Clinical, functional and genetic characterization of 16 patients suffering from chronic granulomatous disease variants - identification of 11 novel mutations in CYBB.

Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The most common form is the X-linked CGD (X91-CGD), caused by mutations in the CYBB gene. Clinical, functional and genetic characterizations of 16 CGD cases of male patients and their relatives were performed. We classified them as suffering from different variants of CGD (X910 , X91- or X91+ ), according to NADPH oxidase 2 (NOX2) expression and NADPH oxidase activity in neutrophils. Eleven mutations were novel (nine X910 -CGD and two X91- -CGD). One X910 -CGD was due to a new and extremely rare double missense mutation Thr208Arg-Thr503Ile. We investigated the pathological impact of each single mutation using stable transfection of each mutated cDNA in the NOX2 knock-out PLB-985 cell line. Both mutations leading to X91- -CGD were also novel; one deletion, c.-67delT, was localized in the promoter region of CYBB; the second c.253-1879A>G mutation activates a splicing donor site, which unveils a cryptic acceptor site leading to the inclusion of a 124-nucleotide pseudo-exon between exons 3 and 4 and responsible for the partial loss of NOX2 expression. Both X91- -CGD mutations were characterized by a low cytochrome b558 expression and a faint NADPH oxidase activity. The functional impact of new missense mutations is discussed in the context of a new three-dimensional model of the dehydrogenase domain of NOX2. Our study demonstrates that low NADPH oxidase activity found in both X91- -CGD patients correlates with mild clinical forms of CGD, whereas X910 -CGD and X91+ -CGD cases remain the most clinically severe forms.

[Starvation ketosis in a breastfeeding woman]. / Cétose de jeûne chez une femme allaitante.

INTRODUCTION: Bovine ketosis is a rare cause of metabolic acidosis. It is a starvation ketosis that appears in lactating woman. CASE REPORT: A 29-year-old woman had a previous gastric surgery one month ago while breastfeeding her 6-month child. She presented to emergency with dyspnea, fatigue, weight loss and anorexia. The explorations revealed a serious metabolic acidosis with a high anion gap, for which all other causes have been eliminated. CONCLUSION: A restrictive diet in lactating patients is a major risk of ketosis or bovine ketosis. Medico-surgical treatment of obesity during lactation seems unreasonable. Breastfeeding should be systematically sought before a medical and surgical management of obesity. With the spread of bariatric surgery, starvation ketosis is a cause of metabolic acidosis not to ignore.

[One case of type 1 auto-immune polyendocrinopathy or APECED]. / Polyendocrinopathie auto-immune de type 1 ou syndrome APECED.

INTRODUCTION: autoimmune polyendocrinopathy with candidiasis and ectodermal dystrophy (APECED) is a rare autosomal recessive disorder caused by mutations in the autoimmune regulator gene (AIRE). We report the case of a young girl with APECED. CASE REPORT: an 18 year-old girl born to consanguineous parents consulted for diffuse alopecia. Dermatological examination showed nail and dental enamel dystrophy and angular cheilitis. She had a history of mineralocorticoid deficiency (Addison's disease), hypoparathyroidism, hypogonadism and Biermer's disease, and she had also had chronic mucocutaneous candidiasis since childhood. She was presenting APECED with autoimmune endocrine failure, chronic mucocutaneous candidiasis and abnormalities of ectoderm-derived tissue. Analysis of mutation in the AIRE gene showed the c.769C>T homozygous mutation in exon 6. DISCUSSION: APECED, a rare autosomal recessive disorder, is a potentially life-threatening autoimmune disease. Chronic mucocutaneous candidiasis is a common and early feature in children. Dermatologists are likely to be the first physicians to diagnose this syndrome.

Dermatofibrosarcoma protuberans: a population-based cancer registry descriptive study of 66 consecutive cases diagnosed between 1982 and 2002.

