Glycoprotein Characterization of the Gelatinous Matrix in the Root-knot Nematode Meloidogyne javanica

Proteinaceous components of freshly formed gelatinous matrix (GM) of the root-knot nematode Metoidogyne javanica were analyzed. Under reducing conditions, the prominent protein fragments had molecular weights of 26 to 66 kDa and 150 to >200 kDa, and most were glycosylated. Most of the fragments were digested by proteinase K, and fewer by trypsin. The lectins soybean agglutinin (SBA), Ulex europaeus agglutinin, and wheat germ agglutinin labeled the higher molecular weight bands (i.e., >200 kDa). SBA labeled additional protein fractions between 26 and 66 kDa. Although Bandeiraea simplicifolia lectin and Concanavalin A did not label bands on the Western blot, they did label the GM in the dot blot technique. Analysis of amino acids and amino sugars in the GM revealed an unusually high amount of ammonia and galactosamine moieties.     

Embryo and larva development in common dentex (Dentex dentex), a pelagophil teleost: The quantitative composition of egg-free amino acids and their interrelations

Free amino acids (FAAs) play a key role in the physiology of marine teleosts (eggs, embryos, and larvae). However, the relationship between the egg FAAs content and the production of viable embryos and larvae (at different developmental stages) in batch spawner pelagophils has not yet comprehensively been investigated. Viable eggs of common dentex, Dentex dentex, were obtained from captive broodstocks. Egg wet weight (WW), dry weight (DW), and water content (%W) and viability parameters, or VPs (egg floating rate [FR], hatching rate [HR], and larval survival rate [SR] at days 0 to 5 posthatch) were determined for 45 egg batches. The egg batches were classified according to their HR magnitude. Twelve egg batches with the same WW, DW, and %W were taken from the same broodstock and at the same developmental stage to determine the qualitative and quantitative composition of FAAs. The total FAA (TFAA) content, glutamic acid (Glu), asparagine (Asn), glutamine (Gln), and arginine (Arg) were correlated with VPs. The Glu was significantly correlated with HR and SR at 0 day posthatch (dph), the Asn with SR at 1 dph, and the Gln and Arg with FR and HR. Of the 361 ratios made based on the absolute concentrations of FAAs, 24 ratios were correlated with VPs (P < 0.005) through 42 simple regression models (R² = 0.641 to 0.846). Of the 42 significant relationships found ∼10%, ∼28%, ∼12%, ∼30%, ∼8%, ∼4%, ∼2%, ∼2%, and ∼2% of the models show the relations of the egg FAAs ratios with FR, HR, SR at days 1 to 5 posthatch, and %W, respectively. A path coefficient in combination with a Pearson's correlation coefficient provided a series of statistical evidences to show the effects of the egg FAAs interrelations on the relationships found between quantitative composition of a FAA and a VP.     

Changes of soluble proteins in leaf and thylakoid exposed in high saline condition of a mangrove taxa Bruguiera gymnorrhiza

One-year-old seedlings of Bruguiera gymnorrhiza (L) Savingay were exposed to 500 mM NaCl for 6d under hydroponic culture condition to characterize the changes in leaf and thylakoid protein profiles in response to short-term salt exposures. Significant changes in leaf dry mass, chlorophylls and soluble leaf proteins were observed in short term of salt exposures, as it happens under tidal situations in nature. Chlorophyll a/b ratio showed decrease of light harvesting efficiency in salt treatment. Total soluble proteins in leaves were extracted from control and NaCl-treated plants at 2d intervals and were analyzed by SDS-PAGE. Intensity of several protein bands of different molecular mass of leaf protein profile ranging from 10 to 86 kDa (10, 16, 23, 33, 37, 42, 44, 50 and 86 kDa) were decreased due to high salt treatment. Out of these, 16, 23 and 33 kDa protein bands decreased dramatically from 1–3 fold but recovered in 7d growth, except the 33 kDa band. SDSPAGE profile of thylakoid protein revealed that both number and the intensity of several protein bands got altered by salt concentration. However, 33 kDa protein band of thylakoid reappeared in recovery that might not be of the same characteristics with same molecular mass as shown in total leaf protein profile. The numbers of major bands found in SDS-PAGE were reduced when analyzed in urea-SDS-PAGE to minimize protein aggregations by high salt. It was noted that 47 kDa disappeared while some proteins of apparent molecular mass like 23 kDa, 33 kDa, 37 kDa and 50 kDa degraded to minor bands. Partial restoration of protein bands occurred when the salt-treated plants were brought back to initial growth condition. These results clearly demonstrate that short term high salt concentration could cause major alterations to photosynthetic apparatus of a true non salt-secreting tree mangrove Bruguiera gymnorrhiza and adapted against fluctuation of salinity by altering leaf protein pool relatively more than the thylakoid proteins.Key words: Bruguiera gymnorrhiza, Mangrove, Polypeptides, Salt shock, Sodium chloride, Thylakoid     

