Antipeptide antibodies to the 141-1141-1141-1receptor confirm the extracellular orientation of the amino-terminus and the putative first extracellular loop

Summary We developed site-directed rabbit antisera against synthetic peptides selected from the deduced amino acid sequence of the hamster lung ₂-adrenergic receptor (amino acids 16–31 and 174–189, respectively). All antisera directed against peptide 1 (four of four rabbits) as well as two antisera directed against peptide 2 (two of four rabbits) recognized the purified ₂-adrenergic receptor in immunoblot conditions when used at a dilution of 1500. Antisera directed against peptide 1 as well as peptide 2 were able to immunoprecipitate iodinated as well as¹²⁵I-cyanopindolol tabeled ₂-adrenergic receptor. This last result implies that the recognized epitopes do not contain the¹²⁵I-cyanopindolol binding domain of the ₂-adrenergic receptor. Immunoblot experiments performed on membrane fractions from hamster lung tissue showed that immunoreactive bands at 64,000, 57,000, 47,000, 44,000 and 38,000 daltons were specifically detected. When purified ₂-adrenergic receptor was iodinated and submitted to glycolytic and/or tryptic treatments, species with similar molecular weights could be recovered. Then, the immunoreactive bands probably correspond to native ₂-adrenergic receptor and to degradative or nonglycosylated species of this molecule. The antisera were also able to detect immunoreactive molecules in murine and human cell lines, suggesting conservation of the probed sequences between these species. Enzymatic linked immunosorbent assay tests on intact cells and immunofluorescence studies confirmed that the amino-terminus and putative first extracellular loop are extracellularly located. Immunofluorescence studies on mouse brain primary cultures showed that cells expressing ₂-adrenergic receptor-like molecules exhibited a neuronal phenotype.     

Hormonal regulation of insulin-like growth factor I secretion by bovine adrenal cells

The role of insulin-like growth factor I (IGF-I) on the specific function of several steroidogenic cells has been recently reported. Since IGF-I is produced by several tissues, we have investigated whether bovine adrenal cells secrete this peptide. Purification of conditioned medium from adrenal cells incubated with [35S]methionine through affinity chromatography (monoclonal anti-IGF-I antibody), high pressure liquid chromatography, and polyacrylamide gel electrophoresis revealed a single band of similar Mr as pure recombinant IGF-I. Moreover, the purified adrenal-secreted IGF-I displaced bound 125I-IGF-I to its adrenal receptors, and pretreatment of adrenal cells with the purified peptide enhanced the acute corticotropin (ACTH)-induced cAMP production as recombinant IGF-I. The basal secretion of IGF-I (6 +/- 1 ng/48 h/10(6) cells) was stimulated 3-, 4.5-, and 9.5-fold by fibroblast growth factor, angiotensin II (A-II), and ACTH, respectively, but not by growth hormone. The stimulatory effects of A-II and ACTH were dose-dependent (ED50 congruent to 2.5 x 10(-8) and 1.5 x 10(-10) M, respectively), and the effects of both hormones were additive. Glucocorticoids were not the mediators of the effect of the two hormones on IGF-I secretion, since inhibition of their steroidogenic action by aminoglutethimide did not significantly modify IGF-I secretion. An immunoreactive IGF-I material was also secreted by mouse adrenal tumor cell line Y-1, but the stimulatory effect of ACTH was only 2-fold, and there was no effect of A-II. Since bovine adrenal cells contain specific IGF-I receptors and this peptide is required for the maintenance of some adrenal cell-specific function, the present data suggest that IGF-I may act in an autocrine fashion to stimulate adrenal cell differentiation stimulated by ACTH and A-II.     

Regulation of expression of the 3β-hydroxysteroid dehydrogenases of human placenta and fetal adrenal

The appropriate expression of 3β-hydroxysteroid dehydrogenase/Δ^5→4-isomerase (3β-HSD) is vital for mammalian reproduction, fetal growth and life maintenance. Several isoforms of 3β-HSD, the products of separate genes, have been identified in various species including man. Current investigations are targeted toward defining the processes that regulate the levels of specific isoforms in various steroidogenic tissues of man. High levels of expression of 3β-HSD were observed in placental tissues. It has been generally considered that the multinucleated syncytiotrophoblastic cells are the principal sites of 3β-HSD expression and, moreover, that 3β-HSD expression is intimately associated with cyclic AMP-promoted formation of syncytia. Herein we report the presence of 3β-HSD immunoreactive and mRNA species in uninucleate cytotrophoblasts in the chorion laeve, similar to that in syncytia but not cytotrophoblast placenta. In vitro, 3β-HSD levels in chorion laeve cytotrophoblasts were not increased with time nor after treatment with adenylate cyclase activators, whereas villous cytotrophoblasts spontaneously demonstrated progressive, increased 3β-HSD expression. Moreover, 3β-HSD synthesis appeared to precede morphologic syncytial formation. Thus high steroidogenic enzyme expression in placenta is not necessarily closely linked to formation of syncytia. Both Western immunoblot and enzymic activity analyses also indicated that the 3β-HSD expressed in these cytotrophoblastic populations was the 3β-HSD type I gene product (M_r, 45K) and not 3β-HSD type II (M_r, 44K) expressed in fetal testis. In cultures of fetal zone and definitive zone cell of human fetal adrenal, 3β-HSD expression was not detected until ACTH was added. ACTH, likely acting in a cyclic AMP-dependent process, induced 3β-HSD type II activity and mRNA expression. The higher level of 3β-HSD mRNA in definitive zone compared with fetal zone cells was associated with parallel increases in cortisol secretion relative to dehydroepiandrosterone sulfate formation.     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Stimulates Adenylyl Cyclase and Phospholipase C Activity in Rat Cerebellar Neuroblasts