BACKGROUND: Dermatofibrosarcoma protuberans (DFSP) is a rare malignant tumour of the skin, with an estimated incidence of 0.8 to five cases per 1 million people per year. OBJECTIVE: To study epidemiological, immunohistochemical and clinical features, delay in diagnosis, type of treatment and outcome of DFSP from 1982 to 2002. METHODS: Using data from the population-based cancer registry, 66 patients with pathologically proved DFSP were included (fibrosarcomatous DFSP were excluded). Each patient lived in one of the four departments of Franche-Comté (overall population of 1 million people) at the time of diagnosis. The main data sources came from public and private pathology laboratories and medical records. The rules of the International Agency for Research on Cancer were applied. RESULTS: The estimated incidence of DFSP in Franche-Comté was about three new cases per 1 million people per year. Male patients were affected 1.2 times as often as female patients were. The trunk (45%) followed by the proximal extremities (38%) were the most frequent locations. DFSP occurred mainly in young adults between 20 and 39 years of age. Mean age at diagnosis was 43 years, and the mean delay in diagnosis was 10.08 years. Our 66 patients initially underwent a radical local excision. Among them, 27% experienced one or more local recurrences during 9.6 years of follow-up. There was one regional lymph node recurrence without visceral metastases. These recurrences were significantly related to the initial peripheral resection margins. We observed a local recurrence rate of 47% for margins less than 3 cm, vs. only 7% for margins ranging from 3 to 5 cm [P=0.004; OR=0.229 (95%, CI=0.103-0.510)]. The mean time to a first local recurrence was 2.65 years. Nevertheless, there was no death due to the DFSP course at the end of the follow-up, and the final outcome was favourable. CONCLUSION: Our study emphasizes the importance of wide local excision with margins of at least 3 cm in order to prevent local recurrence. However, the recent development of inhibitors of signal transduction by the PDGFB pathway should soon modify the surgical strategy, which is often too mutilating.

[Incomplete expression of PHACE (S) syndrome: facial hemangioma associated with absence of the internal carotid artery]. / Syndrome PHACE (S) d'expression incomplète: hémangiome facial associé à une agénésie complète d'une artère carotide interne.

INTRODUCTION: Hemangiomas are the most common benign tumors in childhood. In rare cases, they can be associated with dysmorphic malformations. The acronym, the PHACE (S) syndrome, was recently described by Frieden et al. in 1996 as a large facial or cervical Hemangioma, associated with one or more of the following systemic abnormalities including:Posterior fossa malformation, Arterial abnormalities, Coarctation of the aorta and/or cardiac defects, Eye abnormalities and Sternal clefts. CASE REPORT: A 2 year-old girl presented with a large left hemifacial hemangioma. The rest of the clinical examination was normal. Initially, simple clinical surveillance was scheduled. The outcome was good with almost complete regression of the hemangioma by the age of 8. However, there were remains to the left of the upper lip and plastic surgery was scheduled. Pre-operative conventional arteriography revealed the complete, asymptomatic, absence of the ipsilateral internal carotid artery. Cerebral MRI and cardiac ultrasonography were normal. In the absence of somatic manifestations, regular clinical surveillance was decided on. DISCUSSION: Large facial or cervical hemagiomas can be associated with one or more systemic abnormalities described by the PHACE (S) acronym. Its prevalence is unknown, but is shows marked female preponderance. Among the systemic abnormalities, neurological and cardiac malformations predominate. Hence the PHACE (S) syndrome must be recognized. Moreover, in patients presenting with large facial or cervical hemangioma, the following examinations should be performed: neurological examination and cerebral MRI to rule out abnormality of the posterior fossa, completed by a sequence of angio-MRI in the search for cerebral artery malformations; cardiovascular exploration, completed by echocardiography in the case of doubt and examination of the eyes and sternum. Lastly, the enhanced risk of laryngeal sub-glottis hemangioma should be kept in mind.

Acid prohormone sequence determines size, shape, and docking of secretory vesicles in atrial myocytes.

How vesicles are born in the trans-Golgi network and reach their docking sites at the plasma membrane is still largely unknown and is investigated in the present study on live, primary cultured atrial cardiomyocytes. Secretory vesicles (n=422) are visualized by expressing fusion proteins of proatrial natriuretic peptide (proANP) and green fluorescent protein. Myocytes expressing fusion proteins with intact proANP display two populations of fluorescent vesicles with apparent diameters of 120 and 175 nm, moving at a top velocity of 0.3 microm/s. The number of docked vesicles is significantly correlated with the number of mobile vesicles (r=0.71, P<0.0005). The deletion of the acidic N-terminal proANP[1-44] or point mutations (glu(23,24)-->gln(23,24)) change size and shape-but not velocity-of the vesicles, and, strikingly, abolish their docking at the plasma membrane. The shapes thus change from spheres to larger, irregular floppy bags or vesicle trains. Deletion of the C-terminal proANP[45-127], where the ANP and its disulfide bond reside, does not change size, shape, docking, or velocity of the mobile vesicles. The N-terminal acid calcium-binding sequence of proANP is known to cause protein aggregation at the high calcium concentration prevailing in the trans-Golgi network. Therefore, these results indicate that amino acid residues favoring cargo aggregation are critically important in shaping the secretory vesicles and determining their fate-docking or not docking-at the plasma membrane. The full text of this article is available at http://www.circresaha.org.