百合花瓣衰老过程中的蛋白质变化研究

以5个百合品种为材料,对百合切花花瓣衰老过程中可溶性蛋白的变化进行了研究.结果表明,5个品种的百合切花都具有相同的蛋白质条带,如49.9 kDa、45 kDa、32.8 kDa、22.1 kDa;但品种问也有明显差异,如38.4 kDa是'黄色风暴'特有的;40 kDa为'铁炮百合'特有;'金百合'没有26.5 kDa的条带,但是相同的杂交品系之间蛋白条带的相似性要明显高于不同杂交品系;在百合花花瓣衰老过程中有的蛋白质的含量不断下降,而有的保持不变;在花瓣衰败期有新的蛋白条带出现,如'西伯利亚'、'黄色风暴'和'索邦'均出现58.3 kDa条带、'金百合'出现67 kDa的条带,这些条带可能与花瓣衰老相关,是衰老特异蛋白.     

Affinity labeling of the galactose/N-acetylgalactosamine-specific receptor of rat hepatocytes: preferential labeling of one of the subunits

The galactose/N-acetylgalactosamine-specific receptor (also known as asialoglycoprotein receptor) of rat hepatocytes consists of three subunits, one of which [43 kilodalton (kDa)] exists in a greater abundance (up to 70% of total protein) over the two minor species (52 and 60 kDa). When the receptor on the hepatocyte membranes was photoaffinity labeled with an 125I-labeled high-affinity reagent [a triantennary glycopeptide containing an aryl azide group on galactosyl residues; Lee, R. T., & Lee, Y. C. (1986) Biochemistry 25, 6835-6841], the labeling occurred mainly (51-80%) on one of the minor bands (52 kDa). Similarly, affinity-bound, N-acetylgalactosamine-modified lactoperoxidase radioiodinated the same 52-kDa band preferentially. In contrast, both the photoaffinity labeling and lactoperoxidase-catalyzed iodination of the purified, detergent-solubilized receptor resulted in a distribution of the label that is comparable to the Coomassie blue staining pattern of the three bands; i.e., the 43-kDa band was the major band labeled. These and other experimental results suggest that the preferential labeling of the minor band and inefficient labeling of the major band on the hepatocyte membrane resulted from a specific topological arrangement of these subunits on the membranes. We postulate that in the native, membrane-bound state of the receptor, the 52-kDa minor band is topologically prominent, while the major (43 kDa) band is partially masked. This partial masking may result from a tight packing of the receptor subunits on the membranes to form a lattice work [Hardy, M. R., Townsend, R. R., Parkhurst, S. M., & Lee, Y. C. (1985) Biochemistry 24, 22-28].     

Comparative binding of Cry1Ab and Cry1F Bacillus thuringiensis toxins to brush border membrane proteins from Ostrinia nubilalis, Ostrinia furnacalis and Diatraea saccharalis (Lepidoptera: Crambidae) midgut tissue

The European (Ostrinia nubilalis Hübner) and Asian corn borers (Ostrinia furnacalis Guenée) are closely related and display similar sensitivity to Cry1 toxins. In this study, we compared the binding patterns of Cry1Ab and Cry1F toxins between both Ostrinia spp., as well as the expression of putative cadherin- and aminopeptidase-N (APN)-like protein receptors. Additionally, cDNA sequences of these putative toxin receptors from both Ostrinia species were compared. Ligand blots for both species indicated a similar binding pattern for Cry1Ab with the strongest immunoreactive band at 260 kDa in both species. In addition, similar expression of the putative cadherin- and APN-like protein receptors were observed at 260 and 135 kDa, respectively. A high degree of similarity (98% amino acid sequence identity) of cDNA sequences for both putative receptor sequences was observed. The Cry1F ligand blot revealed that O. furnacalis and O. nubilalis BBMV exhibited slightly different binding patterns, with strong binding to putative proteins at 150 and 140 kDa, respectively. Both proteins appeared to also bind Cry1Ab, although the signal intensity was much reduced with Cry1Ab. O. furnacalis showed an additional but weaker band at 210 kDa relative to the 150 kDa band. Diatraea saccharalis (Fabricius), which was used as an outgroup species, exhibited different binding patterns than either Ostrinia species, with both Cry1Ab and Cry1F toxins binding to a 210 kDa protein. These results support the previous experiments indicating that O. nubilalis and O. furnacalis share similar patterns of susceptibility to Cry toxins.     

