Oral clonidine provocative test in the diagnosis of growth hormone deficiency in childhood: should we make the timing uniform?

INTRODUCTION: Oral clonidine is one of the most frequent drugs used for the diagnosis of growth hormone deficiency (GHD), but the duration of the test, depending on which European centres use it, is not uniform and can vary from 120 to 150 min or even 180 min. SUBJECTS AND METHODS: To standardize this test, evaluating the possibility to shorten it to 90 min, we investigated the response of GH to the oral clonidine test in 291 children evaluated for short stature (height <-2 SD). Of these, 164 were diagnosed as idiopathic short stature (ISS) and 127 as GHD. In these patients, we calculated: (1) the frequency distribution of the GH peaks to clonidine in GHD and in ISS at various times; (2) the percentage of GH peaks to clonidine before and after 90 min in all and in ISS children; (3) the percentage of the first GH value >or=10 ng/ml before 90 min and after 90 min in ISS. RESULTS: GH peak distribution varied between 30 and 180 min, even though the vast majority of peaks occurred between 30 and 60 min. There was no significant difference (p > 0.05) in the peak distribution between ISS and GHD children. The percentages of GH peaks within 90 min were 92.1% in all children and 95.7% in ISS. If considering the first value of GH >or=10 ng/ml this last percentage reaches 96.3%. CONCLUSION: Our study suggests that the oral clonidine test can be administered for only 90 min without significantly changing its validity. This test should be standardized at 90 min in European protocols just as in those currently used in the USA in order to reduce the discomfort of patients and the cost of this diagnostic procedure.     

HIV-1 Tat regulates endothelial cell cycle progression via activation of the Ras/ERK MAPK signaling pathway

Tat, the transactivator of HIV-1 gene expression, is released by acutely HIV-1-infected T-cells and promotes adhesion, migration, and growth of inflammatory cytokine-activated endothelial and Kaposi's sarcoma cells. It has been previously demonstrated that these effects of Tat are due to its ability to bind through its arginine-glycine-aspartic (RGD) region to the alpha5beta1 and alphavbeta3 integrins. However, the signaling pathways linking Tat to the regulation of cellular functions are incompletely understood. Here, we report that Tat ligation on human endothelial cells results in the activation of the small GTPases Ras and Rac and the mitogen-activated protein kinase ERK, specifically through its RGD region. In addition, we demonstrated that Tat activation of Ras, but not of Rac, induces ERK phosphorylation. We also found that the receptor proximal events accompanying Tat-induced Ras activation are mediated by tyrosine phosphorylation of Shc and recruitment of Grb2. Moreover, Tat enabled endothelial cells to progress through the G1 phase in response to bFGF, and the process is linked to ERK activation. Taken together, these data provide novel evidence about the ability of Tat to activate the Ras-ERK cascade which may be relevant for endothelial cell proliferation and for Kaposi's sarcoma progression.     

CD38 and CD157 as receptors of the immune system: a bridge between innate and adaptive immunity

This paper reviews some of the results and the speculations presented at the Torino CD38 Meeting in June, 2006 and focused on CD38 and CD157 seen as a family of molecules acting as surface receptors of immune cells. This partisan view was adopted in the attempt to combine the enzymatic functions with what the immunologists consider key functions in different cell models. At the moment, it is unclear whether the two functions are correlated, indifferent, or independent. Here we present conclusions inferred exclusively on human cell models, namely T and B lymphocytes, dendritic cells, and granulocytes. As an extra analytical tool, we try to follow in the history of life when the enzymatic and receptorial functions were generated, mixing ontogeny, membrane localization, and cell anchorage.     

Organophosphate-resistant forms of acetylcholinesterases in two scallops--the Antarctic Adamussium colbecki and the Mediterranean Pecten jacobaeus

We describe the acetylcholinesterase polymorphisms of two bivalve molluscs, Adamussium colbecki and Pecten jacobaeus. The research was aimed to point out differences in the expression of pesticide-resistant acetylcholinesterase forms in organisms living in different ecosystems such as the Ross Sea (Antarctica) and the Mediterranean Sea. In A. colbecki, distinct acetylcholinesterase molecular forms were purified and characterized from spontaneously soluble, low-salt-soluble and low-salt-Triton extracts from adductor muscle and gills. They consist of two non-amphiphilic acetylcholinesterases (G(2), G(4)) and an amphiphilic-phosphatidylinositol-membrane-anchored form (G(2)); a further amphiphilic-low-salt-soluble G(2) acetylcholinesterase was found only in adductor muscle. In the corresponding tissues of P. jacobaeus, we found a non-amphiphilic G(4) and an amphiphilic G(2) acetylcholinesterase; amphiphilic-low-salt-soluble acetylcholinesterases (G(2)) are completely lacking. Such results are related with differences in cell membrane lipid compositions. In both scallops, all non-amphiphilic AChEs are resistant to used pesticides. Differently, the adductor muscle amphiphilic forms are resistant to carbamate eserine and organophosphate diisopropylfluorophosphate, but sensitive to organophoshate azamethiphos. In the gills of P. jacobaeus, amphiphilic G(2) forms are sensitive to all three pesticides, while the corresponding forms of A. colbecki are sensitive to eserine and diisopropylfluorophosphate, but resistant to azamethiphos. Results indicate that organophosphate and/or carbamate resistant AChE forms are present in species living in far different and far away environments. The possibility that these AChE forms could have ensued from a common origin and have been spread globally by migration is discussed.     