Pituitary adenylate cyclase-activating polypeptide activates K(ATP) current in rat atrial myocytes.

Because the electrophysiological effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on the heart are little known, we studied the regulation of the atrial ATP-sensitive K(+) (K(ATP)) current by PACAP on primary cultured neonatal rat atrial myocytes. PACAP-38 stimulates cAMP production with EC(50) = 0.28 nmol/l (r = 0.92, P < 0.02). PACAP-38 and PACAP-27 (10 nmol/l) have similar maximal effects, whereas 100 nmol/l vasoactive intestinal polypeptide (VIP) is 2.7 times less effective (P < 0.05). RT-PCR shows the presence of cloned PACAP receptors PAC(1) (> or =2 isoforms), VPAC(1), and VPAC(2). PACAP-38 dose dependently activates the whole cell atrial K(ATP) current with EC(50) = 1-3 nmol/l (n = 44). Maximal effects occur at 10 nmol/l (91 +/- 15 pA/pF, n = 18). Diazoxide further increases the PACAP-activated current by 78% (P < 0.05; n = 6). H(89) (500 nmol/l), a protein kinase A (PKA) inhibitor, reduces the PACAP-activated K(ATP) current to 17.8 +/- 9.6% (n = 5) of the maximal diazoxide-induced current and totally inhibits the cAMP-induced K(ATP) current. A protein kinase C (PKC) inhibitor peptide (50 micromol/l) in the pipette reduces the PACAP-38-induced K(ATP) current to 33 +/- 17 pA/pF (P < 0.05, n = 6) without significantly affecting the currents induced by cAMP or VIP. The results suggest that: 1) PAC(1), VPAC(1), and VPAC(2) are present in atrial myocytes; and 2) PACAP-38 activates the atrial K(ATP) channels through both PKA and PKC pathways.

Soluble TNF receptors partially protect injured motoneurons in the postnatal CNS.

There is accumulating evidence that cytokines are involved in the functioning of the brain and the spinal cord. However, it has been controversial whether they exert a neurotoxic or a neuroprotective effect. To address this question in vivo, we have examined the survival of injured motoneurons in a line of transgenic mice that overexpress the soluble form of tumour necrosis factor receptor-1 (sTNFR1). In these animals, all of the circulating TNF and lymphotoxin-alpha are neutralized by the continuous expression of the soluble receptor. Following axotomy of the facial nerve in 7-day-old control mice, we observed a loss of approximately 90% of the motoneurons at two weeks survival. In the transgenic mice under the same conditions, the percentage of motoneuron survival was increased two-fold (515 vs. 224) and varied as a function of the level of the circulating receptor. These results indicate that neutralization of endogenous TNF and lymphotoxin-alpha by means of overexpression of the soluble receptor can decrease cell death of injured motoneurons and suggest that these cytokines may play an important role in neuronal degeneration in the CNS following a lesion.

IAP family proteins delay motoneuron cell death in vivo.

Neuronal apoptosis inhibitory protein (NAIP), and human inhibitors of apoptosis 1 and 2 (HIAP1 and HIAP2) are three members of the mammalian family of antiapoptosis proteins called 'inhibitors of apoptosis' (IAP). These molecules can prevent apoptosis in vitro and the over-expression of NAIP can decrease ischemic damage in the hippocampus. The goal of our experiments was to determine whether administration of NAIP, HIAP1 and HAIP2 could rescue motoneurons following axotomy of a peripheral nerve. In young rats, an adenoviral gene transfer technique was used to deliver and express these proteins in motoneurons; a fluorescent tracer was simultaneously added as a means for quantitatively assessing the rescue of fluorescently labelled motoneurons in serial sections of the lumbar spinal cord. Control experiments using adenoviral vectors (adv) expressing the lacZ gene showed that 14% of the sciatic motoneuron pool could be transfected indicating the existence of a subpopulation of spinal motoneurons susceptible to this class of viral vectors. The administration of an adv-NAIP, adv-HIAP1 and adv-HIAP2 rescued 30-40% of motoneurons at one week after sciatic axotomy. The efficiency of these proteins was similar to that of two neurotrophic factors, ciliary neurotrophic factor and brain-derived neurotrophic factor, administrated by the same viral technique. The effect of the IAP proteins on motoneuron survival decreased with time but was still present after 4 weeks postaxotomy; the duration of the response was dependent upon the viral titre. These experiments demonstrate that IAP family proteins can prevent motoneuron cell death in vivo and may offer a new therapeutic approach for motoneuron diseases.

