Metabolism and Trafficking of N-Type Voltage-Operated Calcium Channels in Neurosecretory Cells

The N-type voltage-operated calcium channel has been characterized over the years as a high-threshold channel, with variable inactivation kinetics, and a unique ability to bind with high affinity and specificity -conotoxin GVIA and related toxins. This channel is particularly expressed in some neurons and endocrine cells, where it participates in several calcium-dependent processes, including secretion. -conotoxin GVIA was instrumental not only for the biophysical and pharmacological characterization of N-type channels but also for the development of in vitro assays for studying N-type VOCC subcellular localization, biosynthesis, turnover, as well as short-and long-term regulation of its expression. We here summarize our studies on N-type VOCC expression in neurosecretory cells, with a major emphasis on recent data demonstrating the presence of N-type channels in intracellular secretory organelles and their recruitment to the cell surface during regulated exocytosis.     

Release-Regulating Serotonin 5-HT1D Autoreceptors in Human Cerebral Cortex

Abstract: Primary cultures of cerebral cortical astrocytes were incubated with [U^-13C]glutamate (0.5 m M ) in modified Dulbecco's medium for 2 h. Perchloric acid (PCA) extracts of the cells as well as redissolved lyophilized media were subjected to NMR spectroscopy to identify ¹³C-labeled metabolites. NMR spectra of the PCA extracts exhibited distinct multiplets for glutamate, aspartate, glutamine, and malate. The culture medium showed peaks for a multitude of compounds released from the astrocytes, among which lactate, glutamine, alanine, and citrate were readily identifiable. For the first time incorporation of label into lactate from glutamate was clearly demonstrated by doublet formation in the C-3 position and two doublets in the C-2 position of lactate. This labeling pattern can only occur by incorporation from glutamate, because natural abundance will only produce singlets in proton-decoupled ¹³C spectra. Glutamine, released into the medium, was labeled uniformly to a large extent, but the C-3 position not only showed the expected apparent triplet but also a doublet due to ¹³C incorporation into the C-4 position of glutamine. The doublet accounted for 11% of the total label in the glutamine synthesized and released within the incubation period. The corresponding labeling pattern of [¹³C]glutamate in the PCA extracts showed that 19% of the glutamate contained ¹²C. Labeling of lactate, citrate, malate, and aspartate as well as incorporation of ¹²C into uniformly labeled glutamate and glutamine could only arise via the tricarboxylic acid cycle. The relative amount of glutamate metabolized via this route is at least 70% as calculated from the areas of the C-3 resonances of these compounds. Only a maximum of 30% was converted to glutamine directly.     

Delayed-type hypersensitivity response in an isogenic murine model of paracoccidioidomycosis

The specific delayed-type hypersensitivity (DTH) response was evaluated in resistant (A/SN) and susceptible (B10.A) mice intraperitoneally infected with yeasts from a virulent (Pb18) or from a non-virulent (Pb265)Paracoccidioides brasiliensis isolates. Both strains of mice were footpad challenged with homologous antigens. Pb18 infected A/SN mice developed an evident and persistent DTH response late in the course of the disease (90th day on) whereas B10.A animals mounted a discrete and ephemeral DTH response at the 14th day post-infection. A/SN mice infected with Pb265 developed cellular immune responses whereas B10.A mice were almost always anergic. Histological analysis of the footpads of infected mice at 48 hours after challenge showed a mixed infiltrate consisting of predominantly mononuclear cells. Previous infection of resistant and susceptible mice with Pb18 did not alter their DTH responses against heterologous unrelated antigens (sheep red blood cells and dinitrofluorobenzene) indicating that the observed cellular anergy was antigen-specific. When fungal related antigens (candidin and histoplasmin) were tested in resistant mice, absence of cross-reactivity was noted. Thus, specific DTH responses againstP. brasiliensis depend on both the host's genetically determined resistance and the virulence of the fungal isolate.Abbreviations DTH delayed-type hypersensitivity - DNFB dinitrofluorobenzene - FN18 Fava Netto's antigen obtained from isolate Pb18 - FN265 Fava Netto's antigen obtained from isolate Pb265 - SRBC sheep red blood cells     

Organ-specific modulation of gene expression in transgenic plants using antisene RNA

We have shown leaf-specific inhibition GUS gene expression in transgenic Nicotiana plants using an antisense RNA with a 41-base homology spanning the translation start codon of the gene. GUS was expressed from the nominally constitutive 35S promoter and the antisense RNA was expressed from the light-regulated ca/b promoter of Arabidopsis thaliana. A range of GUS inhibition from 0 to 100% was obtained by screening a small population of transgenic plants and the specific levels of inhibition observed were stably inherited in two generations. An antiGUS gene dosage effect was observed in plants which were homozygous for antiGUS. RNA detection results suggest that duplex formation with the 41 base pair antiGUS RNA destabilized the GUS mRNA and that an excess of antisense. RNA was not required. Our results demonstrate the potential of antisense RNA as a strategy for obtaining plant mutants, especially down mutations in essential genes where only a short 5 sequence of the mRNA is required. They also suggest that the position effect on gene expression could be used in conjunction with an antisense RNA strategy to provide a versatile approach for crop improvement.     

