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Biomedical subjects

D Aunis

Publications and source records attributed to D Aunis.

At least 37 records · Page 2Linked to original sources
PubMed2000

Cyclic AMP-elevating agents induce the expression of MAP kinase phosphatase-1 in PC12 cells.

Stimulation of pheochromocytoma PC12 cells by cAMP-elevating agents caused the induction of the immediate early gene 3CH134, which encodes MAP kinase phosphatase-1 (MKP-1). Forskolin was as potent as serum in stimulating MKP-1 gene expression, whereas dibutyryl-cAMP and neuropeptide PACAP were less effective. Induction of the MKP-1 gene was accompanied by neo-synthesis of MKP-1 protein. MAP kinase activation was not involved in the cAMP-induced MKP-1 gene expression. The MAP kinase inactivation, that would result from MKP-1 induction in response to increased intracellular cAMP level, contributes to explain how hormones or neurotransmitters signaling through cAMP influence cell growth and differentiation.

Animals↗
PubMed2000

Immunohistochemical studies of the localization of neurons containing the enzyme that synthesizes dopamine, GABA, or gamma-hydroxybutyrate in the rat substantia nigra and striatum.

gamma-Hydroxybutyrate (GHB) is an endogenous metabolite of gamma-aminobutyric acid (GABA), which is synthesized in the neuronal compartment of the central nervous system. This substance possesses several properties that support its role as a neurotransmitter/neuromodulator in brain. In particular, it is synthesized by a specific pathway that transforms GABA into succinic semialdehyde via GABA-T activity; then succinic semialdehyde is converted into GHB by a specific succinic semialdehyde reductase (SSR). The last enzyme is considered as a marker for neurons that synthesize GHB. This compound binds in brain to receptors whose distribution, ontogenesis, kinetics, and pharmacology are specific. Endogenous GHB, but also GHB exogenously administered to rats, participate in the regulation of dopaminergic activity of the nigrostriatal pathway. To investigate the distribution of GHB neurons in this pathway and the anatomic relationships between dopaminergic and GHB neurons, immunocytochemical identification of dopamine, GABA, and GHB neurons was carried out in the substantia nigra and striatum of the rat. The following markers for these neurons were used: anti-tyrosine hydroxylase (TH) antibodies for dopamine neurons, anti-glutamate decarboxylase (GAD) antibodies for GABA neurons, and anti-succinic semialdehyde reductase (SSR) antibodies for GHB neurons. GABA neurons were studied because GAD and SSR co-exist frequently in the same neuron, and GABA alone also exerts its own regulatory effects on dopaminergic neurons. This study reveals the co-existence of GAD/SSR and GAD/SSR/TH in numerous neurons of the substantia nigra. However, some neurons appear to be only GAD or SSR positive. In the striatum, TH-positive terminals surround many GHB neurons. GAD innervation is abundant in close contact with unlabeled neurons in the caudate-putamen, whereas distinct SSR-positive punctuates are also present. The existence of SSR-reactive synapses and neurons was confirmed in the striatum at the electron microscopic level. On the basis of these results, a clear anatomo-functional relationship between GHB and dopamine networks cannot be defined; however, we propose the modulation by GHB of striatal intrinsic neurons that could then interfere with the presynaptic control of dopaminergic activity.

Animals↗
PubMed2000

The presence of antibacterial and opioid peptides in human plasma during coronary artery bypass surgery.

Antibacterial peptides, found in both invertebrates and vertebrates, represent a potential innate defense mechanism against microbial infections. However, it is unknown whether this process occurs in humans during surgery. We looked for evidence of release of antibacterial peptides during coronary artery bypass grafting (CABG). We used immunological techniques and antibacterial assays combined with high-performance gel-permeation chromatography, reverse-phase HPLC, N-terminal sequencing and comparison with synthetic standards to characterize the peptide B/enkelytin. We show the presence of anionic antibacterial peptide, the peptide B/enkelytin which correspond to the C-terminal part of proenkephalin A, from the plasma of patients undergoing CABG. Our studies show that peptide B/enkelytin is initially present at low levels in plasma and is then released in increased amounts just after skin incision. Antibacterial assays confirmed that the peptides specifically target gram-positive bacteria. We also demonstrate that peptide B/enkelytin is metabolized in vivo to the opioid peptides methionine-enkephalin-Arg-Phe and methionine-enkephalin, peptides that we show have granulocyte chemotactic activity. These findings suggest that in humans, surgical incision leads to the release of antibacterial peptides. Furthermore, these antibacterial peptides can be metabolized into compounds that have immune-activating properties.

