In vivo and in vitro development of alpha-MSH and ACTH in the embryonic and postnatal rat brain.

The appearance of immunoreactive alpha-melanotropin (alpha-MSH) and adrenocorticotropin (ACTH) during development was studied in 3 areas of the rat brain--cerebral hemispheres, midbrain and hindbrain--from embryonic day (ED) 13-14 until day 21 postnatally. The alpha-MSH content in vivo was always highest in the midbrain; a peak content at birth was followed by a transient decline and a later, higher plateau from postnatal day 7 onwards. The alpha-MSH content in the cerebral hemispheres rose progressively after birth reaching a peak at day 21. Values in the hindbrain rose at day 3 and changed relatively sue taken at ED 15-16 showed a gradual increase in alpha-MSH content over the 20 days. The alpha-MSH content of hindbrain cultures remained at constant low levels, while no alpha-MSH was detectable in cerebral hemisphere cultures. ACTH appeared in vivo earlier than alpha-MSH and was detectable in embryonic brains at ED 13-14. A transient rise was seen at ED 17-18 and major peaks at birth, day 2 and day 3, in the midbrain, hemispheres and hindbrain, respectively. In vitro, the ACTH content increased in all brain regions during the first 5 days in culture and showed no further change thereafter. Comparisons of the in vivo and in vitro development of alpha-MSH and ACTH demonstrate that (i) these two peptide systems are independent in respect to their localization and time of appearance; (ii) they undergo maturation both in vivo and in vitro; (iii) epigenetic factors, such as interactions with other neurotransmitter systems may modulate the developmental pattern of these two peptides.

Rhizosphere properties of rice genotypes as influenced by anoxia and availability of zinc and iron

The objective of this work was to study possible mechanisms involved in root-induced changes of rhizosphere physicochemical properties of rice genotypes, under anoxia and low supply of Zn and Fe. Two rice genotypes, including an upland and a lowland ones, were grown in hydroponic medium under adequate and low supply of Zn and Fe, with or without aeration. Anoxia increased shoot dry weight, root length and uptake of Zn and Fe in lowland Amol genotype, but reduced these parameters in upland Gasrol-Dashti genotype. The amount of oxygen released by roots was statistically higher in 'Amol'. The highest acidification potential of roots was observed in the lowland genotype under low supply of Zn, and in the upland genotype under Fe starvation. The highest oxalate (only organic acid detected) exudation from roots was observed in Zn and Fe deficient Gasrol-Dashti genotype. Zinc deficiency caused reduction of alcohol dehydrogenase and stimulation of lactate dehydrogenase activity, particularly in shoot. The ability to induce changes in the rhizosphere properties has a great contribution for the adaptation of both lowland and upland rice genotypes to specific soil conditions.

A comparison of alpha and gamma spectrometry for environmental natural radioactivity surveys.

An alpha-spectrometry, using automated borate fusion and sequential extraction and exchange chromatography, was used to determine the uranium and thorium based on environmental radioactivity of 20 soil samples. The same set of the samples was analysed using gamma-spectrometry with an HPGe detector. The two data sets were checked for coherence using Z-score and chi2 statistical tests. We show that gamma-spectrometry is a valid alternative to time-consuming alpha-spectrometry for the determination of natural uranium and thorium activity in soil (activity range: 12.5-58.2 Bq/kg). The measured activities were compared with the theoretical activities to ensure secular equilibrium in the 238U and 232Th series. For 226Ra, a special study was made on deconvolution of the 186 keV multiplet with the Levenberg-Marquardt algorithm. Finally, the combined use of Z-score and chi2-tests was found to be a powerful tool for comparing the results obtained with two different methods.

Antagonistic regulation of a proline-rich transcription factor by transforming growth factor beta and tumor necrosis factor alpha.

