Serum 25-hydroxy vitamin D levels in essential hypertension.

Vitamin D may have prognostic value in hypertension patients and, in addition to conventional biomarkers, could be a valuable tool for disease management. The aim of this study was to assess the association of vitamin D status in patients with essential hypertension and to evaluate its prognostic utility. Forty-eight consecutive patients (40 Caucasian and 8 Asian) aged between 30 and 80 years (mean 61.5, range 34-84 years), were enrolled in the study. The main exclusion criteria were age less than 18 years, kidney failure, onco-hematologic disease, hypo-hyperparathyroidism, osteoporosis, treatment with bisphosphonate or 25(OH) vitamin D supplementation. Of the 48 patients included in the study, hyperlipidemia was described in 28, diabetes type 2 in 8, and ischemic heart disease in 14. Serum electrolytes, calcium, sodium, and potassium concentrations were within normal range. Low 25(OH) vitamin D levels inversely correlated with essential hypertension values (p less than 0.001) were considered extremely significant. The determination of 25(OH) vitamin D levels in patients with essential hypertension could improve the research for possible underlying conditions, which should be managed meticulously according to current guidelines.

Protective role for type-1 metabotropic glutamate receptors against spike and wave discharges in the WAG/Rij rat model of absence epilepsy.

Eight-month old WAG/Rij rats, which developed spontaneous occurring absence seizures, showed a reduced function of mGlu1 metabotropic glutamate receptors in the thalamus, as assessed by in vivo measurements of DHPG-stimulated polyphosphoinositide hydrolysis, in the presence of the mGlu5 antagonist MPEP as compared to age-matched non-epileptic control rats. These symptomatic 8-month old WAG/Rij rats also showed lower levels of thalamic mGlu1α receptors than age-matched controls and 2-month old (pre-symptomatic) WAG/Rij rats, as detected by immunoblotting. Immunohistochemical and in situ hybridization analysis indicated that the reduced expression of mGlu1 receptors found in symptomatic WAG/Rij rats was confined to an area of the thalamus that excluded the ventroposterolateral nucleus. No mGlu1 receptor mRNA was detected in the reticular thalamic nucleus. Pharmacological manipulation of mGlu1 receptors had a strong impact on absence seizures in WAG/Rij rats. Systemic treatment with the mGlu1 receptor enhancer SYN119, corresponding to compound RO0711401, reduced spontaneous spike and wave discharges spike-wave discharges (SWDs) in epileptic rats. Subcutaneous doses of 10 mg/kg of SYN119 only reduced the incidence of SWDs, whereas higher doses (30 mg/kg) also reduced the mean duration of SWDs. In contrast, treatment with the non-competitive mGlu1 receptor antagonist, JNJ16259685 (2.5 and 5 mg/kg, i.p.) increased the incidence of SWDs. These data suggest that absence epilepsy might be associated with a reduction of mGlu1 receptors in the thalamus, and that compounds that amplify the activity of mGlu1 receptors might be developed as novel anti-absence drugs. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Switch in the expression of mGlu1 and mGlu5 metabotropic glutamate receptors in the cerebellum of mice developing experimental autoimmune encephalomyelitis and in autoptic cerebellar samples from patients with multiple sclerosis.

Recent evidence suggests that changes in the expression of membrane receptors/ion channels in cerebellar Purkinje cells contribute to the onset of cerebellar motor symptoms in patients with multiple sclerosis (MS). We examined the expression of group-I metabotropic glutamate receptors (mGlu1 and mGlu5 receptors) in the cerebellum of mice developing experimental autoimmune encephalomyelitis (EAE) and in autoptic cerebellar samples of MS patients. EAE was induced in mice by immunization with the 35-55 fragment of MOG (myelin oligodendrocyte glycoprotein). EAE mice showed a progressive loss of mGlu1a receptors in the cerebellum, associated with an increased expression of mGlu5 receptors. These changes were restricted to Purkinje cells and their dendritic arborization, as shown by immunohistochemistry. A reduced expression of mGlu1a receptors in cerebellar Purkinje cells was also found in 7 of 9 MS patients. In addition, a light/moderate to very strong mGlu5 receptor immunoreactivity was detected in Purkinje cells of 8 MS patients, but was always absent in non-MS control patients. In EAE mice, an acute treatment with the mGlu1 receptor enhancer, 9H-xanthene-9-carboxylic acid (4-trifluoromethyl-oxazol-2-yl)-amide (RO0711401), significantly improved motor coordination, whereas treatment with the mGlu5 receptor antagonists, 2-methyl-6-(phenylethynyl)-pyridine (MPEP) and 6-methyl-2-(phenylazo)-3-pyridinol (SIB-1757), had no effect. We conclude that mGlu1 receptor enhancers improve motor symptoms associated with EAE and might be helpful as symptomatic drugs in patients with MS.