Abstract: The presence of receptors for the novel neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been recently demonstrated in the external granule cell layer of the cerebellum, a germinative matrix that generates the majority of cerebellar interneurons. In the present study, we have taken advantage of the possibility of obtaining a culture preparation that is greatly enriched in immature cerebellar granule cells to investigate the effect of PACAP on the adenylyl cyclase and phospholipase C transduction pathways. The two molecular forms of PACAP, i.e., 27-(PACAP27) and 38-(PACAP38) amino-acid forms of PACAP, induced a dose-dependent stimulation of cyclic AMP production in granule cells. The potencies of PACAP27 and PACAP38 were similar (ED₅₀ = 0.12 ± 0.01 and 0.23 ± 0.07 n M , respectively), whereas vasoactive intestinal polypeptide (VIP) was ∼100 times less potent. PACAP27 and PACAP38 also induced a dose-dependent stimulation of polyphosphoinositide breakdown (ED₅₀ = 19.1 ± 6.3 and 13.4 ± 6.0 n M , respectively), whereas VIP had no effect on polyphosphoinositide metabolism. The effect of PACAP38 on inositol phosphate formation was significantly reduced by U-73122 and by pertussis toxin, indicating that activation of PACAP receptors causes stimulation of a phospholipase C through a pertussis toxin-sensitive G protein. In contrast, forskolin and dibutyryl cyclic AMP did not affect PACAP-induced stimulation of inositol phosphates. Taken together, the present results demonstrate that PACAP stimulates independently the adenylyl cyclase and the phospholipase C transduction pathways in immature cerebellar granule cells. These data favor the concept that PACAP may play important roles in the control of proliferation and/or differentiation of cerebellar neuroblasts.     

The role of proline residues in the folding kinetics of the bovine pancreatic trypsin inhibitor derivative RCAM(14–38)

The role of proline residues in the folding of the trypsin inhibitor derivative RCAM(14–38) has been studied by testing for slow-folding species of the unfolded protein, which could result from the introduction of wrong proline isomers after unfolding. The unfolded protein at 25 °C contains chiefly fast-folding (U_F) molecules: they refold with a time constant of 40 milliseconds at pH 6.8 in 1.9 m-guanidinium chloride. At least one minor slow-folding (U_s) species has been found, using fluorescence to monitor refolding. The reaction in which this U_s species is formed after unfolding shows the properties expected for the cis: Irans isomerization of a proline residue. When refolding is monitored by tyrosine absorbance, two minor slow reactions are found. The faster reaction is in the same time range (15 s at 25 °C) as that studied by fluorescence, and the slower reaction is quite slow (200 s at 25 °C). It is not known whether the slower reaction results from a second U_s species. There are four trans proline residues in bovine pancreatic trypsin inhibitor: the proportion of slow-folding molecules (not more than 25% at 25 °C) is smaller than expected if every proline residue can produce a U_s species and if the cis to trans ratio of each residue after unfolding is at least 0.1:0.9.Criteria based on folding kinetics are given for classifying the types of folding reaction shown by unfolded molecules containing a single wrong proline isomer. Levitt (1980) has classified three types of proline residues according to the energy difference (small, intermediate or large) between the native protein and the predicted minimum energy structure containing a wrong proline isomer. He suggests that these three types of proline residues can be recognized by the types of folding reactions they produce. Only type II (intermediate) folding reactions have thus far been characterized by the criteria introduced here. We point out that the type of folding reaction depends also on the folding conditions, and a possible explanation for this effect is given.     

