Cytometric quantification of nitrate reductase by immunolabeling in the marine diatom Skeletonema costatum

BACKGROUND: The uptake of nitrate by phytoplankton is a central issue in biological oceanography due to its importance to primary production and vertical flux of biogenic carbon. Nitrate reductase catalyzes the first step of nitrate assimilation, the reduction of NO(3) to NO(2). A cytometric protocol to detect and quantify relative changes in nitrate reductase (NR) protein content of the marine centric diatom Skeletonema costatum is presented. METHODS: Immunolabeling of NR protein was achieved with polyclonal antibodies raised against S.costatum NR. Antisera specific to a NR protein subunit and to a NR polypeptide sequence were compared, and cytometric results of NR protein abundance were related to Western analyses. Changes in cellular NR abundance and activity were followed during an upwelling simulation experiment in which S. costatum was exposed to a shift from ammonia to nitrate as major nitrogen source. RESULTS: NR protein could be detected in NO(3)-grown cells and at extremely low levels hardly discernible by Western Blot densiometry in NH(4)-grown cells. The protocol allowed observation of early stages of NR induction during an upwelling simulation. NR abundance increased after the nutrient shift to reach a new physiological "steady-state" 96 hrs later. NR activity exhibited diel variation with maxima at mid-day. NR abundance as estimated by both flow cytometry and Western analysis exhibited a hyperbolic relationship to NR activity. This pattern suggests post-translational activation of NR protein. CONCLUSIONS: The presented protocol allows the differentiation of NH(4)- versus NO(3)-grown algae as well as the monitoring of early stages in the induction of nitrate assimilatory capacities.     

Direct intracardiac injection of umbilical cord-derived stromal cells and umbilical cord blood-derived endothelial cells for the treatment of ischemic cardiomyopathy

The development of new therapeutic strategies is necessary to reduce the worldwide social and economic impact of cardiovascular disease, which produces high rates of morbidity and mortality. A therapeutic option that has emerged in the last decade is cell therapy. The aim of this study was to compare the effect of transplanting human umbilical cord-derived stromal cells (UCSCs), human umbilical cord blood-derived endothelial cells (UCBECs) or a combination of these two cell types for the treatment of ischemic cardiomyopathy (IC) in a Wistar rat model. IC was induced by left coronary artery ligation, and baseline echocardiography was performed seven days later. Animals with a left ventricular ejection fraction (LVEF) of ≤40% were selected for the study. On the ninth day after IC was induced, the animals were randomized into the following experimental groups: UCSCs, UCBECs, UCSCs plus UCBECs, or vehicle (control). Thirty days after treatment, an echocardiographic analysis was performed, followed by euthanasia. The animals in all of the cell therapy groups, regardless of the cell type transplanted, had less collagen deposition in their heart tissue and demonstrated a significant improvement in myocardial function after IC. Furthermore, there was a trend of increasing numbers of blood vessels in the infarcted area. The median value of LVEF increased by 7.19% to 11.77%, whereas the control group decreased by 0.24%. These results suggest that UCSCs and UCBECs are promising cells for cellular cardiomyoplasty and can be an effective therapy for improving cardiac function following IC.     

Most nuclear systemic autoantigens are extremely disordered proteins: implications for the etiology of systemic autoimmunity

Patients with systemic autoimmune diseases usually produce high levels of antibodies to self-antigens (autoantigens). The repertoire of common autoantigens is remarkably limited, yet no readily understandable shared thread links these apparently diverse proteins. Using computer prediction algorithms, we have found that most nuclear systemic autoantigens are predicted to contain long regions of extreme structural disorder. Such disordered regions would generally make poor B cell epitopes and are predicted to be under-represented as potential T cell epitopes. Consideration of the potential role of protein disorder may give novel insights into the possible role of molecular mimicry in the pathogenesis of autoimmunity. The recognition of extreme autoantigen protein disorder has led us to an explicit model of epitope spreading that explains many of the paradoxical aspects of autoimmunity – in particular, the difficulty in identifying autoantigen-specific helper T cells that might collaborate with the B cells activated in systemic autoimmunity. The model also explains the experimentally observed breakdown of major histocompatibility complex (MHC) class specificity in peptides associated with the MHC II proteins of activated autoimmune B cells, and sheds light on the selection of particular T cell epitopes in autoimmunity. Finally, the model helps to rationalize the relative rarity of clinically significant autoimmunity despite the prevalence of low specificity/low avidity autoantibodies in normal individuals.     