NGF-induced motoneuron cell death depends on the genetic background and motoneuron sub-type.

Nerve growth factor (NGF) promotes the survival of several neuronal populations, but recently it has also been shown to induce neuronal cell death. Here we report the effects of NGF on lesioned motoneurons. We have analyzed facial and sciatic motoneurons in newborn and adult BALB/c and C57BL/6 mice, in addition to mice deficient in the low-affinity p75 receptor for the neurotrophins (p75NTR). NGF application did not alter survival of lesioned facial motoneurons in any of the strains examined independent of the age of the animals. Only in the adult C57BL/6 mouse strain where the sciatic nerve had been crushed prior to factor application did NGF induce cell death of axotomized sciatic motoneurons. Our results illustrate the importance of the genetic background and the motoneuron sub-type in studies related to cell death and survival of motoneurons in relation to NGF and p75NTR.

A novel K(ATP) current in cultured neonatal rat atrial appendage cardiomyocytes.

The functional and pharmacological properties of ATP-sensitive K(+) (K(ATP)) channels were studied in primary cultured neonatal rat atrial appendage cardiomyocytes. Activation of a whole-cell inward rectifying K(+) current depended on the pipette ATP concentration and correlated with a membrane hyperpolarization close to the K(+) equilibrium potential. The K(ATP) current could be activated either spontaneously or by a hypotonic stretch of the membrane induced by lowering the osmolality of the bathing solution from 290 to 260 mOsm/kg H(2)O or by the K(+) channel openers diazoxide and cromakalim with EC(50) approximately 1 and 10 nmol/L, respectively. The activated atrial K(ATP) current was highly sensitive to glyburide, with an IC(50) of 1.22+/-0.15 nmol/L. Recorded in inside-out patches, the neonatal atrial K(ATP) channel displayed a conductance of 58.0+/-2.2 pS and opened in bursts of 133.8+/-20.4 ms duration, with an open time duration of 1.40+/-0.10 ms and a close time duration of 0.66+/-0.04 ms for negative potentials. The channel had a half-maximal open probability at 0.1 mmol/L ATP, was activated by 100 micromol/L diazoxide, and was inhibited by glyburide, with an IC(50) in the nanomolar range. Thus, pending further tests at low concentrations of K(ATP) channel openers, the single-channel data confirm the results obtained with whole-cell recordings. The neonatal atrial appendage K(ATP) channel thus shows a unique functional and pharmacological profile resembling the pancreatic beta-cell channel for its high affinity for glyburide and diazoxide and for its conductance, but also resembling the ventricular channel subtype for its high affinity for cromakalim, its burst duration, and its sensitivity to ATP. Reverse transcriptase-polymerase chain reaction experiments showed the expression of Kir6.1, Kir6.2, SUR1A, SUR1B, SUR2A, and SUR2B subunits, a finding supporting the hypothesis that the neonatal atrial K(ATP) channel corresponds to a novel heteromultimeric association of K(ATP) channel subunits.

Reactions of 1-methyl-2-phenylindole with malondialdehyde and 4-hydroxyalkenals. Analytical applications to a colorimetric assay of lipid peroxidation.

