sporadic meningiomas, exclusively in tumors that show loss of heterozygosity (LOH) of 22q. These preliminary results indicate that the NF2 gene is involved in the pathogenesis of at least a subset of meningiomas, where it does indeed behave as a tumor suppressor gene. In order to characterize better the role of the NF2 gene in the genesis of meningiomas we have examined the entire coding sequence of the gene in 125 meningiomas by single-strand conformational polymorphism analysis; furthermore, LOH analysis for markers of 22q has been carried out. Inactivating mutations were identified in 30% of our samples, all of which also showed LOH of 22q. The majority of mutations identified were frameshifts and nonsense mutations, which are predicted to produce a truncated or non-functional protein. We also found two missense and three in-frame deletions that may pinpoint specific regions of the protein critical to its function. Furthermore, the distribution of mutations throughout the gene, suggested that exons 2, 3, 5, 11 and 13 are more frequently involved. Our results reconfirm the importance of the NF2 gene in the pathogenesis of meningiomas and also suggest that there may be a nonrandom clustering of mutations throughout the gene.     

Genetic characterization of 12 heterologous microsatellite markers for the giant tropical tree Cariniana legalis (Lecythidaceae)

Twelve microsatellite loci previously developed in the tropical tree Cariniana estrellensis were genetically characterized in Cariniana legalis. Polymorphisms were assessed in 28 C. legalis individuals found between the Pardo and Mogi-Guaçu River basins in the state of São Paulo, Brazil. Of the 12 loci, 10 were polymorphic and exhibited Mendelian inheritance. The allelic richness at each locus ranged from 2-11, with an average of 7 alleles per locus, and the expected heterozygosity ranged from 0.07-0.88. These loci showed a high probability of paternity exclusion. The characteristics of these heterologous microsatellite markers indicate that they are suitable tools for investigating questions concerning population genetics in C. legalis.