Hormonal induction of differentiation in teratocarcinoma stem cells: generation of parietal endoderm by retinoic acid and dibutyryl cAMP

It has previously been shown that retinoic acid induces multiple phenotypic changes in cultures of F9 teratocarcinoma stem cells. In this paper we demonstrate that these retinoid-generated cells can be converted to yet another cell type by compounds that elevate cAMP concentrations. The phenotype of the new cell type is characterized by the synthesis of plasminogen activator, laminin and type IV collagen, and by very low levels of alkaline phosphatase and lactate dehydrogenase. The secretion of plasminogen activator and type IV collagen, and low levels of alkaline phosphatase and lactate dehydrogenase, have been previously shown to be properties of parietal endoderm, an extraembryonic cell which is generated early in mouse embryonesis. We show here that parietal endoderm also synthesizes laminin. The cell type generated by retinoic acid and dibutyryl cAMP treatment is therefore indistinguishable from definitive parietal endoderm. Analysis of the final phenotype indicates that it is not dependent upon the continued presence of either compound, and that cAMP agents are active only on cells that have been treated with retinoic acid.     

Distribution of Serotonin Receptor of Type 6 (5-HT6) in Human Brain Post-mortem. A Pharmacology, Autoradiography and Immunohistochemistry Study

The aim of this study was to investigate the distribution of serotonin (5-HT) receptors of type 6 (5-HT(6)) in postmortem human prefrontal cortex, striatum and hippocampus. The brain samples were obtained from 6 subjects who had died for causes not involving primarily or secondarily the CNS. The 5-HT(6) receptor distribution was explored by the [(125)I]SB-258585 binding to brain membranes followed by the pharmacological characterization, where possible, and by autoradiographic, immunohistochemical and immunofluorescence evaluations. A specific and saturable [(125)I]SB-258585 binding was detected in striatum only, with a pharmacological characterization consistent with that of a 5-HT(6) receptor. The autoradiography showed the presence of a specific [(125)I]SB-258585 binding distributed homogeneously in caudate, putamen and accumbens. The immunohistochemistry, carried out in the striatum only, coupled with the immunofluorescence with glial fibrillary acidic protein (GFAP) and parvalbumin (PV) showed the co-localization of 5-HT(6) receptor with PV, while indicating that this receptor subtype was expressed in neurons and not in astrocytes. Taken together, the present findings showed the presence of a higher density of 5-HT(6) receptors, as labeled by [(125)I]SB-258585, in striatum than in hippocampus and prefrontal cortex, and specifically within the neuronal body. In addition, they would suggest that striatum is one of the major potential CNS targets linked to 5-HT(6) receptor modulation.     

Mutant PrP suppresses glutamatergic neurotransmission in cerebellar granule neurons by impairing membrane delivery of VGCC α(2)δ-1 Subunit

How mutant prion protein (PrP) leads to neurological dysfunction in genetic prion diseases is unknown. Tg(PG14) mice synthesize a misfolded mutant PrP which is partially retained in the neuronal endoplasmic reticulum (ER). As these mice age, they develop ataxia and massive degeneration of cerebellar granule neurons (CGNs). Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective glutamate exocytosis from granule neurons due to impaired calcium dynamics. We found that mutant PrP interacts with the voltage-gated calcium channel α(2)δ-1 subunit, which promotes the anterograde trafficking of the channel. Owing to ER retention of mutant PrP, α(2)δ-1 accumulates intracellularly, impairing delivery of the channel complex to the cell surface. Thus, mutant PrP disrupts cerebellar glutamatergic neurotransmission by reducing the number of functional channels in CGNs. These results link intracellular PrP retention to synaptic dysfunction, indicating new modalities of neurotoxicity and potential therapeutic strategies.     

Establishment of keratinocyte colonies from small-sized cervical intraepithelial neoplasia specimens

The size of human cervical intraepithelial neoplasia (CIN) biopsies is usually very small and standard methods do not allow an adequate number of keratinocytes to be isolated for culturing purposes. In this study, a new approach to establish keratinocyte cultures from small CIN a tissue fragments was developed. Neoplastic specimens and corresponding normal tissues, which were used as controls, were digested with collagenase. Tissue‐derived fibroblasts and keratinocytes were co‐cultured in calcium and serum medium. Single keratinocyte colonies from primary cultures were expanded using a culture medium optimized in our laboratory. Primary keratinocyte colonies, as well as expanded colonies, were tested for epithelial and cervical markers such as 5, 14, 17, and 19 keratins, and p63 by immunofluorescence. Our results indicate that a variable number of primary keratinocyte colonies could be detected in neoplastic cultures, depending on the grade of cervical lesions from which the colonies originated. Single colonies, when cultured with our new medium, grew at a high rate with uniform size and morphology for some passages. Epithelial and p63 markers were expressed in keratinocyte colonies, as well as in expanded colonies. In conclusion, our study reports a rapid and easy culturing system which enables keratinocyte colonies from minute cervical tumor tissues to be obtained. Moreover, using the new culture medium, keratinocyte colonies can be expanded at a high proliferative rate. J. Cell. Physiol. 227: 3787–3795, 2012. © 2012 Wiley Periodicals, Inc.