Expression of the Wnt inhibitor Dickkopf-1 is required for the induction of neural markers in mouse embryonic stem cells differentiating in response to retinoic acid.

Cultured mouse D3 embryonic stem (ES) cells differentiating into embryoid bodies (EBs) expressed several Wnt isoforms, nearly all isotypes of the Wnt receptor Frizzled and the Wnt/Dickkopf (Dkk) co-receptor low-density lipoprotein receptor-related protein (LRP) type 5. A 4-day treatment with retinoic acid (RA), which promoted neural differentiation of EBs, substantially increased the expression of the Wnt antagonist Dkk-1, and induced the synthesis of the Wnt/Dkk-1 co-receptor LRP6. Recombinant Dkk-1 applied to EBs behaved like RA in inducing the expression of the neural markers nestin and distal-less homeobox gene (Dlx-2). Recombinant Dkk-1 was able to inhibit the Wnt pathway, as shown by a reduction in nuclear beta-catenin levels. Remarkably, the antisense- or small interfering RNA-induced knockdown of Dkk-1 largely reduced the expression of Dlx-2, and the neuronal marker beta-III tubulin in EBs exposed to RA. These data suggest that induction of Dkk-1 and the ensuing inhibition of the canonical Wnt pathway is required for neural differentiation of ES cells.

Activation of Fas receptor is required for the increased formation of the disialoganglioside GD3 in cultured cerebellar granule cells committed to apoptotic death.

Apoptosis was induced in cultured cerebellar granule cells by lowering extracellular K+ concentrations (usually from 25 to 10 mM). The apoptotic phenotype was preceded by an early and transient increase in the intracellular levels of the disialoganglioside, GD3, which behaves as a putative pro-apoptotic factor. We examined whether activation of Fas receptor mediates the increase in GD3 formation in granule cells committed to die. Degenerating granule cells showed increased expression of both Fas receptor and its ligand (Fas-L), at times that coincided with the increase in GD3 levels and the induction of GD3 synthase mRNA. Addition of neutralizing anti-Fas-L antibodies reduced the extent of 'low-K+'-induced apoptosis and abolished the increase in GD3 levels and GD3 synthase mRNA. Similar reductions were observed in cultures prepared from gld or lpr mice, which harbor loss-of-function mutations of Fas-L and Fas receptor, respectively. In addition, exogenous application of soluble Fas-L further enhanced both the increase in GD3 formation and cell death in cultured granule cells switched from 25 into 10 mM K+. We conclude that activation of Fas receptor is entirely responsible for the increase in GD3 levels and contributes to the development of apoptosis by trophic deprivation in cultured cerebellar granule cells.

The mGlu5 metabotropic glutamate receptor is expressed in zones of active neurogenesis of the embryonic and postnatal brain.

Metabotropic glutamate (mGlu) receptors have been implicated in the regulation of developmental plasticity. Here, we examined the expression of mGlu1a-b, -2, -3, -4a-b, and -5a receptor subtypes from embryonic day 12 (E12) to the early and late postnatal life. While all transcripts (with the exception of mGlu4 mRNA) were detected prenatally, only the mGlu5 receptor protein was found in detectable amounts in the embryonic brain. Immunohistochemical analysis showed that the mGlu5 receptor was mainly expressed by cells surrounding the ventricles at E15, whereas it was more diffusely expressed at E18. In the postnatal life, besides its classical expression sites, the mGlu5 receptor was found in zones of active neurogenesis such as the external granular layer (EGL) of the cerebellar cortex and the subventricular zone. In these regions, the presence of actively proliferating progenitor cells was detected by BrdU staining. No other subtype (among those we have examined) was found to be expressed in regions enriched of BrdU(+) cells. These data suggest a role for mGlu5 receptors in the early brain development and in basic cellular processes such as proliferation and/or differentiation.

