Effects of ceramide on aggrecanase activity in rabbit articular cartilage

Ceramide participates in signal transduction of IL-1 and TNF, two cytokines likely involved in cartilage degradation in osteoarthritis. We previously showed that ceramide stimulates proteoglycan degradation, mRNA expression of matrix metalloproteinase (MMP)-1, -3, and -13, and pro-MMP-3 production in rabbit cartilage. Since aggrecan, the main cartilage proteoglycan, can be cleaved by metalloproteinases both of MMP and aggrecanase type, the aim of this study was to determine if ceramide stimulates aggrecanase action and, if that is the case, in which measure aggrecanase mediates the degradative effect of ceramide. To this end, antibodies were used against the C terminal aggrecan neoepitopes generated by aggrecanases (NITEGE(373)) and MMPs (DIPEN(341)). Ceramide C(2) at 10(-5) to 10(-4) M dose-dependently increased NITEGE signal, without changing that of DIPEN, in cultured explants of rabbit cartilage. The effects of 10(-4) M C(2) on NITEGE signal and proteoglycan degradation were similarly antagonized by the metalloproteinase inhibitor batimastat, with return to the basal level at 10(-6) M. These results show that, similarly to IL-1 and TNF, ceramide-induced aggrecan degradation is mainly due to aggrecanases. That no increase of MMP activity was detected, despite stimulation of MMP expression, was probably due to lack of proenzyme conversion to mature form, since addition of a MMP activator to C(2)-treated cartilage increased both DIPEN signal and proteoglycan degradation. These findings support the hypothesis that cytokine-induced ceramide could play a mediatory role in situations of increased degradation of cartilage matrix.     

Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome

Identification and characterization of anion channel genes in plants represent a goal for a better understanding of their central role in cell signaling, osmoregulation, nutrition, and metabolism. Though channel activities have been well characterized in plasma membrane by electrophysiology, the corresponding molecular entities are little documented. Indeed, the hydrophobic protein equipment of plant plasma membrane still remains largely unknown, though several proteomic approaches have been reported. To identify new putative transport systems, we developed a new proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis cell suspensions a highly purified plasma membrane fraction and characterized it in detail by immunological and enzymatic tests. Using complementary methods for the extraction of hydrophobic proteins and mass spectrometry analyses on mono-dimensional gels, about 100 proteins have been identified, 95% of which had never been found in previous proteomic studies. The inventory of the plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, one-third displaying at least four transmembrane segments. The plasma membrane localization was confirmed for several proteins, therefore validating such proteomic strategy. An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic, and metabolism. This analysis also reveals 10 proteins that display structural properties compatible with transport functions and will constitute interesting targets for further functional studies.     

Proteomics of chloroplast envelope membranes

Proteomics is a very powerful approach to link the information contained in sequenced genomes, like Arabidopsis, to the functional knowledge provided by studies of plant cell compartments, such as chloroplast envelope membranes. This review summarizes the present state of proteomic analyses of highly purified spinach and Arabidopsis envelope membranes. Methods targeted towards the hydrophobic core of the envelope allow identifying new proteins, and especially new transport systems. Common features were identified among the known and newly identified putative envelope inner membrane transporters and were used to mine the complete Arabidopsis genome to establish a virtual plastid envelope integral protein database. Arabidopsis envelope membrane proteins were extracted using different methods, that is, chloroform/methanol extraction, alkaline or saline treatments, in order to retrieve as many proteins as possible, from the most to the less hydrophobic ones. Mass spectrometry analyses lead to the identification of more than 100 proteins. More than 50% of the identified proteins have functions known or very likely to be associated with the chloroplast envelope. These proteins are (a) involved in ion and metabolite transport, (b) components of the protein import machinery and (c) involved in chloroplast lipid metabolism. Some soluble proteins, like proteases, proteins involved in carbon metabolism or in responses to oxidative stress, were associated with envelope membranes. Almost one third of the newly identified proteins have no known function. The present stage of the work demonstrates that a combination of different proteomics approaches together with bioinformatics and the use of different biological models indeed provide a better understanding of chloroplast envelope biochemical machinery at the molecular level.     