Transforming growth factor beta (TGF-beta) and tumor necrosis factor alpha (TNF-alpha) often exhibit antagonistic actions on the regulation of various activities such as immune responses, cell growth, and gene expression. However, the molecular mechanisms involved in the mutually opposing effects of TGF-beta and TNF-alpha are unknown. Here, we report that binding sites for the transcription factor CTF/NF-I mediate antagonistic TGF-beta and TNF-alpha transcriptional regulation in NIH3T3 fibroblasts. TGF-beta induces the proline-rich transactivation domain of specific CTF/NF-I family members, such as CTF-1, whereas TNF-alpha represses both the uninduced as well as the TGF-beta-induced CTF-1 transcriptional activity. CTF-1 is thus the first transcription factor reported to be repressed by TNF-alpha. The previously identified TGF-beta-responsive domain in the proline-rich transcriptional activation sequence of CTF-1 mediates both transcriptional induction and repression by the two growth factors. Analysis of potential signal transduction intermediates does not support a role for known mediators of TNF-alpha action, such as arachidonic acid, in CTF-1 regulation. However, overexpression of oncogenic forms of the small GTPase Ras or of the Raf-1 kinase represses CTF-1 transcriptional activity, as does TNF-alpha. Furthermore, TNF-alpha is unable to repress CTF-1 activity in NIH3T3 cells overexpressing ras or raf, suggesting that TNF-alpha regulates CTF-1 by a Ras-Raf kinase-dependent pathway. Mutagenesis studies demonstrated that the CTF-1 TGF-beta-responsive domain is not the primary target of regulatory phosphorylations. Interestingly, however, the domain mediating TGF-beta and TNF-alpha antagonistic regulation overlapped precisely the previously identified histone H3 interaction domain of CTF-1. These results identify CTF-1 as a molecular target of mutually antagonistic TGF-beta and TNF-alpha regulation, and they further suggest a molecular mechanism for the opposing effects of these growth factors on gene expression.

Insulin resistance in mice lacking neuronal nitric oxide synthase is related to an alpha-adrenergic mechanism

Rapport de synthèse : Le monoxyde d'azote (NO) joue un rôle important dans la régulation de l'homéostasie du système cardiovasculaire et du glucose. Les souris déficientes pour le gène codant l'isoforme neuronale de la synthase de monoxyde d'azote (nNOS) sont résistantes à l'insuline, mais les mécanismes sous-jacents sont inconnus. Le manque de NO produit par la nNOS pourrait être à l'origine d'une diminution de la perfusion du muscle squelettique et ainsi d'une diminution de l'apport de substrat. Alternativement, le déficit de nNOS normalement hautement exprimé dans le tissu musculaire squelettique pourrait directement y perturber la consommation de glucose. Finalement l'absence de l'action sympatholytique du NO neuronal pourrait diminuer la sensibilité à l'insuline. Afin de tester ces hypothèses nous avons étudié, chez des souris déficientes en nNOS et des souris-contrôle, la consommation corporelle totale de glucose et le flux musculaire squelettique pendant des clamps hyperinsulinémiques euglycémiques in vivo, ainsi que la consommation de glucose dans le muscle squelettique in vitro. De plus nous avons analysé les effets d'une inhibition alpha-adrénergique sur la consommation de glucose pendant les clamps hyperinsulinémiques euglycémiques in vivo. Le taux de perfusion de glucose pendant les clamps était grossièrement 15 pourcent plus bas (P<0.001) chez les souris déficientes en nNOS que chez les souris-contrôle. Cette résistance à l'insuline chez les souris déficientes en nNOS n'était due ni à une stimulation déficiente du flux sanguin musculaire par l'insuline ni à un défaut intrinsèque de la consommation de glucose du muscle (qui étaient comparables dans les deux groupes), mais à un mécanisme alpha-adrénergique, car l'administration de phentolamine rétablissait la sensibilité à l'insuline chez les souris déficientes en nNOS. Ces résultats suggèrent qu'une hyperactivité sympathique, potentiellement due à la perte de l'inhibition neuronale centrale du flux sympathique par le NO provenant de nNOS, contribue à la résistance à l'insuline des souris déficientes en nNOS. Par ailleurs ces résultats tendent à prouver qu'un défaut de production de NO provoquerait une résistance à l'insuline par des mécanismes différents selon l'isoforme de NO synthase déficiente (par exemple chez les souris déficientes pour la forme endothéliale de NO synthase, il a été montré que la résistance à l'insuline est due à un défaut de stimulation de la perfusion musculaire par l'insuline et à un défaut du signalling de l'insuline dans la cellule musculaire squelettique). Chez l'être humain il est établi que les états de résistance à l'insuline sont associés à une synthèse défectueuse et/ou une mauvaise biodisponibilité du NO, ainsi qu'à une hyperactivité sympathique. Nous spéculons que la perte d'inhibition centrale du flux sympathique représente un mécanisme contribuant à la résistance à l'insuline et ses complications cardiovasculaires chez l'être humain.

Co- and posttranslational modification of the alpha(1B)-adrenergic receptor: effects on receptor expression and function.