Native group-III metabotropic glutamate receptors are coupled to the mitogen-activated protein kinase/phosphatidylinositol-3-kinase pathways.

We used cultured cerebellar granule cells to examine whether native group-III metabotropic glutamate (mGlu) receptors are coupled to the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI-3-K) pathways. Cultured granule cells responded to the group-III mGlu receptor agonist, L-2-amino-4-phosphonobutanoate (l-AP4), with an increased phosphorylation and activity of MAPKs (ERK-1 and -2) and an increased phosphorylation of the PI-3-K target, protein kinase B (PKB/AKT). These effects were attenuated by the group-III antagonists, alpha-methyl-serine-O -phosphate (MSOP) and (R,S )-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG), or by pretreatment of the cultures with pertussis toxin. l-AP4 also induced the nuclear translocation of beta-catenin, a downstream effector of the PI-3-K pathway. To assess the functional relevance of these mechanisms we examined the ability of l-AP4 to protect granule cells against apoptosis by trophic deprivation, induced by lowering extracellular K(+) from 25 to 10 mm. Neuroprotection by l-AP4 was attenuated by MSOP and abrogated by the compounds PD98059 and UO126, which inhibit the MAPK pathway, or by the compound LY294002, which inhibits the PI-3-K pathway. Taken together, these results show for the first time that native group-III mGlu receptors are coupled to MAPK and PI-3-K, and that activation of both pathways is necessary for neuroprotection mediated by this particular class of receptors.

An early increase in the disialoganglioside GD3 contributes to the development of neuronal apoptosis in culture.

We induced apoptosis in primary cultures of cerebellar granule neurons by switching the growing medium into a medium containing lower concentrations of K(+) (5 or 10 mM instead of 25 mM) or, alternatively, by addition of staurosporine. The apoptotic phenotype was always preceded by an early increase in the intracellular levels of the disialoganglioside GD3, which peaked at 2-6 h and returned back to normal at 12 h. GD3 synthase, the enzyme that forms GD3 from the monosialoganglioside GM3, was also induced at early times after the induction of apoptosis in granule cells. Immunofluorescent staining showed that GD3 increased in neuronal cell bodies and neurites, but was never localized in cell nuclei. In cultures switched into a low K(+)-containing medium, exogenously applied GD3, but not the disialoganglioside GD1a, accelerated the development of neuronal apoptosis. In contrast, the antisense-induced knock-down of GD3 synthase was protective against granule cell death induced by lowering extracellular K(+) from 25 to 10 - but not 5 - mM. These results demonstrate that an early and transient increase in GD3 synthesis is one of the factors that contribute to the induction of neuronal apoptosis in culture.

Evidence for anticoagulant activity and beta2-GPI accumulation in late endosomes of endothelial cells induced by anti-LBPA antibodies.

This investigation was undertaken to test whether anti-LBPA antibodies and IgG from patients with APS interfere with intracellular beta2GPI distribution in EAhy926 endothelial cells and with the coagulation system. Cell incubation with anti-LBPA MoAb or with patients' IgG resulted in antibody binding to late endosomes and caused beta2GPI redistribution and accumulation within perinuclear vesicular structures reminiscent of late endosomes. This finding suggests that aPI may contribute to the pathogenic mechanisms of APS by modifying the intracellular traffic of proteins, by interactions between aPl and LBPA, beta2GPI and/or LBPA-beta2GPI complexes. The anticoagulant activity of anti-LBPA MoAb was analyzed in a sensitized activated partial thromboplastin time (aPTT) system and in a dilute Russell's viper venom time (dRVVT). A significant, concentration-dependent effect of the antibody on both aPTT and dRVVT prolongation was found. These observations suggest that LBPA is an important lipid target for aPl with potential functional implications for the immunopathogenesis of APS.

GD3 glycosphingolipid contributes to Fas-mediated apoptosis via association with ezrin cytoskeletal protein.