Amino Acid Sequence↗
PubMed2000

SNAP-25 regulation during adrenal gland development: comparison with differentiation markers and other SNAREs.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is one of a limited number of soluble N-ethylmaleimide-sensitive fusion attachment protein receptors (SNAREs) that play a major role in membrane docking of synaptic vesicles and secretory granules during regulated exocytosis. We have previously shown that SNAP-25 levels differ between noradrenergic and adrenergic chromaffin cell populations of the adult adrenal gland. We examine SNAP-25 expression by immunofluoresence in cells of the sympathoadrenal lineage in the rat during late embryonic and postnatal development. In parallel, tyrosine hydroxylase was used to identify sympathoadrenal cells, phenylethanolamine N-methyltransferase to distinguish adrenergic from noradrenergic chromaffin cells, and chromogranin A to define the presence of secretory granules. In addition, SNAP-25 protein and mRNA levels were followed in adrenal gland extracts by immunoblotting and reverse transcription-polymerase chain reaction (RT-PCR). Protein levels were compared with those of other molecules also implicated in organelle trafficking, including syntaxin 1 and vesicle-associated membrane protein (VAMP-2) and the nonneuronal analogues SNAP-23 and cellubrevin. This study provides evidence that SNAP-25 is expressed early during development in sympathoadrenal neurons and migrating cells. It is detected in intra-adrenal chromoblasts as soon as they enter the adrenal primordium. Its differential expression between catecholamine chromaffin cell phenotypes is already evident from the 17th embryonic day, future noradrenergic cells appearing to express higher levels than adrenergic cells. The granule maturation marker chromogranin A is expressed in chromaffin cells later than SNAP-25. Both SNAP-25 protein and mRNA increased rapidly in the adrenal gland in the perinatal period to peak during the first postnatal week, after which levels dropped dramatically to adult values. In contrast, levels of both syntaxin and SNAP-23 appeared to remain fairly constant throughout adrenal gland development. VAMP-2 expression increased gradually around birth to reach maximal levels during the first two postnatal weeks, and then decreased slightly. Cellubrevin levels also appeared to increase gradually until adult values were attained by the end of the second postnatal week. The threefold increase of SNAP-25 mRNA shortly after birth compared to the low adult levels suggests that during this period SNAP-25 is implicated in additional functions than regulated secretion, possibly associated with cellular growth or maturation.

Adrenal Glands↗
PubMed2000

Antibacterial and antifungal activities of vasostatin-1, the N-terminal fragment of chromogranin A.

Vasostatin-1, the natural N-terminal 1-76 chromogranin A (CGA)-derived fragment in bovine sequence, has been purified from chromaffin secretory granules and identified by sequencing and matrix-assisted laser desorption time-of-flight mass spectrometry. This peptide, which displays antibacterial activity against Gram-positive bacteria at micromolar concentrations, is also able to kill a large variety of filamentous fungi and yeast cells in the 1-10 microM range. We have found that the C-terminal moiety of vasostatin-1 is essential for the antifungal activity, and shorter active peptides have been synthesized. In addition, from the comparison with the activity displayed by related peptides (human recombinant and rat synthetic fragments), we could determine that antibacterial and antifungal activities have different structural requirements. To assess for such activities in vivo, CGA and CGA-derived fragments were identified in secretory material released from human polymorphonuclear neutrophils upon stimulation. Vasostatin-1, which is stored in a large variety of cells (endocrine, neuroendocrine, and neurons) and which is liberated from stimulated chromaffin and immune cells upon stress, may represent a new component active in innate immunity.

Amino Acid Sequence↗
PubMed2000

Proenkephalin A-derived peptides in invertebrate innate immune processes.