We have characterized the maturation, co- and posttranslational modifications, and functional properties of the alpha(1B)-adrenergic receptor (AR) expressed in different mammalian cells transfected using conventional approaches or the Semliki Forest virus system. We found that the alpha(1B)-AR undergoes N-linked glycosylation as demonstrated by its sensitivity to endoglycosidases and by the effect of tunicamycin on receptor maturation. Pulse-chase labeling experiments in BHK-21 cells demonstrate that the alpha(1B)-AR is synthesized as a 70 kDa core glycosylated precursor that is converted to the 90 kDa mature form of the receptor with a half-time of approximately 2 h. N-Linked glycosylation of the alpha(1B)-AR occurs at four asparagines on the N-terminus of the receptor. Mutations of the N-linked glycosylation sites did not have a significant effect on receptor function or expression. Surprisingly, receptor mutants lacking N-linked glycosylation migrated as heterogeneous bands in SDS-PAGE. Our findings demonstrate that N-linked glycosylation and phosphorylation, but not palmitoylation or O-linked glycosylation, contribute to the structural heterogeneity of the alpha(1B)-AR as it is observed in SDS-PAGE. The modifications found are similar in the different mammalian expression systems explored. Our findings indicate that the Semliki Forest virus system can provide large amounts of functional and fully glycosylated alpha(1B)-AR protein suitable for biochemical and structural studies. The results of this study contribute to elucidate the basic steps involved in the processing of G protein-coupled receptors as well as to optimize strategies for their overexpression.

Tumor necrosis factor-alpha mediates changes in tissue protein turnover in a rat cancer cachexia model.

Rats bearing the Yoshida AH-130 ascites hepatoma showed enhanced fractional rates of protein degradation in gastrocnemius muscle, heart, and liver, while fractional synthesis rates were similar to those in non-tumor bearing rats. This hypercatabolic pattern was associated with marked perturbations of the hormonal homeostasis and presence of tumor necrosis factor in the circulation. The daily administration of a goat anti-murine TNF IgG to tumor-bearing rats decreased protein degradation rates in skeletal muscle, heart, and liver as compared with tumor-bearing rats receiving a nonimmune goat IgG. The anti-TNF treatment was also effective in attenuating early perturbations in insulin and corticosterone homeostasis. Although these results suggest that tumor necrosis factor plays a significant role in mediating the changes in protein turnover and hormone levels elicited by tumor growth, the inability of such treatment to prevent a reduction in body weight implies that other mediators or tumor-related events were also involved.

Enrichment of human CD4+ V(alpha)24/Vbeta11 invariant NKT cells in intrahepatic malignant tumors.

Invariant NKT cells (iNKT cells) recognize glycolipid Ags via an invariant TCR alpha-chain and play a central role in various immune responses. Although human CD4(+) and CD4(-) iNKT cell subsets both produce Th1 cytokines, the CD4(+) subset displays an enhanced ability to secrete Th2 cytokines and shows regulatory activity. We performed an ex vivo analysis of blood, liver, and tumor iNKT cells from patients with hepatocellular carcinoma and metastases from uveal melanoma or colon carcinoma. Frequencies of Valpha24/Vbeta11 iNKT cells were increased in tumors, especially in patients with hepatocellular carcinoma. The proportions of CD4(+), double negative, and CD8alpha(+) iNKT cell subsets in the blood of patients were similar to those of healthy donors. However, we consistently found that the proportion of CD4(+) iNKT cells increased gradually from blood to liver to tumor. Furthermore, CD4(+) iNKT cell clones generated from healthy donors were functionally distinct from their CD4(-) counterparts, exhibiting higher Th2 cytokine production and lower cytolytic activity. Thus, in the tumor microenvironment the iNKT cell repertoire is modified by the enrichment of CD4(+) iNKT cells, a subset able to generate Th2 cytokines that can inhibit the expansion of tumor Ag-specific CD8(+) T cells. Because CD4(+) iNKT cells appear inefficient in tumor defense and may even favor tumor growth and recurrence, novel iNKT-targeted therapies should restore CD4(-) iNKT cells at the tumor site and specifically induce Th1 cytokine production from all iNKT cell subsets.

An overview of L-2-hydroxyglutarate dehydrogenase gene (L2HGDH) variants: a genotype-phenotype study.