Specific association of increased vascular endothelial growth factor expression and its receptors with macrophage differentiation of HL-60 leukemia cells

We investigated the gene expression profiles of vascular endothelial growth factor (VEGF) and its receptors in HL-60 leukemia cells. In the VEGF family, both mRNA and protein expression of VEGF-C were up-regulated in phorbol myristate acetate (PMA)-differentiated HL-60 cells. We detected two bands of ∼31 and ∼60 kDa in cell lysates, and the higher expression of ∼31 kDa band was further increased after stimulation with tumor necrosis factor (TNF)-α and lipopolysaccharide (LPS). A ∼31 kDa VEGF-C protein was also detected in conditioned media from PMA-differentiated HL-60 cells after LPS stimulation. The mRNA expression of VEGFR-1, VEGFR-2, and neuropilin-1 (NRP-1) was markedly up-regulated in PMA-differentiated HL-60 cells, corresponding to the results from VEGF binding studies, in which VEGF binding activity was increased in PMA-differentiated HL-60 cells. These did not occur in dimethylsulfoxide (DMSO)-differentiated HL-60 cells. The expression of VEGF-C and VEGF receptors is regulated specifically in HL-60 cells during macrophage differentiation.     

Surface Coat of Meloidogyne incognita

The nematode surface coat is defined as an extracuticular component on the outermost layer of the nematode body wall, visualized only by electron microscopy. Surface coat proteins of Meloidogyne incognita race 3 infective juveniles were characterized by electrophoresis and Western blotting of extracts from radioiodine and biotin-labeled nematodes. Extraction of labeled nematodes with cetyltrimethylammonium bromide yielded a principal protein band larger than 250 kDa and, with water soluble biotin, several faint bands ranging from 31 kDa to 179 kDa. The pattern of labeling was similar for both labeling methods. Western blots of unlabeled proteins were probed with a panel of biotin-lectin conjugates, but only Concanavalin A bound to the principal band. Nematodes labeled with radioiodine and biotin released ¹²⁵I and biotin-labeled molecules into water after 20 hours incubation, indicating that surface coat proteins may be loosely attached to the nematode. Antiserum to the partially purified principal protein bound to the surface of live nematodes and to several proteins on Western blots. Differential patterns of antibody labeling were obtained on immuno-blots of extracts from M. incognita race 1, 2, and 3; Meloidogyne hapla race 2; and Meloidogyne arenaria cytological race B.     

Aven is dynamically regulated during Xenopus oocyte maturation and is required for oocyte survival

We have analyzed the expression and function of the cell death and cell cycle regulator Aven in Xenopus. Analysis of Xenopus Aven expression in oocytes and embryos revealed a band close to the predicted molecular weight of the protein (36 kDa) in addition to two bands of higher molecular weight (46 and 49 kDa), one of which was determined to be due to phosphorylation of the protein. The protein is primarily detected in the cytoplasm of oocytes and is tightly regulated during meiotic and mitotic cell cycles. Progesterone stimulation of oocytes resulted in a rapid loss of Aven expression with the protein levels recovering before germinal vesicle breakdown (GVBD). This loss of Aven is required for the G2–M1 cell cycle transition. Aven morpholino knockdown experiments revealed that early depletion of the protein increases progesterone sensitivity and facilitates GVBD, but prolonged depletion of Aven results in caspase-3 activation and oocyte death by apoptosis. Phosphorylated Aven (46 kDa) was found to bind Bcl-x_L in oocytes, but this interaction was lost in apoptotic oocytes. Thus, Aven alters progesterone sensitivity in oocytes and is critical for oocyte survival.     