Water stress effects on the content and organization of chlorophyll in mesophyll and bundle sheath chloroplasts of maize

The consequences of drought stress on the organization of chlorophyll into photosynthetic units and on the chlorophyll-protein composition of mesophyll and bundle sheath chloroplasts of Zea mays L. were studied. It was found that the majority of chlorophyll lost in response to water stress occurs in the mesophyll cells with a lesser amount being lost from the bundle sheath cells. All of the chlorophyll loss could be accounted for by reduction in the lamellar content of the light-harvesting chlorophyll a/b-protein, a rather specific target for water stress. The decreased content of this chlorophyll-protein accounts for the elevated chlorophyll a/b ratios and the reduced photosynthetic unit sizes of the two cell types in stressed plants. The implications of the selective catabolism of this major membrane component are discussed.     

EFFECTS OF HUMAN ACTIVITIES ON STRUCTURE AND COMPOSITION OF WOODY SPECIES OF THE NOKREK BIOSPHERE RESERVE OF MEGHALAYA,NORTHEAST INDIA

Aims Our study was conducted in the Nokrek Biosphere Reserve (NBR) in the Garo hills districts of Meghalaya, Northeast India. Our aim was to assess the effects of human activities on plant diversity,population structure and regeneration.Methods We selected a representative 1.2 hm2 stand in both the core and buffer zones of NBR. Structure and composition were determined by randomly sampling square quadrats, population structure was assessed by determining age structure, and regeneration was assessed by measuring densities of seedling, sapling and adult trees.Important findings More woody species were recorded from the core zone than the buffer zone (87 vs. 81 species), and there were a large number of tropical, temperate, and Sino-Himalayan, Burma-Malaysian and Malayan elements, primitive families and primitive genera. The trees were distributed in three distinct strata,canopy, subcanopy and sapling. Subcanopy and sapling layers had the highest species richness (81％ -88％ ). Lauraceae and Euphorbiaceae were the dominant families in terms of the number of species, and a large number of families were represented by single species. Most woody species (57 ％ - 79 ％ ) were contagiously distributed and had low frequency ( ＜ 20％ ). Although stand density was high in the buffer zone, its basal area was low compared to the stand in the core zone. Low similarity and high β-diversity indicate marked differences in species composition of the stands. Shannon diversity index was high in both the stands, while Simpson dominance index was low. The diameter-class distribution for dominant species revealed that the most had a large number of young individuals in their populations. Preponderance of tree seedlings, followed by a steep decline in population density of saplings and adult trees, indicated that the seedling to sapling stage was the most critical in the life cycle of the tree populations. Most species (42 ％ - 48 ％ ) had no regeneration,25 ％ - 35 ％ had good/fair regeneration, and the rest had poor regeneration or reoccurred as immigrants.     