L-2-Hydroxyglutaric aciduria (L2HGA) is a rare, neurometabolic disorder with an autosomal recessive mode of inheritance. Affected individuals only have neurological manifestations, including psychomotor retardation, cerebellar ataxia, and more variably macrocephaly, or epilepsy. The diagnosis of L2HGA can be made based on magnetic resonance imaging (MRI), biochemical analysis, and mutational analysis of L2HGDH. About 200 patients with elevated concentrations of 2-hydroxyglutarate (2HG) in the urine were referred for chiral determination of 2HG and L2HGDH mutational analysis. All patients with increased L2HG (n=106; 83 families) were included. Clinical information on 61 patients was obtained via questionnaires. In 82 families the mutations were detected by direct sequence analysis and/or multiplex ligation dependent probe amplification (MLPA), including one case where MLPA was essential to detect the second allele. In another case RT-PCR followed by deep intronic sequencing was needed to detect the mutation. Thirty-five novel mutations as well as 35 reported mutations and 14 nondisease-related variants are reviewed and included in a novel Leiden Open source Variation Database (LOVD) for L2HGDH variants (http://www.LOVD.nl/L2HGDH). Every user can access the database and submit variants/patients. Furthermore, we report on the phenotype, including neurological manifestations and urinary levels of L2HG, and we evaluate the phenotype-genotype relationship.

Functions of the peroxisome proliferator-activated receptor (PPAR) alpha and beta in skin homeostasis, epithelial repair, and morphogenesis.

The three peroxisome proliferator-activated receptors (PPAR alpha, PPAR beta, and PPAR gamma) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily. They are regarded as being sensors of physiological levels of fatty acids and fatty acid derivatives. In the adult mouse skin, they are found in hair follicle keratinocytes but not in interfollicular epidermis keratinocytes. Skin injury stimulates the expression of PPAR alpha and PPAR beta at the site of the wound. Here, we review the spatiotemporal program that triggers PPAR beta expression immediately after an injury, and then gradually represses it during epithelial repair. The opposing effects of the tumor necrosis factor-alpha and transforming growth factor-beta-1 signalling pathways on the activity of the PPAR beta promoter are the key elements of this regulation. We then compare the involvement of PPAR beta in the skin in response to an injury and during hair morphogenesis, and underscore the similarity of its action on cell survival in both situations.

Two cases of interferon-alpha-induced sarcoidosis koebnerized along venous drainage lines : new pathogenic insights and review of the literature of interferon-induced sarcoidosis

La sarcoïdose est une affection inflammatoire granuiomateuse systémique d'origine inconnue touchant le plus fréquemment les poumons, le système lymphoïde, le foie, les yeux et la peau. Dans cet article, nous rapportons deux cas de sarcoïdose cutanée touchant les avant-bras de deux patients anciens toxicomanes traités par interféron-a et ribavirine pour une hépatite C chronique. Nous procédons à une revue de la littérature de la sarcoïdose induite par l'interféron et élaborons une nouvelle hypothèse pathogénique de l'effet Koebner dans la sarcoïdose cutanée. Dans le cas des deux patients que nous décrivons, la distribution des lésions cutanées coïncide avec les anciens sites d'injection d'héroïne le long des trajets veineux des deux avant- bras. Cette distribution unique de l'atteinte cutanée suggère que les dommages tissulaires induits par la répétition d'injections percutanées puissent représenter un terrain favorisant au développement local d'une sarcoïdose cutanée. Fait intéressant, il a été récemment démontré que les cellules dendritiques plasmacytoïdes - sous-type de cellules dendritiques généralement absent de la peau - infiltrent rapidement les sites de peau lésée. Ces cellules sont la source d'une production endogène d'interféron-a, cytokine connue pour promouvoir le processus de cicatrisation, mais également pour favoriser le développement de sarcoïdose chez des individus prédisposés. Ainsi, nous postulons que les lésions de sarcoïdose cutanée limitées le long des trajets veineux - sites préalables d'injection percutanée de drogues - peuvent résulter d'une expression locale supplémentaire d'interféron-a. Celle-ci serait en outre favorisée par le traitement de ribavirine dans le cadre de l'hépatite C, connu pour renforcer la production endogène d'interféron-a. L'identification de nombreuses cellules dendritiques plasmacytoïdes circonscrivant l'inflammation granuiomateuse sur la biopsie cutanée de l'un de nos patients semble être un argument dans ce sens, conforté par l'absence de corps étranger détecté en microscopie par lumière polarisée. Cette observation semble pouvoir représenter un point crucial dans la compréhension des mécanismes physiopathologiques à la base de l'infiltration des cicatrices cutanées par la sarcoïdose. Des investigations supplémentaires doivent encore être effectuées afin de confirmer cette hypothèse.