Lipopolysaccharides (LPS) injection into the coelomic fluid of the leech Theromyzon tessulatum stimulates release of proenkephalin A (PEA)-derived peptides as determined by immunoprecipitation and Western blot analyses. This release occurs in the first 15 min after LPS exposure and yields a 5.3-kDa peptide fragment corresponding to the C-terminal part of the precursor. This fragment is then cleaved to free an antibacterial peptide related to mammals arginine phenylalanine extended enkelytin: the peptide B. These PEA processing peptides were characterized using a combination of techniques including reversed-phase HPLC, microsequencing and mass spectrometry. The isolated invertebrate peptide B presents a high sequence homology with the bovine's and the same activity against Gram+bacteria. Titrations revealed the simultaneous appearance of Methionine-enkephalin (ME) and peptide B in invertebrates after stimulation by LPS (in a dose-dependent manner), surgical trauma or electrical stimulations to neural tissues of the mussel. Furthermore, peptide B processing in vitro yields Methionine-enkephalin arginine phenylalanine (MERF), which exhibits via the delta receptors, immunocyte excitatory properties, i.e., movement and conformational changes, but no antibacterial activity. We surmise that this unified response to the various stimuli is a survival strategy for organism by providing immediate antibacterial activity and immunocyte stimulation, thereby reducing any immune latency period needed for an adequate immune response.

Amino Acid Sequence↗
PubMed2000

The nuclear receptor chicken ovalbumin upstream promoter transcription factor interacts with HIV-1 Tat and stimulates viral replication in human microglial cells.

Human immunodeficiency virus type 1 (HIV-1) infects the central nervous system and plays a direct role in the pathogenesis of AIDS dementia. However, the molecular mechanisms underlying HIV-1 expression in the central nervous system are poorly understood. We have recently reported that the nuclear receptor chicken ovalbumin upstream promoter transcription factor (COUP-TF), an orphan member of the nuclear receptor superfamily, is an activator of HIV-1 gene transcription. Here, our results show that COUP-TF stimulates HIV-1 transcription in primary cultured human microglial cells, the primary target for HIV-1 infection in brain. Run-on assays indicated that COUP-TF acts on the initiation step of transcription. Results from reverse transcription-polymerase chain reaction and immunocytochemistry analysis further revealed the importance of this factor by demonstrating that overexpression of COUP-TF leads to initiation of viral replication in primary HIV-infected human microglia. In addition, COUP-TF is able to physically interact and cooperate with the viral transactivator Tat. The combination of COUP-TF and Tat leads to NF-kappaB- and Sp1-independent enhanced transcriptional stimulation. In vitro binding studies showed that COUP-TF interacts with Tat through amino acids within the N-terminal DNA-binding domain of COUP-TF. Amino acids 48-72 in the basic and C-terminal regions of Tat are required for the binding of Tat to COUP-TF. These results suggest that COUP-TF is an essential transcription factor involved in HIV-1 expression in microglia and reveal a novel interplay of Tat and COUP-TF during regulation of viral expression.

Animals↗
PubMed2000

Immunohistochemical expression of chromogranins A and B, prohormone convertases 2 and 3, and amidating enzyme in carcinoid tumors and pancreatic endocrine tumors.

Although chromogranin A (CgA) is widely distributed in neuroendocrine tumors, the distribution of chromogranin B (CgB) has not been elucidated. Hormones produced by tumors are sometimes prohormones and not necessarily bioactive hormones. Prohormones have to be processed into bioactive peptides by prohormone convertases (PCs), and some of them have to be amidated by peptidylglycine a-amidating monooxygenase (PGM). Whether PCs and PGM are present or not in tumors may explain why some tumors are functioning and some are nonfunctioning. We investigated 45 carcinoids and 16 pancreatic endocrine tumors. Of the carcinoids, CgA was expressed in most of the tumors, except for the rectal and ovarian carcinoids, which expressed CgB strongly. The expressions of PC2, PC3, and PGM were 31%, 100%, and 87%, respectively. In the pancreatic tumors, CgA was expressed in all tumors, whereas CgB was not expressed in any tumor. The expressions of PC2, PC3, and PGM were 63%, 88%, and 63%, respectively. PC3 was expressed in all of the functioning tumors but not in two of the four nonfunctioning tumors. PC2 and PGM were not expressed in three of the four nonfunctioning tumors. In conclusion, expression of CgA and CgB was different depending on the tumor location. High frequency of PCs and PGM may explain why even nonfunctioning tumors produce some inconspicuous peptides.

Adenoma, Islet Cell↗
PubMed2000

C-fos and egr-1 immediate-early gene induction by cocaine and cocaethylene in rat brain: a comparative study.