Negative evidence in vivo of DNA-damaging, mutagenic and chromosomal effects of eugenol

The DNA-damaging, mutagenic and chromosomal effects of eugenol were assayed by the DNA alkaline elution technique, the granuloma pouch assay and the bone marrow micronucleus test in rats. With all the techniques used, eugenol did not show any genotoxic activity.     

Proteomic analysis of human low‐density lipoprotein reveals the presence of prenylcysteine lyase,a hydrogen peroxide‐generating enzyme

The molecular mechanisms underlying the relationship between low‐density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information on the protein composition of LDL may help to reveal its role in atherogenesis. Liquid‐phase IEF has been used to resolve LDL proteins into well‐defined fractions on the basis of pI, which improves the subsequent detection and resolution of low abundance proteins. Besides known LDL‐associated proteins, this approach revealed the presence of proteins not previously described to reside in LDL, including prenylcysteine lyase (PCL1), orosomucoid, retinol‐binding protein, and paraoxonase‐1. PCL1, an enzyme crucial for the degradation of prenylated proteins, generates free cysteine, isoprenoid aldehyde and hydrogen peroxide. Addition of the substrate farnesylcysteine to lipoprotein resulted in a time‐dependent generation of H₂O₂ which was stronger in very low density lipoprotein (VLDL) than in LDL or HDL, reflecting the greater protein content of PCL1 in VLDL. Farnesol, a dead end inhibitor of the PCL1 reaction, reduced H₂O₂ generation by VLDL. PCL1 is generated along with nascent lipoprotein, as shown by its presence in the lipoprotein secreted by HepG2 cells. The finding that an enzyme associated with atherogenic lipoproteins can itself generate an oxidant suggests that PCL1 may play a significant role in atherogenesis.     

Induction of autophagic cell death by a novel molecule is increased by hypoxia

Adaptation to hypoxia through activation of the hypoxia inducible factor-1 (HIF-1) is crucial for tumor cells survival. Here we describe the antitumoral effects of the new molecule CR 3294 on tumor cells in the presence of hypoxia. Treatment of the breast carcinoma cell line MDA-MB-231 with CR 3294 in 1% O(2) resulted in an in vivo and in vitro inhibition of tumor growth. CR 3294 induced accumulation of autophagosomes in hypoxic MDA-MB-231 cells as assessed by both transmission electron microscopy (TEM) and the autophagic marker LC3-II. TEM analysis revealed the presence of invaginations of the cytoplasm into the nucleus. Autophagosomes were present in such invaginations. Moreover, CR 3294 inhibited both the DNA binding of HIF-1alpha and VEGF mRNA synthesis. Immunoprecipitation and immunofluorescence studies showed an interaction between LC3 and HIF-1alpha. We next detailed the effect of inhibitors and activators of autophagy on both HIF-1alpha and LC3. In particular, 3 methyladenine (3MA) and wortmannin, two macroautophagic inhibitors, prevented both the decrease of HIF-1alpha protein levels and LC3 processing in cells treated with CR 3294. Bafilomycin and leupeptin, inhibitors of lysosomes, prevented HIF-1alpha decrease without affecting LC3 processing. By contrast, treating hypoxic MDA-MB-231 cells with trifluoperazine (TFP) or serum withdrawal (SW), two activators of autophagy, diminished HIF-1alpha levels and stimulated LC3 processing. These results indicate that activation of the autophagic pathway in hypoxic cells by the new molecule CR 3294, as well as by TFP or SW, can have potentially important implications for cancer treatment.     

Transcriptome of hypoxic immature dendritic cells: modulation of chemokine/receptor expression

Hypoxia is a condition of low oxygen tension occurring in inflammatory tissues. Dendritic cells (DC) are professional antigen-presenting cells whose differentiation, migration, and activities are intrinsically linked to the microenvironment. DCs will home and migrate through pathologic tissues before reaching their final destination in the lymph node. We studied the differentiation of human monocytes into immature DCs (iDCs) in a hypoxic microenvironment. We generated iDC in vitro under normoxic (iDCs) or hypoxic (Hi-DCs) conditions and examined the hypoxia-responsive element in the promoter, gene expression, and biochemical KEGG pathways. Hi-DCs had an interesting phenotype represented by up-regulation of genes associated with cell movement/migration. In addition, the Hi-DC cytokine/receptor pathway showed a dichotomy between down-regulated chemokines and up-regulated chemokine receptor mRNA expression. We showed that CCR3, CX3CR1, and CCR2 are hypoxia-inducible genes and that CCL18, CCL23, CCL26, CCL24, and CCL14 are inhibited by hypoxia. A strong chemotactic response to CCR2 and CXCR4 agonists distinguished Hi-DCs from iDCs at a functional level. The hypoxic microenvironment promotes the differentiation of Hi-DCs, which differs from iDCs for gene expression profile and function. The most prominent characteristic of Hi-DCs is the expression of a mobility/migratory rather than inflammatory phenotype. We speculate that Hi-DCs have the tendency to leave the hypoxic tissue and follow the chemokine gradient toward normoxic areas where they can mature and contribute to the inflammatory process.