Efficiency of Fas-mediated apoptosis of lymphoid cells is regulated, among other means, by a mechanism involving its association with ezrin, a cytoskeletal protein belonging to the 4.1 family of proteins. In the present work, we provide evidence for a further molecule that associates to ezrin in Fas-triggered apoptosis, the disialoganglioside GD3. In fact, as an early event, GD3 redistributed in membrane-associated domains in uropods and co-localized with ezrin. Co-immunoprecipitation analyses confirmed this result, indicating a GD3-ezrin association. Altogether, these results are suggestive for a role of GD3 in Fas/ezrin-mediated apoptosis, supporting the view that uropods contain a multimolecular signaling complex involved in Fas-mediated apoptosis.

Evidence for cell surface association between CXCR4 and ganglioside GM3 after gp120 binding in SupT1 lymphoblastoid cells.

CXCR4 (fusin) is a chemokine receptor which is involved as a coreceptor in gp120 binding to the cell surface. In this study we provide evidence that binding of gp120 triggers CXCR4 recruitment to glycosphingolipid-enriched microdomains. Scanning confocal microscopy showed a nearly complete localization of CXCR4 within GM3-enriched plasma membrane domains of SupT1 cells and coimmunoprecipitation experiments revealed that CXCR4 was immunoprecipitated by IgG anti-GM3 after gp120 pretreatment. These findings reveal that gp120 binding induces a strict association between CXCR4 and ganglioside GM3, supporting the view that GM3 and CXCR4 are components of a functional multimolecular complex critical for HIV-1 entry.

Effects of long-term treatment with the anti-androgen bicalutamide on human testis: an ultrastructural and morphometric study.

AIMS: To assess the effects of more than 4 years' treatment with the anti-androgen bicalutamide on human testis by clinical, ultrastructural and morphometric analysis. METHODS AND RESULTS: Two patients (aged 74 and 69 years) with prostate cancer were treated for more than 4 years with bicalutamide 50 mg daily. Clinical characterization and follow-up included luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone and prostate-specific antigen (PSA) measurements and clinical response of the tumours. Due to progression of the disease, patients underwent surgical orchidectomy as a further androgen withdrawal therapy. Testis biopsies were studied by light and electron microscopy and analysed by morphometry. Control samples were obtained from the normal testis of two patients with testicular cancer who underwent orchidectomy. Clinical follow-up showed a good response in the control of tumour growth and serum PSA decreased to < 4 ng/mL; concentrations of serum LH, FSH and testosterone were within the normal range. Testicular morphology of treated patients was unexpectedly well preserved; the organization of seminiferous tubules was normal with all the germ line elements and mature spermatozoa present. In some areas, a net increase of peritubular connective tissue was evident which may be a consequence of the age of the patients. CONCLUSIONS: Long-term bicalutamide (50 mg) treatment appears to have very little impact on testis ultrastructure and sperm maturation, while it is effective in the control of androgen-dependent prostatic tumours.

Hepatitis C virus structural proteins reside in the endoplasmic reticulum as well as in the intermediate compartment/cis-Golgi complex region of stably transfected cells.

The intracellular localization of hepatitis C virus structural proteins was analyzed by confocal immunofluorescence microscopy, cell fractionation, and immunoelectron microscopy in stably transfected cells that do not overexpress the viral proteins. The results strongly suggest that at steady state the structural proteins reside not only in the endoplasmic reticulum but also in the intermediate compartment and cis-Golgi complex region. By analogy with other viral systems, this finding raises the possibility that the intermediate compartment and cis-Golgi complex play a role in the assembly and budding of hepatitis C virus.

Role of galectin-3 as a receptor for advanced glycosylation end products.