Amphibian albumins as members of the albumin, alpha-fetoprotein, vitamin D-binding protein multigene family.

The Xenopus laevis 68-kd and 74-kd albumin amino acid sequences are examined with respect to their relationship to the other known members of the albumin/alpha-fetoprotein/vitamin D-binding protein gene family. Each of the three members of this family presents a unique pattern of conserved regions indicating a differential selective pressure related to specific functional characteristics. Furthermore, an evolutionary tree of these genes was deduced from the divergence times calculated from direct nucleotide sequence comparisons of individual gene pairs. These calculations indicate that the vitamin D-binding protein/albumin separation occurred 560-600 million years (Myr) ago and the albumin/alpha-fetoprotein divergence 280 Myr ago. This observation leads to the hypothesis according to which the albumin/alpha-fetoprotein gene duplication occurred shortly after the amphibian/reptile separation. Consequently, and unlike mammals, amphibians and fishes should lack an alpha-fetoprotein in their serum at larval stages, which is consistent with a recent analysis of serum proteins in Xenopus laevis larvae. This hypothesis now will have to be tested further in additional lower vertebrates.

Simultaneous determination of gross alpha, gross beta and Ra-226 in natural water by liquid scintillation counting.

The determination of gross alpha, gross beta and 226Ra activity in natural waters is useful in a wide range of environmental studies. Furthermore, gross alpha and gross beta parameters are included in international legislation on the quality of drinking water [Council Directive 98/83/EC].1 In this work, a low-background liquid scintillation counter (Wallac, Quantulus 1220) was used to simultaneously determine gross alpha, gross beta and 226Ra activity in natural water samples. Sample preparation involved evaporation to remove 222Rn and its short-lived decay daughters. The evaporation process concentrated the sample ten-fold. Afterwards, a sample aliquot of 8 mL was mixed with 12 mL of Ultima Gold AB scintillation cocktail in low-diffusion vials. In this study, a theoretical mathematical model based on secular equilibrium conditions between 226Ra and its short-lived decay daughters is presented. The proposed model makes it possible to determine 226Ra activity from two measurements. These measurements also allow determining gross alpha and gross beta simultaneously. To validate the proposed model, spiked samples with different activity levels for each parameter were analysed. Additionally, to evaluate the model's applicability in natural water, eight natural water samples from different parts of Spain were analysed. The eight natural water samples were also characterised by alpha spectrometry for the naturally occurring isotopes of uranium (234U, 235U and 238U), radium (224Ra and 226Ra), 210Po and 232Th. The results for gross alpha and 226Ra activity were compared with alpha spectrometry characterization, and an acceptable concordance was obtained.

Methionine-321 in the C-terminal alpha-helix of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa is important for positive homotropic cooperativity.

Pseudomonas aeruginosa has a pair of distinct ornithine carbamoyltransferases. The anabolic ornithine carbamoyltransferase encoded by the argF gene catalyzes the formation of citrulline from ornithine and carbamoylphosphate. The catabolic ornithine carbamoyltransferase encoded by the arcB gene promotes the reverse reaction in vivo; although this enzyme can be assayed in vitro for citrulline synthesis, its unidirectionality in vivo is determined by its high concentration at half maximum velocity for carbamoylphosphate ([S]0.5) and high cooperativity toward this substrate. We have isolated mutant forms of catabolic ornithine carbamoyltransferase catalyzing the anabolic reaction in vivo. The corresponding arcB mutant alleles on a multicopy plasmid specifically suppressed an argF mutation of P. aeruginosa. Two new mutant enzymes were obtained. When methionine 321 was replaced by isoleucine, the mutant enzyme showed loss of homotropic cooperativity at physiological carbamoylphosphate concentrations. Substitution of glutamate 105 by lysine resulted in a partial loss of the sigmoidal response to increasing carbamoylphosphate concentrations. However, both mutant enzymes were still sensitive to the allosteric activator AMP and to the inhibitor spermidine. These results indicate that at least two residues of catabolic ornithine carbamoyltransferase are critically involved in positive carbamoylphosphate cooperativity: glutamate 105 (previously known to be important) and methionine 321. Mutational changes in either amino acid will affect the geometry of helix H2, which contains several residues required for carbamoylphosphate binding.