Clostridium chauvoei: Immunological characterization of Antigenic Preparations

Clostridium chauvoei is an anaerobic and sporulated bacterium which produces blackleg in cattle, sheep and other ungulates. Elsewhere we have studied the immunogenic power of the following antigenic preparations in mice: cellular extract (CE), a suspension of flagella (F), formolinized cells (FC) and heated cells (ΘC), and the immunogenic power of the CE in guinea pigs. The aim of this work was to characterize these immunogenic preparations by SDS-PAGE and immunoblotting. When SDS-PAGE was carried out F showed four protein bands of 86, 59, 51 and 47 kDa; CE showed 10 major bands of 86, 75, 62, 55, 47, 41, 30, 28, 25 and 16 kDa; FC six major bands of 86, 75, 62, 53, 46 and 16 kDa; and ΘC 11 bands, the major ones having 80, 41 and 16 kDa. All four preparations share the flagellar protein band of 47 kDa except ΘC. The formol-treatment reduces the number of the protein bands when it is compared with those of CE. The heat-treatment also reduces the number of the bands compared with CE and FC. When immunoblotting is analysed it can be seen that CE induces the antibodies that react with the 47 kDa protein band, the same ones are induced by FC but not by ΘC. On the other hand, CE induces antibodies that react with the 86 kDa protein band, which is believed to be a polymeric form of a flagellin monomer. According to these results and those obtained in the protection tests, we can conclude that: (1) flagella are in part, responsible for the immunogenicity of the strain; (2) the buffer used to obtain the CE extracts flagellar and somatic antigens; (3) the formol-treatment reduces the protein bands compared with CE, but retains their immunogenic power; and (4) the heat-treatment reduces immunogenic power which is seen by its minor protein bands number and by its lower immunoprotective power.     

Comparative Disc-Electrophoretic Protein Analyses of Selected Meloidogyne,Ditylenchus, Heterodera and Aphelenchus spp.

Disc-electrophoretic separation of soluble proteins from whole nematode homogenates yielded band profiles useful for distinguishing selected species of Meloidogyne and Ditylenchus, and the genera Heterodera, and Aphelenchus. Certain protein bands were common to all the species of Meloidogyne, whereas other bands were specific. Meloidogyne spp. and Heterodera glycines shared some protein similarities, but other genera differed distinctly. Protein profiles of Meloidogyne spp. were not significantly altered by the host on which the nematode was cultured.     

Desiccation induced oxidative stress and its biochemical responses in intertidal red alga Gracilaria corticata (Gracilariales, Rhodophyta)

Intertidal alga Gracilaria corticata growing in natural environment experiences various abiotic stresses during the low tides. The aim of this study was to determine whether desiccation exposure would lead to oxidative stress and its effect varies with exposure periods. This study gives an account of various biochemical changes in G. corticata following the exposure to desiccation for a period of 0 (control), 1, 2, 3 and 4 h under controlled conditions. During desiccation, G. corticata thalli showed dramatic loss of water by almost 47% when desiccated for 4 h. The enhanced production of reactive oxygen species (ROS) and increased lipid peroxidation observed during the exposure of 3-4 h were chiefly contributed by higher lipoxygenase (LOX) activity with the induction of two new LOX isoforms (LOX-2, ∼85 kDa; LOX-3, ∼65 kDa). The chlorophyll, carotenoids and phycobiliproteins (phycoerythrin and phycocyanin) were increased during initial 2 h exposure compared to control and thereafter declined in the succeeding exposure. The antioxidative enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), glutathione peroxidase (GPX) and the regeneration rate of reduced ascorbate (AsA) and glutathione (GSH) increased during desiccation up to 2-3 h. Further, the isoforms of antioxidant enzymes Mn-SOD (∼150 kDa), APX-4 (∼110 kDa), APX-5 (∼45 kDa), GPX-1 (∼80 kDa) and GPX-2 (∼65 kDa) responded specifically to the desiccation exposure. Compared to control, a relative higher content of both free and bound insoluble putrescine and spermine together with enhanced n-6 PUFAs namely C20:4(n-6) and C20:3(n-6) fatty acids found during 2 h exposure reveals their involvement in defence reactions against the desiccation induced oxidative stress.     

Immunoreactivity of the corpuscles of Stannius of the garpike,Lepisosteus osseus,to antisera against salmon and trout stanniocalcins

Stanniocalcin-immunoreactive cells were localized in the corpuscles of Stannius of a holostean fish, the garpike (Lepisosteus osseus), using antisera against salmon and trout stanniocalcins and the peroxidase-antiperoxidase and protein A-gold immunohistochemical methods. The stanniocalcin-immunoreactive cells were periodic acid-Schiff-positive, and antibody staining was abolished if the antiserum was preabsorbed with corpuscle homogenate. Immunocytochemistry revealed two reactive cell types in the glandular parenchyma, and immunoreactivity was confined to the secretory granules. Staining of the granules was also abolished when the antisera were blocked with crude corpuscle homogenate. When corpuscle extracts from garpike were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis, a single dense band was evident with a molecular weight of 68 kDa under non-reducing conditions, whereas three bands were observed (29, 31, and 34 kDa) under reducing conditions. Staining of all bands disappeared following preabsorption of the antiserum with salmon stanniocalcin, trout stanniocalcin, or garpike corpuscle extract. The results are compared with stanniocalcins from another extant holostean, the bowfin (Amia calva), and from more modern bony fishes, the teleosts.     