Under acidic and mild-temperature conditions, 1-methyl-2-phenylindole was found to react with malondialdehyde (MDA) and 4-hydroxyalkenals to yield a stable chromophore with intense maximal absorbance at 586 nm. The use of methanesulfonic acid results in optimal yields of chromophore produced from MDA as well as from 4-hydroxynonenal. By contrast, the use of hydrochloric acid results in an optimal yield of chromophore produced from MDA and a negligible reaction of 4-hydroxynonenal. Taking advantage of such chromogenic reactions, we developed a new colorimetric assay of lipid peroxidation. Using a methanesulfonic acid-based medium, MDA and 4-hydroxyalkenals can be measured at the 586 nm wavelength. However, the presence of endogenous inhibitors of the reaction with 4-hydroxyalkenals is common, and this means that the latter may be underestimated in some biological samples. The assay performed in a hydrochloric acid-based medium enables the specific measurement of MDA in the presence of 4-hydroxyalkenals. Upon hydrolysis of Schiff bases in hydrochloric acid (pH 1.5), either assay can be used to specifically measure the amount of total MDA in biological samples because 4-hydroxyalkenals undergo an irreversible cyclization reaction under the hydrochloric acid-based conditions of hydrolysis. The two assays were applied to the determination of the amount of MDA alone and of MDA and 4-hydroxyalkenals in an in vitro model of lipid peroxidation. This methodology was also used to clarify complex patterns of tissue-specific MDA production in vivo, following hydrolysis of Schiff bases, in rodents treated with doxorubicin.

Reactions of N-methyl-2-phenylindole with malondialdehyde and 4-hydroxyalkenals. Mechanistic aspects of the colorimetric assay of lipid peroxidation.

Under specific acidic conditions, both malondialdehyde (1, MDA) and 4-hydroxynonenal (2, 4-HNE) react with N-methyl-2-phenylindole (3) to give the same chromophoric cyanine 4 with maximal absorbance at 586 nm. Under such conditions, the reaction of 3 with 4-HNE (2) as well as with alkanals yields a second chromophoric cyanine 10 with maximal absorbance at 505 nm. The influence of different acids, iron(III), and oxygen on the reaction of 3 with such aldehydes was studied in detail. Under anaerobic conditions, the acid-induced reaction of 4-HNE with 3 afforded three rapidly interconverting intermediates, 5-7. Their subsequent fragmentation to 4 and hexanal in the presence of iron(III) and oxygen is consistent with the tandem beta-fragmentation of an indolyl radical cation. 1-Indolylalkenes were identified as essential intermediates in the acid-induced reaction of 3 with alkanals. A very mild iron(III)-catalyzed fragmentation of these intermediates afforded the corresponding 3-formylindole 11 as the direct precursor of the 505 nm chromophore 10. Such reactions were markedly influenced by the nature of the acid. Contrary to the rapid chromogenic reaction of 4-HNE which was observed in the presence of methanesulfonic acid, the HCl-induced reaction of 4-HNE with 3 did not afford the 586 nm chromophore. Furthermore, hexanal did not yield the 505 nm chromophore 10 upon reaction with 3 in the presence of HCl, again in contrast with the rapid chromogenic reaction which was observed in the presence of methanesulfonic acid. Comparison of the reaction mixtures under the two assay conditions confirmed that the same intermediates were formed. We conclude that the nature of the acid plays a crucial role in the oxidative fragmentation of intermediates into chromophores, allowing the selective assay of MDA in the presence of 4-HNE, using HCl acidic conditions.

Pituitary adenylate cyclase-activating polypeptide stimulates proenkephalin gene transcription through AP1- and CREB-dependent mechanisms.

The effects of the pituitary adenylase cyclate-activating peptides (PACAP) 27 and 38 on proenkephalin (PENK) gene transcription were examined in PC12 (rat pheochromocytoma) cells using transient transfection assays. Both ligands stimulated PENK gene transcription in a dose-dependent manner, with an apparent ED50 close to 5 x 10(-11) M. Inactivation of cAMP dependent-protein kinase (PKA) with a dominant inhibitory mutant strongly reduced PACAP-stimulated PENK transcription. Using reporter genes driven by either the minimal TPA-responsive element (TRE: TGACTCA) or cAMP-responsive element (CRE: TGACGTCA), we showed that the two PACAPs activate transcription through both regulatory sequences. These effects could result from direct post-translational activation of Jun and CREB, as shown using GAL4-Jun or GAL4-CREB fusion proteins. Expression of a dominant inhibitory mutant of CREB decreased by 60% the response to PACAP, suggesting that CREB is implicated in PENK transactivation. Similarly, expression of c-fos antisense RNA reduced by 80% the stimulatory effects of PACAP. Taken together, these results indicate that PACAP stimulates PENK transcription by members of both the AP1 and the CREB families. However, AP1 by itself is not sufficient to increase PENK transcription, as insulin-like growth factor 1 (IGF1), which stimulates AP1 activity but not cAMP production, is unable to stimulate PENK transcription. These results indicate a cooperative effect of AP1 and CREB on PENK transcription.