PHOTOSYNTHETIC LIGHT-HARVESTING FUNCTION OF VIOLAXANTHIN IN NANNOCHLOROPSIS SPP. (EUSTIGMATOPHYCEAE)1

Whole cell absorption spectra of the Eustigmatophycean algae Nannochloropsis salina Bourrelly and Nannochloropsis sp. reveal the presence of a distinct absorption peak at 490 nm. The lack of chlorophylls b and c in these species indicates that this peak must be attributed to carotenoid absorption. In vivo fluorescence excitation spectra for chlorophyll a emission show a corresponding maximum at 490 nm. This peak is more clearly resolved than carotenoid maxima in other algal classes due to the absence of accessory chlorophylls. The carotenoid composition of the two Nannochloropsis species shows that violaxanthin and vaucheriaxanthin are the main contributors to 490 nm absorption. Violaxanthin accounts for approximately 60% of the total carotenoid in both clones. We conclude that light absorption by violaxanthin, and possibly by vaucheriaxanthin, is coupled in energy transfer to chlorophyll a and that violaxanthin is the major light-harvesting pigment in the Eustigmatophyceae. This is the first report of the photosynthetic light-harvesting function of this carotenoid.     

PHYLOGENETIC DISTRIBUTION OF THE MAJOR DIATOM LIGHT-HARVESTING PIGMENT-PROTEIN DETERMINED BY IMMUNOLOGICAL METHODS 1

Monospecific, polyclonal antibodies raised against the apoprotein of the major light-harvesting pigment-protein of Phaeodactylum tricornutum Bohlin UTEX 646 were used to determine (1) whether this complex was common to the class Bacillariophyceae, whose members contain chlorophylls a and c and fucoxanlhin; (2) whether antigenically-related apoproteins were present in other chlorophyll c-containing groups, and (3) whether there was immunological homology with the light-hanvsting chlorophyll a/b protein of similar photosynthetic function in the Chlorophyta and vascular plants. We have used protein blotting techniques to show that antibodies against the two P. tricornutum light-harvesting complex polypeptides cross-reacted with one or two polypeptides of similar molecular weight (17–21 kD) in all ten diatom species examined, representing two orders and six families. No cross-reactivity was obtained with total membrane polypeptides from isolated representatives of three chromophyte algal divisions (Chrysophyta, Cryptophyta, Pyrrophyta), all of which contained chlorophyll c. No cross-reactivity was observed with membrane Polypeptides isolated from members of two classes of Chlorophte algae. These data suggest that the Bacillariophyceae may be monophyletic, and that the primary structure of the diatom light-harvesting complex is not closely related to pigment-protein complexes with similar function in other chlorophyll c-containing unicellular algal groups. Lastly, it may be possible to use the antibodies to the diatom light-harvesting polypeptides as specific markers for diatoms in natural phytoplankton assemblages.     

SEASONAL PATTERNS OF PHOTOSYNTHESIS AND LIGHT-INDEPENDENT CARBON FIXATION IN MARINE MACROPHYTES1

The contribution of light-independent carbon fixation (LICF) to the overall carbon gain and the seasonal patterns of maximum photosynthesis (P_max and LICF were characterized in a broad taxonomic range of macrophytes from Monterey Bay, California. P_max and LICF rates (nmol C.g filtered seawater^?1.min^?1) varied among species and taxonomic groups examined, and as a function of tissue type in the phaeophyte Laminaria setchellii Silva (Phaeophyceae). On average, P_max values were higher in the Rhodophyta, whereas LICF rates were greater in the Phaeophyceae. LICF rates were generally less than 5% of P_max in the marine macrophytes studied and, as a consequence, cannot fully compensate for respiratory carbon losses, which usually are greater than 10% of P_max. All species studied possessed the highest P_max and LICF rates when irradiance levels were highest and decreased during periods of low incident irradiance. Seasonal patterns of P_max and LICF in most of the macrophytes from the stenothermal environment of Monterey Bay were strongly correlated with photosynthetic photon flux rather than seawater temperature. The concomitant decrease of LICF and P_max rates in all species examined argues against LICF playing a major role in carbon acquisition under light-limiting conditions as suggested previously. Rather, the strong positive correlation of P_max and LICF indicates the direct coupling of photosynthate (e.g. 3-phosphoglyceric acid) generation with production of substrates for LICF reactions. Our results also suggest that LICF might be a useful indicator of photosynthetic metabolism in marine macrophytes.