The induction of immediate-early genes can now be considered as a tool to study neuronal activation in different brain structures. These genes, which are rapidly and transiently induced in response to diverse extracellular stimulation, coordinate alterations in gene expression underlying neuronal plasticity. Using in situ hybridization, we found that acute i.p. cocaine (20 mg/kg) injection produced a strong expression of egr-1 and c-fos genes in the nucleus accumbens, caudate-putamen, and frontal cortex in the rat. Cocaethylene is an active metabolite of cocaine that is formed when cocaine is consumed together with ethyl alcohol. Injection of cocaethylene at a dose equivalent to cocaine induced the expression of the two immediate-early genes in the same brain structures, but to a lesser extent. A high dose of ethanol increased egr-1 and c-fos expression in the frontal cortex and in the lateral part of the caudate-putamen. Since cocaine is known to potently inhibit both dopamine and serotonin transporters, whereas cocaethylene only inhibits the dopamine transporter, our results strongly suggest that the serotonergic system participates in the mode of action of cocaine in its ability to trigger immediate-early gene transcription.

Animals↗
PubMed2000

Functional interactions between C/EBP, Sp1, and COUP-TF regulate human immunodeficiency virus type 1 gene transcription in human brain cells.

Human immunodeficiency virus type 1 (HIV-1) infects the central nervous system (CNS) and plays a direct role in the pathogenesis of AIDS dementia. However, mechanisms underlying HIV-1 gene expression in the CNS are poorly understood. The importance of CCAAT/enhancer binding proteins (C/EBP) for HIV-1 expression in cells of the immune system has been recently reported. In this study, we have examined the role and the molecular mechanisms by which proteins of the C/EBP family regulate HIV-1 gene transcription in human brain cells. We found that NF-IL6 acts as a potent activator of the long terminal repeat (LTR)-driven transcription in microglial and oligodendroglioma cells. In contrast, C/EBPgamma inhibits NF-IL6-induced activation. Consistent with previous data, our transient expression results show cell-type-specific NF-IL6-mediated transactivation. In glial cells, full activation needs the presence of the C/EBP binding sites; however, NF-IL6 is still able to function via the minimal -40/+80 region. In microglial cells, C/EBP sites are not essential, since NF-IL6 acts through the -68/+80 LTR region, containing two binding sites for the transcription factor Sp1. Moreover, we show that functional interactions between NF-IL6 and Sp1 lead to synergistic transcriptional activation of the LTR in oligodendroglioma and to mutual repression in microglial cells. We further demonstrate that NF-IL6 physically interacts with the nuclear receptor chicken ovalbumin upstream promoter transcription factor (COUP-TF), via its DNA binding domain, in vitro and in cells, which results in mutual transcriptional repression. These findings reveal how the interplay of NF-IL6 and C/EBPgamma, together with Sp1 and COUP-TF, regulates HIV-1 gene transcription in brain cells.

Brain↗
PubMed1999

CREB and COUP-TF mediate transcriptional activation of the human immunodeficiency virus type 1 genome in Jurkat T cells in response to cyclic AMP and dopamine.

Infection of lymphocytes by the human immunodeficiency virus type 1 (HIV-1) is associated with an increase in intracellular cAMP levels. Recent studies have shown that lymphocytes are able to synthesize and bind the dopamine, known to affect multiple cellular pathways, such as the cAMP pathway. Here we have investigated the molecular mechanisms by which cAMP and dopamine regulate HIV-1 gene transcription in Jurkat T cells. Transient expression experiments revealed that dopamine and forskolin lead to a synergistic stimulation of long terminal repeat (LTR)-driven transcription. This action is mediated through the cAMP response element binding (CREB) protein and chicken ovalbumin upstream promoter transcription factor (COUP-TF). CREB and COUP-TF act indirectly through the minimal -40/+80 and -68/+80 LTR region, respectively. We have previously demonstrated that COUP-TF stimulates HIV-1 transcription via the -68/+29 LTR region without direct DNA binding. Here, gel supershift experiments show that CREB does not directly bind to the -45/+85 proximal LTR sequences. Moreover, our data reveal novel functional interactions between COUP-TF and CREB, which lead to synergistic cAMP- and dopamine-induced transactivation of the HIV-1 LTR. These findings reveal that dopamine-induced signals and the cAMP pathway stimulate HIV-1 gene transcription in lymphocytes by converging to the minimal -68/+80 LTR region, through the transcription factors CREB and COUP-TF.