The advanced glycosylation end product (AGE)-binding proteins identified so far include the components of the AGE-receptor complex p60, p90 and galectin-3, receptor for advanced glycosylation end products (RAGE), and the macrophage scavenger receptor types I and II. Galectin-3 interacts with beta-galactoside residues of several cell surface and matrix glycoproteins through the carbohydrate recognition domain and is also capable of peptide-peptide associations mediated by its N-terminus domain. These structural properties enable galectin-3 to exert multiple functions, including the modulation of cell adhesion, the control of cell cycle, and the mRNA splicing activity. Moreover, in macrophages, astrocytes, and endothelial cells, galectin-3 has been shown to exhibit a high-affinity binding for AGEs; the lack of a transmembrane anchor sequence or signal peptide suggests that it associates with other AGE-receptor components rather than playing an independent role as AGE-receptor. In tissues that are targets of diabetic vascular complications, such as the mesangium and the endothelium, galectin-3 is not expressed or only weakly expressed under basal conditions, at variance with p90 and p60 but becomes detectable with aging and is induced or up-regulated by the diabetic milieu, which only slightly affects the expression of p90 or p60. This (over)expression of galectin-3 may in turn modulate AGE-receptor-mediated events by modifying the function of the AGE-receptor complex, which could play a role in the pathogenesis of target tissue injury. Up-regulated galectin-3 expression may also exert direct effects on tissue remodeling, independently of AGE ligands, by virtue of its adhesive and growth regulating properties.

The diabetic milieu modulates the advanced glycation end product-receptor complex in the mesangium by inducing or upregulating galectin-3 expression.

Nonenzymatic glycation has been implicated in the pathogenesis of the dysregulated tissue remodeling that characterizes diabetic glomerulopathy, via the formation of advanced glycation end products (AGEs) and their binding to cell surface receptors. Several AGE-binding proteins have been identified so far, including p60, p90, and the adhesive and growth-regulating lectin galectin-3 (Gal-3), the components of the so-called AGE-receptor complex. This study aimed to evaluate the mesangial expression of the AGE-receptor complex and its modulation by the diabetic milieu, both in vivo, in non-diabetic versus streptozotocin-induced diabetic rats, and in vitro, in mesangial cells exposed to either normal glucose (NG) levels (5.5 mmol/l), as compared with high glucose (HG) levels (30 mmol/l) and iso-osmolar mannitol (M), or to native bovine serum albumin (BSA), as compared with glycated BSA with AGE formation (BSA-AGE) and glycated BSA in which AGE formation was prevented by aminoguanidine (BSA-AM). In vivo, Gal-3 protein and mRNA were not detectable in glomeruli from nondiabetic rats until 12 months after initiating the study. On the contrary, in diabetic rats, Gal-3 expression was observed at 2 months of disease duration, and it increased thereafter. Both p60 and p90 immunoreactivities were observed at the glomerular level with slightly increased expression of p90, but not p60, in diabetic versus nondiabetic animals. In vitro, Gal-3 was not detectable in mesangial cells cultured in NG (although it became evident after a certain number of passages in culture), whereas Gal-3 was detectable in cells grown on BSA. Prolonged exposure (2-4 weeks) of mesangial cells to HG but not to M, as well as growing cells on BSA-AGE and, to a lesser extent, BSA-AM, induced or significantly increased the expression of Gal-3, both protein (up to 2.65-fold) and mRNA (up to 3.10-fold) and its secretion in the medium (by approximately 50%). Both p60 and p90 were demonstrated in mesangial cells under NG conditions, and the expression of p90, but not p60, was upregulated by approximately 20% by HG or BSA-AGE. These results indicate that 1) under basal conditions, Gal-3, unlike p90 and p60, is not detectable in the mesangium but becomes expressed with aging and 2) the diabetic milieu induces or upregulates Gal-3 production, whereas it increases only slightly the expression of p90, but not p60. Gal-3 expression or overexpression may modulate the AGE-receptor-mediated events by modifying the function of the AGE-receptor complex. Additionally, it may exert direct effects on tissue remodeling by virtue of its adhesive and growth-regulating properties.

Surface expression of the IFN-gamma R2 chain is regulated by intracellular trafficking in human T lymphocytes.

The surface and cytoplasmic expressions of the transducing chain (IFN-gamma R2) of the heterodimeric IFN-gamma receptor on human T lymphocytes have been investigated. We show that its surface expression is low, whereas high cytoplasmic levels are found in both resting and PHA-activated T lymphocytes. This low expression does not prevent activated T cells from responding to IFN-gamma, because it induces IFN-regulatory factor 1 expression. Low surface IFN-gamma R2 expression appears to be due to recycling between cytoplasmic stores and the cell surface, which does not depend on signals mediated by endogenous IFN-gamma, because IFN-gamma R2 surface expression is low, and its internalization is equally observed in patients with inherited IFN-gamma R1 gene deficiency and in healthy donors. Moreover, IFN-gamma R2 internalization in T lymphoblasts from healthy donors was not affected by the presence of anti-IFN-gamma-neutralizing or anti-IFN-gamma R1-blocking mAb. In conclusion, these data illustrate a new mechanism whereby human T cells limit the surface expression of IFN-gamma R2 in a ligand-independent manner.