Controlled bioassay systems for determination of lethal infective doses of tissue homogenates containing Taura syndrome or white spot syndrome virus

In vivo bioassay is the predominant method for evaluating the infectivity of materials potentially harboring viable shrimp pathogens and determining the relative susceptibility of shrimp species to viral infections. A controlled bioassay system for white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) was developed utilizing 260 ml tissue culture flasks modified with an air exchange vent. Individual shrimp (1.00 +/- 0.25 g) were placed in separate flasks containing artificial seawater (100 to 150 ml) and held in an incubator at 27 degrees C. After a 48 h acclimation period, shrimp were either injected intramuscularly with viral inoculum or exposed to virus-laden water. Water was exchanged and shrimp were fed a commercial food pellet daily except 24 h post-infection (p.i.). Bioassays were performed with serial dilutions of stock viral preparations and shrimp mortality was recorded for 7 d p.i. Mortality rates of test animals permitted the estimation of the lethal infective doses, LD50 and LD90. The LD50 of the TSV injection preparation was estimated at viral dilutions of 1:7.692 x 10(7) (Trial 1) and 1:6.667 x 10(7) (Trial 2). The LD50s of 2 different WSSV injection preparations were estimated at 1:4.444 x 10(6) and 1:4.505 x 10(6). The LD50 for the TSV waterborne challenge was 1:9916 (Trial 1) and 1:15 710 (Trial 2) at 20 degrees C and 1:1272 at 27 degrees C. A second waterborne TSV inoculum challenge at 27 degrees C produced an LD50 of 1:2857. WSSV doses used in the waterborne challenge only reached 39% mortality, which did not allow for the estimation of effective lethal doses. Bioassay by injection proved to be a more reliable method of estimating viral infectivity compared to waterborne method. The dose-response curves developed can serve as a basis for controlled comparisons of relative levels of viral infectivity of specific tissue preparations and for controlled comparisons of relative susceptibility of shrimp species or stocks to viral pathogens.     

Effect of adrenergic blockade on lymphocyte cytokine production at rest and during exercise

To examine the effect of exercise andadrenergic blockade on lymphocyte cytokine production, six men ingestedeither a placebo (control) or an - (prazosin hydrochloride) and-adrenoceptor antagonist (timolol malate) capsule (blockade, or BLK)2 h before performing 19 ± 1 min of supine bicycle exerciseat 78 ± 3% peak pulmonary uptake. Blood was collected before andafter exercise, stimulated with phorbol 12-myristate 13-acetate andionomycin, and surface stained for T (CD3⁺) and naturalkiller [NK (CD3CD56⁺)] lymphocyte surfaceantigens. Cells were permeabilized, stained for the intracellularcytokines interleukin (IL)-2 and interferon (IFN)-, and analyzedusing flow cytometry. BLK had no effect on the resting concentration ofstimulated cytokine-positive T and NK lymphocytes or the amount ofcytokine they were producing. Exercise resulted in an increase (P< 0.05) in the concentration of stimulated T and NK lymphocytesproducing cytokines in the circulation, but these cells produced less(P < 0.05) cytokine post- compared with preexercise.BLK attenuated (P < 0.05) the elevation in theconcentration of lymphocytes producing cytokines during exercise;however, BLK did not affect the amount of IL-2 and IFN- produced.These results suggest that adrenergic stimulation contributes to theexercise-induced increase in the concentration of lymphocytes in thecirculation; however, it does not appear to be responsible for theexercise-induced suppression in cytokine production.

     

A review of chemically-induced alterations in thyroid and vitamin A status from field studies of wildlife and fish

This paper reviews 22 published field studies that have found an association between exposure to environmental contaminants and alterations in thyroid gland structure, circulating thyroid hormones and vitamin A (retinoid) status in free-ranging populations of wildlife and fish. Vitamin A and thyroid hormones play critical roles during development, growth and function 'throughout life. Studies of captive wildlife and laboratory studies support a relationship between alterations in thyroid hormones and vitamin A status and exposure to dioxins, furans, and planar polychlorinated biphenyls, which bind to the aryl hydrocarbon receptor. Some studies have found adverse health effects in wildlife associated with exposure to polyhalogenated aromatic hydrocarbons and altered thyroid and retinoid status including: decreased reproductive success, immune system changes, dermatologic abnormalities and developmental deformities. A direct causal relationship between these effects and thyroid and retinoid changes has not been demonstrated. Field researchers studying the responses to these synthetic chemicals in wildlife and fish should include measurement of thyroid hormones and retinoids and histological examination of the thyroid gland in their study design as biomarkers of exposure to these chemicals in the environment.     