Binding Sites↗
PubMed1999

Dopamine stimulates expression of the human immunodeficiency virus type 1 via NF-kappaB in cells of the immune system.

Recent studies have reported that lymphocytes produce, transport and bind dopamine present in plasma. However, the action of dopamine on HIV-1 gene expression in cells of the immune system has not yet been examined. Here, we have investigated the regulation of HIV-1 expression by dopamine in Jurkat T cells and in primary blood mononuclear cells (PBMC). HIV-1 replication was increased by dopamine, which correlated with the increased levels of HIV-1 transactivation. Our transient expression data revealed that dopamine stimulated transcription through the NF-kappaB element present in the long terminal repeat. The importance of NF-kappaB sites was confirmed by using vectors containing wild-type or mutant kappaB sites in a heterologous promoter. Consistent with the role of NF-kappaB in mediating dopamine responsiveness, the proteasome inhibitor MG132 abolished dopamine-induced transcriptional activation. We further explored the effect of dopamine in the presence of phorbol esters or tumor necrosis factor-alpha (TNF-alpha) known to activate NF-kappaB. The combination of dopamine and TNF-alpha led to a stimulation of HIV-1 transcription and replication. However, in contrast with TNF-alpha, dopamine treatment did not affect NF-kappaB DNA binding activity nor the concentrations of p50, p65 and IkappaB-alpha proteins, which suggests a distinct NF-kappaB activation mechanism. These results reveal a new link between the dopamine system, cytokine signaling pathway and regulation of gene expression via the involvement of NF-kappaB in T cells and PBMC.

Dopamine↗
PubMed1999

Cultured glial cells express the SNAP-25 analogue SNAP-23.

Astrocytes release glutamate and aspartate in response to elevated intracellular calcium levels, and it has been proposed that this occurs by a vesicular release mechanism, in which SNARE proteins are implicated. Although syntaxin, synaptobrevin, and cellubrevin have been shown to be expressed by cultured astrocytes, SNAP-25 has not been detected. By using immunocytochemical, immunoblotting, and polymerase chain reaction techniques, the present study demonstrates that SNAP-23, an analogue of SNAP-25, is expressed by astrocytes both in culture and in rat cerebellum. These findings provide additional evidence that astrocytes release excitatory amino acids by a vesicular mechanism involving SNARE proteins. SNAP-23 and also syntaxin 1 and cellubrevin were found to be expressed in glial precursor cells, oligodendrocytes, and microglia. These data suggest that the t-SNAREs SNAP-23 and syntaxin 1 and the v-SNARE cellubrevin participate in general membrane insertion mechanisms involved in diverse glial cell functions such as secretion, phagocytosis, and myelinogenesis.

Animals↗
PubMed1999

A new human chromogranin A (CgA) immunoradiometric assay involving monoclonal antibodies raised against the unprocessed central domain (145-245).

Chromogranin A (CgA), a major protein of chromaffin granules, has been described as a potential marker for neuroendocrine tumours. Because of an extensive proteolysis which leads to a large heterogeneity of circulating fragments, its presence in blood has been assessed in most cases either by competitive immunoassays or with polyclonal antibodies. In the present study, 24 monoclonal antibodies were raised against native or recombinant human CgA. Their mapping with proteolytic peptides showed that they defined eight distinct epitopic groups which spanned two-thirds of the C-terminal part of human CgA. All monoclonal antibodies were tested by pair and compared with a reference radioimmunoassay (RIA) involving CGS06, one of the monoclonal antibodies against the 198-245 sequence. It appears that CgA C-terminal end seems to be highly affected by proteolysis and the association of C-terminal and median-part monoclonal antibodies is inadequate for total CgA assessment. Our new immunoradiometric assay involves two monoclonal antibodies, whose contiguous epitopes lie within the median 145-245 sequence. This assay allows a sensitive detection of total human CgA and correlates well with RIA because dibasic cleavage sites present in the central domain do not seem to be affected by degradation. It has been proved to be efficient in measuring CgA levels in patients with neuroendocrine tumours.

Amino Acid Sequence↗