Expression of Reg and cytokeratin 20 during ductal cell differentiation and proliferation in a mouse model of autoimmune diabetes.

OBJECTIVE: To evaluate the existence of beta-cell differentiation and proliferation in the low-dose streptozotocin (ld-STZ) mouse model of autoimmune diabetes. DESIGN: We studied the expression of Reg protein and cytokeratin 20 (CK20), the presence of proliferative phenomena (judged by the incorporation of bromodeoxyuridine (BrdU)), and the co-expression of Reg, CK20 or BrdU with insulin. MATERIALS AND METHODS: Diabetes was induced in male C57Bl6/J mice by administration of ld-STZ. The animals were killed at days 10 and 23 from the beginning of the induction of disease. Five animals were used at each time point and each group was evaluated for blood glucose concentrations, insulitis, expression of Reg and CK20 pancreatic proteins and BrdU incorporation, together with staining for insulin by immunohistochemistry and laser confocal microscopy. RESULTS: All mice treated with ld-STZ were hyperglycemic and histological investigation showed a mild or severe insulitis both at day 10 and at day 23. At day 10, immunochemistry revealed an intense expression of Reg and CK20 in pancreatic ducts in ld-STZ mice, but not in control mice. Reg and CK20 immunoreactive cells were also positive for insulin. In contrast, at day 23, pancreatic sections reacted weakly with anti-Reg and anti-CK20 antibody; co-localization with insulin was observed for both Reg and CK20. The incorporation of BrdU was observed only in insulin-positive cells in pancreatic sections from mice killed at day 10. CONCLUSIONS: These observations show an islet regeneration mechanism in response to an autoimmune attack, and that the ld-STZ mouse is a suitable model in which to evaluate intervention strategies.

Nitric oxide synthases in normal and benign hyperplastic human prostate: immunohistochemistry and molecular biology.

The expression of nitric oxide synthase (NOS) isoforms has been investigated in normal (three subjects) and benign hyperplastic prostate (ten patients) by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). The inducible NOS (iNOS or NOS-2) is not detected in normal prostate, while it is expressed in the prostate of all benign prostatic hyperplasia (BPH) patients, even in the absence of prostatitis or systemic signs of an inflammatory condition. This suggests that sex hormones may be involved in iNOS induction and that there may be a role for NO in the pathogenesis of BPH. Constitutive NOSs (nNOS and eNOS) are expressed in both normal and hyperplastic prostate and are co-expressed in epithelial cells. eNOS, however, is present mainly in the basal layer cells; nNOS seems abundantly expressed in the more superficial cells of the affected prostate. This indicates that the switching between the two constitutive isoforms may be part of the usual process of cell differentiation from the basal to the secretory layer of the epithelium.

Eps15 is recruited to the plasma membrane upon epidermal growth factor receptor activation and localizes to components of the endocytic pathway during receptor internalization.

Eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (EGFR) and is characterized by the presence of a novel protein:protein interaction domain, the EH domain. Eps15 also stably binds the clathrin adaptor protein complex AP-2. Previous work demonstrated an essential role for eps15 in receptor-mediated endocytosis. In this study we show that, upon activation of the EGFR kinase, eps15 undergoes dramatic relocalization consisting of 1) initial relocalization to the plasma membrane and 2) subsequent colocalization with the EGFR in various intracellular compartments of the endocytic pathway, with the notable exclusion of coated vesicles. Relocalization of eps15 is independent of its binding to the EGFR or of binding of the receptor to AP-2. Furthermore, eps15 appears to undergo tyrosine phosphorylation both at the plasma membrane and in a nocodazole-sensitive compartment, suggesting sustained phosphorylation in endocytic compartments. Our results are consistent with a model in which eps15 undergoes cycles of association:dissociation with membranes and suggest multiple roles for this protein in the endocytic pathway.