Pituitary adenylate cyclase-activating polypeptide triggers dual transduction signaling in CATH.a cells and transcriptionally activates tyrosine hydroxylase and c-fos expression.

We used a catecholaminergic neuron-like cell line (CATH.a cells) as a model system to investigate the likelihood that pituitary adenylate cyclase-activating polypeptide (PACAP) may participate in the regulation of specific gene expression in catecholaminergic neurons. Analysis by reverse transcriptase-PCR amplification revealed the presence in these cells of type I PACAP receptors, with a short isoform, together with a heavier so-called Hop splice variant. PACAP38 and PACAP27 enhanced, in a dose-dependent manner, both cyclic AMP formation and phosphoinositide breakdown, with EC50 values of, respectively, 0.6 x 10(-10) and 2 x 10(-9) M. These peptides, in addition, also elevated [Ca2+]i by mobilizing intracellular calcium pools. Vasoactive intestinal peptide (VIP) was approximately 1,000-fold less potent in stimulating cyclic AMP (with EC50 = 2 x 10(-7) M) and failed to change the turnover of phosphoinositides and to alter [Ca2+]i. Both forms of PACAP, as well as forskolin, stimulated transcriptional induction of tyrosine hydroxylase (TH) and c-fos promoters fused to a chloramphenicol acetyltransferase (CAT) reporter gene in transiently transfected cells (p < 0.01 vs. controls). Induction of CAT activity linked to both TH and c-fos promoters was obliterated upon coexpression of a dominant inhibitory mutant (Mt-RAB) of cyclic AMP-dependent protein kinase. We conclude that CATH.a cells do express functional PACAP type I receptors, the activation of which impinges on TH and c-fos transcription according to a process that is primarily dependent on the cyclic AMP-PKA pathway.

PACAP type I receptor activation promotes cerebellar neuron survival through the cAMP/PKA signaling pathway.

The molecular nature, transduction pathways, and neurotrophic functions of pituitary adenylate cyclase activating peptide (PACAP) receptors were studied in primary culture of rat cerebellar granule cells. We show that cerebellar neurons express several PACAP type I receptor (PVR I) isoforms, including the short (PVR Is) and the Hop (PVR I-Hop) splice variants, the latter being restricted to neurons and not found in cerebellar glial cell cultures. In vitro, cerebellar granule cells die rapidly in the absence of a high concentration of K+ (25 mM), as demonstrated by TUNEL histochemistry, which shows that K+ deprivation induces massive neuronal apoptosis within 12 hr. This effect was reversed by PACAP 27 and 38. Both forms of PACAP prevent DNA fragmentation and allow long-term neuronal survival in the absence of high K+ (as shown by MAP2 immunostaining) and stimulate a reporter gene driven by the full-length c-fos promoter. These effects of PACAP are fully abolished upon transient transfection of cells with a dominant inhibitory mutant of the cAMP-dependent protein kinase (PKA). Taken together, these results show that in cerebellar granule neurons, PACAP type I receptors regulate gene expression and promote neuronal survival through the cAMP/PKA pathway.

Evidence for the presence of receptors for pituitary adenylate cyclase-activating polypeptide in the neurohypophysis that are positively coupled to cyclic AMP formation and neurohypophyseal hormone secretion.

This study examines the neural lobe of the pituitary gland for the presence of receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) and their possible involvement in the regulation of neurosecretion. The presence of PACAP receptors of type I was revealed in the neural lobe, as well as in anterior and intermediate lobes, by means of RT-PCR amplification using selective oligonucleotide pairs of primers. They appeared to be expressed in the tissues as a short form together with an isoform of heavier molecular weight. Activation of receptors in the presence of PACAP stimulated both formation of cyclic AMP (cAMP) and secretion of arginine vasopressin (AVP) in neural lobes, in a dose-related fashion, with half-maximum (EC50) values of 1.0 +/- 0.2 x 10(-9) M and 1.4 +/- 0.3 x 10(-8) M, respectively. Parallel with AVP, PACAP also stimulated oxytocin (OXT) output, with an EC50 value of 0.6 +/- 0.1 x 10(-8) M. In an attempt to localize receptors on cells (mainly astrocyte-like glials or pituicytes) and/or on nerve fibers of the gland, we used cultures of neural lobe cells and explants (in which nerve fibers undergo degeneration), as well as isolated nerve endings. In both cells and nerve terminals, PACAP enhanced accumulation of cAMP, while it triggered AVP secretion from the latter. The stimulatory effect of PACAP on both AVP and OXT release was mimicked by dbcAMP and blocked by H89, an inhibitor of cAMP-dependent protein kinase. We conclude that in the neural lobe, PACAP receptors are localized on both nerve terminals and pituicytes, which participate in the modulation of secretion of neurohypophyseal hormones in an interactive way and mainly through the cAMP signalling route.