Changes in radial tree growth for Picea abies, Larix decidua, Pinus cembra and Pinus uncinata near the alpine timberline since 1750

Changes in radial growth of the four coniferous species growing in the French Alps near the upper treeline are investigated. Thirty-seven populations of Norway spruce [Picea abies (L.) Karst.], European larch (Larix decidua Mill.), Swiss stone pine (Pinus cembra L.) and mountain pine (Pinus uncinata Mill. ex Mirb.) were sampled by taking 1320 cores and analysing tree-ring widths. Sites were chosen in various climatic conditions (macroclimate and aspect) and on two kinds of bedrock in order to take into account the ecological behaviour of these species. Belledonne, Moyenne-Tarentaise, Haute-Maurienne and Briançonnais areas were sampled along increasing gradients of summer aridity and winter continentality. The calculation of time series after removing the age trend brings strong evidence for an increase in radial growth during the two last centuries, but with different stages and fluctuations for each species. This growth trend is significantly enhanced since 1860 for the spruce, and since 1920 for the two pine species. Furthermore, it also appears on Larix decidua with the same pattern despite periodical growth reduction due to attacks of the larch bud moth (Zeiraphera diniana Gn.). The analysis of ring-widths at a given cambial age reveals that this enhanced phenomenon is observed especially during the tree’s early years (25–75 years). The analysis of four regional climatic series, and three longer series of temperature (in farther single sites) reveals synchronous decadal fluctuations and an evident secular increase in minimum temperatures (especially in January and from July to October), that may be involved in tree-growth enhancement. Thermic amplitudes are significantly reduced during the whole growing period, what is more pronounced in Belledonne, the most oceanic region. Long term growth changes are well described by stepwise regression models, especially for the pine species. These models involved both a linear trend (CO₂ concentration or N-deposition) and low frequency of Turin monthly temperatures. However, they show different patterns than those observed from response functions at a yearly scale.     

Chloroplast envelope membranes: a dynamic interface between plastids and the cytosol

Chloroplasts are bounded by a pair of outer membranes, the envelope, that is the only permanent membrane structure of the different types of plastids. Chloroplasts have had a long and complex evolutionary past and integration of the envelope membranes in cellular functions is the result of this evolution. Plastid envelope membranes contain a wide diversity of lipids and terpenoid compounds serving numerous biochemical functions and the flexibility of their biosynthetic pathways allow plants to adapt to fluctuating environmental conditions (for instance phosphate deprivation). A large body of knowledge has been generated by proteomic studies targeted to envelope membranes, thus revealing an unexpected complexity of this membrane system. For instance, new transport systems for metabolites and ions have been identified in envelope membranes and new routes for the import of chloroplast-specific proteins have been identified. The picture emerging from our present understanding of plastid envelope membranes is that of a key player in plastid biogenesis and the co-ordinated gene expression of plastid-specific protein (owing to chlorophyll precursors), of a major hub for integration of metabolic and ionic networks in cell metabolism, of a flexible system that can divide, produce dynamic extensions and interact with other cell constituents. Envelope membranes are indeed one of the most complex and dynamic system within a plant cell. In this review, we present an overview of envelope constituents together with recent insights into the major functions fulfilled by envelope membranes and their dynamics within plant cells. Special Issue of Photosynthesis Research in honor of Andrew A. Benson.     

Association of specific expansins with growth in maize leaves is maintained under environmental, genetic, and developmental sources of variation

We aimed to evaluate whether changes in maize (Zea mays) leaf expansion rate in response to environmental stimuli or developmental gradients are mediated by common or specific expansins, a class of proteins known to enhance cell wall extensibility. Among the 33 maize expansin or putative expansin genes analyzed, 19 were preferentially expressed at some point of the leaf elongation zone and these expansins could be organized into three clusters related to cell division, maximal leaf expansion, and cell wall differentiation. Further analysis of the spatial distribution of expression was carried out for three expansins in leaves displaying a large range of expansion rates due to water deficit, genotype, and leaf developmental stage. With most sources of variation, the three genes showed similar changes in expression and consistent association with changes in leaf expansion. Moreover, our analysis also suggested preferential association of each expansin with elongation, widening, or both of these processes. Finally, using in situ hybridization, expression of two of these genes was increased in load-bearing tissues such as the epidermis and differentiating xylem. Together, these results suggest that some expansins may be preferentially related to elongation and widening after integrating several spatial, environmental, genetic, and developmental cues.