Native tree regeneration in native tree plantations: understanding the contribution of Araucaria angustifolia to biodiversity conservation in the threatened Atlantic Forest in Argentina

Deforestation is a global process that has strongly affected the Atlantic Forest in South America, which has been recognised as a threatened biodiversity hotspot. An important proportion of deforested areas were converted to forest plantations. Araucaria angustifolia is a native tree to the Atlantic Forest, which has been largely exploited for wood production and is currently cultivated in commercial plantations. An important question is to what extent such native tree plantations can be managed to reduce biodiversity loss in a highly diverse and vulnerable forest region . We evaluated the effect of stand age, stand basal area, as a measure of stand density, and time since last logging on the density and richness of native tree regeneration in planted araucaria stands that were successively logged over 60 years, as well as the differences between successional groups in the response of plant density to stand variables. We also compared native tree species richness in planted araucaria stands to neighbouring native forest. Species richness was 71 in the planted stands (27 ha sampled) and 82 in native forest (18 ha sampled) which approximate the range of variation in species richness found in the native forests of the study area. The total abundance and species richness of native trees increased with stand age and time since last logging, but ecological groups differed in their response to such variables. Early secondary trees increased in abundance with stand age 3–8 times faster than climax or late secondary trees. Thus, the change in species composition is expected to continue for a long term. The difference in species richness between native forest and planted stands might be mainly explained by the difference in plant density. Therefore, species richness in plantations can contribute to local native tree diversity if practices that increase native tree density are implemented.     

Contrasting Gene Decay in Subterranean Vertebrates: Insights from Cavefishes and Fossorial Mammals

Evolution sometimes proceeds by loss, especially when structures and genes become dispensable after an environmental shift relaxes functional constraints. Subterranean vertebrates are outstanding models to analyze this process, and gene decay can serve as a readout. We sought to understand some general principles on the extent and tempo of the decay of genes involved in vision, circadian clock, and pigmentation in cavefishes. The analysis of the genomes of two Cuban species belonging to the genus Lucifuga provided evidence for the largest loss of eye-specific genes and nonvisual opsin genes reported so far in cavefishes. Comparisons with a recently evolved cave population of Astyanax mexicanus and three species belonging to the Chinese tetraploid genus Sinocyclocheilus revealed the combined effects of the level of eye regression, time, and genome ploidy on eye-specific gene pseudogenization. The limited extent of gene decay in all these cavefishes and the very small number of loss-of-function mutations per pseudogene suggest that their eye degeneration may not be very ancient, ranging from early to late Pleistocene. This is in sharp contrast with the identification of several vision genes carrying many loss-of-function mutations in ancient fossorial mammals, further suggesting that blind fishes cannot thrive more than a few million years in cave ecosystems.     

Serum level of adiponectin is a surrogate independent biomarker of radiographic disease progression in early rheumatoid arthritis: results from the ESPOIR cohort

Introduction

Adipokines such as adiponectin, leptin, and visfatin/nicotinamide phosphoribosyltransferase (NAMPT) have recently emerged as pro-inflammatory mediators involved in the pathophysiology of rheumatoid arthritis (RA). We aimed to determine whether serum adipokine levels independently predicted early radiographic disease progression in early RA.

Methods

In total, 791 patients were included from the prospective Etude et Suivi des POlyarthrites Indifférenciées Récentes (ESPOIR) cohort who met the American College of Rheumatology-European League Against Rheumatism criteria for RA (n = 632) or had undifferentiated arthritis (UA) (n = 159). Enzyme-linked immunosorbent assay (ELISA) was used to assess baseline serum levels of adiponectin, leptin, and visfatin/NAMPT. In the RA group, we tested the association of serum adipokine levels and (a) baseline radiographic damage and (b) radiographic disease progression, defined as a change >0 or ≥5 in total Sharp-van der Heijde Score (∆SHS) between inclusion and 1 year (∆SHS ≥1 or rapid radiographic progression: ∆SHS ≥5), adjusting for confounders (age, sex, body-mass index, insulin resistance, C-reactive protein level, Disease Activity Score in 28 joints, Health Assessment Questionnaire score, autoantibody status, steroid use, and radiographic evidence of RA damage at inclusion).

Results

Adiponectin level was independently associated with baseline total SHS (adjusted β = 0.12; P = 0.006). It was also associated with ∆SHS ≥1 (adjusted odds ratio (aOR) = 1.84 (1.25 to 2.72)) involving erosive as well as narrowing disease progression (aOR = 1.73 (1.17 to 2.55) and 1.93 (1.04 to 3.57), respectively). Serum adiponectin level predicted ∆SHS ≥5 (aOR = 2.0 (1.14 to 3.52)). Serum leptin level was independently associated only with ∆SHS >0 (aOR = 1.59 (1.05 to 2.42)). Conversely, serum visfatin/NAMPT level and radiographic disease progression were unrelated. Considering the receiver-operated characteristic curves, the best adiponectin cut-offs were 4.14 μg/ml for ∆SHS ≥1 and 6.04 μg/ml for ∆SHS ≥5, with a good specificity (58% and 75% for ∆SHS ≥1 and ∆SHS ≥5, respectively) and high negative predictive values (75% and 92% for ∆SHS ≥1 or ∆SHS ≥5, respectively).

Conclusion

Serum adiponectin level is a simple useful biomarker associated with early radiographic disease progression in early RA, independent of RA-confounding factors and metabolic status.