Pituitary adenylate cyclase-activating polypeptide transduces through cAMP/PKA and PKC pathways and stimulates proopiomelanocortin gene transcription in mouse melanotropes.

Pituitary adenylate cyclase-activating polypeptide (PACAP) receptors were characterized and their function investigated in mouse pituitary neurointermediate lobe explants. We show that mouse neurointermediate lobes can be maintained for up to 1 month in defined medium. After 8 days in culture, these explants are devoid of any of the original tyrosine hydroxylase or glutamate decarboxylase immunoreactive fibers, which in situ innervate the melanotropes. Under these culture conditions, no mitotic activity is detectable in melanotropes and these cells remain sensitive to physiological regulation such as dopamine and corticotropin-releasing hormone. Using in situ hybridization and polymerase chain reaction, we show that in situ and in neurointermediate lobe explants, melanotropes express PACAP receptor type I isoforms that transduce through the cAMP and inositol phosphate pathways. In neurointermediate lobe explants, PACAP 27 and PACAP 38 (10(-8) M) stimulate cAMP accumulation whereas PACAP 38 but not PACAP 27 stimulates inositol phosphate breakdown. However, both ligands are potent stimulators of proopiomelanocortin (POMC)-derived peptides exocytosis and POMC gene transcription. In addition, stimulation of POMC gene transcription is mediated both by cAMP and by inositol phosphate pathways. Taken together, our data suggest that PACAP is a major regulator of melanotrope functions.

[Metabolism and antioxidant function of glutathione]. / Métabolisme et fonction antioxydante du glutathion.

Glutathione (gamma-glutamyl-cysteinyl-glycine or GSH) is a cysteine-containing tripeptide with reducing and nucleophilic properties which play an important role in cellular protection from oxidative damage of lipids, proteins and nucleic acids. GSH regulates the metabolism of proteins and their activities by means of thiol-disulfide exchange. During oxidative stress, GSH plays a key role of protection and detoxification as a cofactor of glutathione peroxidases and glutathione-S-transferases. There are synergistic interactions between GSH and other components of the antioxidant defense system such as vitamin C, vitamin E and superoxide dismutases.

Corticotropin-releasing hormone stimulates proopiomelanocortin transcription by cFos-dependent and -independent pathways: characterization of an AP1 site in exon 1.

The POMC gene, encoding a hormonal precursor protein, is primarily expressed in the pituitary in a tissue-specific manner. The POMC gene is transcriptionally regulated by a variety of hormones and neuropeptides and the second messengers cAMP and Ca++. Using the corticotrope-derived AtT20 cell line, we have previously shown that overexpression of cFos stimulates POMC transcription. The aim of this work was to analyze whether cFos directly interacts with the POMC gene in basal and corticotropin-releasing hormone (CRH) stimulated cells. Using progressively deleted POMC promoter sequences or heterologous promoter constructs coupled to the chloramphenicol acetyl transferase reporter gene, we demonstrate the existence of a major cFos- responsive sequence within the first exon of the POMC gene. This sequence, TGACTAA, appears functionally indistinguishable from the canonical AP1 binding site. When fused to a minimal promoter, this sequence confers inducibility by cFos and CRH. Gel shift analyses with CRH-stimulated AtT20 nuclear extracts or in vitro synthesized proteins revealed that this sequence efficiently binds Fos and Jun. Expression of c-fos anti-sense mRNA reduced CRH-stimulated POMC transcription, thus indicating that, at least in part, cFos mediates the effect of CRH on POMC transcription. However, deletion of this major exonic AP1 site from the POMC constructs greatly reduced the effect of c-fos overexpression but did not suppress POMC stimulation by CRH, indicating that CRH stimulates POMC transcription by one or more cFos-independent mechanism(s).