Spatio-temporal variation in energy content of the periwinkle Littorina littorea, along a pollution gradient in the western Scheldt estuary, The Netherlands

Energy reserves, expressed as lipid, glycogen and protein fractions, were measured in the periwinkle L. littorea after 4 weeks of translocation from a relatively clean, marine (i.e. Westkapelle) to an intermediate (i.e. Ellewoutsdijk) and a highly heavy metal polluted, brackish site (i.e. Hansweert) in the Western Scheldt estuary (The Netherlands). Energy reserves of resident periwinkles from all three sites were assessed over time as well (i.e. 16/03/05, 23/03/05, 30/03/05, 13/04/05 and 11/05/05). None of the energy fractions reflected the upstream decreasing salinity and increasing heavy metal gradients. Glycogen and protein fractions decreased significantly in the translocated specimens, despite no differences were detected among the resident populations, suggesting adaptation and/or acclimation effects at the upstream parts of the estuary. The temporal data revealed a consistent post-winter, pre-spawning increase of glycogen and protein levels and a subsequent drop after spawning. Compared to glycogen and proteins, lipids follow a slightly different pattern, which may be explained by sex related and site specific interactions between biotic and abiotic factors.     

Differential tissue distribution and specificity of phenoloxidases from the Pacific oyster Crassostrea gigas

Phenoloxidases (POs) play a key role in melanin production, are involved in invertebrate immune mechanisms, and have been detected in different bivalves. Recently, we identified catecholase- and laccase-like PO activities in plasma and haemocyte lysate supernatant (HLS) of the Pacific oyster Crassostrea gigas. To go further in our investigations, the aims of this study were (i) to determine the tissue distribution of PO activities in C. gigas, and (ii) to identify and characterise the different sub-classes of POs (i.e. tyrosinase, catecholase and/or laccase) involved in these oxido-reductase activities. With dopamine and p-phenylenediamine (PPD) but not with l-tyrosine used as substrates, PO-activities were detected by spectrophotometry in the gills, digestive gland, mantle, and muscle. These results suggest the presence of catecholase and laccase but not of tyrosinase activities in oyster tissues. The highest activity was recovered in the digestive gland. PO-like activities were all inhibited by 1-phenyl-2-thiourea (PTU) and by the specific laccase inhibitor, cethyltrimethylammonium bromide (CTAB). With dopamine as substrate, the catecholase inhibitor 4-hexylresorcinol (4-HR) only inhibited PO in the muscle. SDS-PAGE zymographic assays with dopamine and PPD elicited a unique ~40kDa protein band in the muscle. In the other tissues, laccase-like activities could be related to ~10kDa and/or ~200kDa protein bands. The ~10kDa protein band was also detected in plasma and HLS, confirming the presence of a laccase in the later compartments, and probably in most of the tissues of C. gigas. This is the first time to our knowledge that a ~10kDa protein band is associated to a laccase-like activity in a mollusc species, contributing to the characterisation of phenoloxidase activities in marine bivalves.     

A comparison of photographic analyses used to quantify zooxanthella density and pigment concentrations in Cnidarians

Many cnidarians exist in an obligatory mutualism with dinoflagellates commonly called zooxanthellae. When these symbioses are stressed, zooxanthella densities often decrease (i.e., bleaching), resulting in reduced host fitness or mortality. Because zooxanthellae play a prominent role in the coloration of hosts, several analyses of reflected spectra from photographic images have been developed to quantify zooxanthella densities and serve as a proxy for invasive sampling methods. To date these techniques have not been compared. In this study, global information system (GIS) tools, commonly used with aerial and satellite images, and photographs of healthy and bleached sea anemones, Aiptasia pallida (Verrill), were used to compare these image analysis methods. Zooxanthella densities and chlorophyll-a concentrations were correlated with image brightness (i.e., digital number) in: the red, green, and blue bands (RGB); the average of the three RGB bands (RGB/3); intensity and saturation bands (IHS); and using a principal components analysis (PCA) of the RGB bands. RGB brightness correlations with zooxanthella densities and chlorophyll-a concentrations were highest using the blue band, followed by green, then red. Using any one band within RGB, however, restricts comparisons to similar color morphs. RGB/3, IHS or PCA transformations enable intra and inter-specific comparisons where colors may vary. Among these transformations, PCA and intensity had higher correlations, followed by RGB/3, then saturation. RGB/3 and IHS, unlike PCA, ignore the correlations between the three RGB bands, treating each pixel independently. PCA uses these correlations, and in doing so lessens the effects of heteroscedasticity in the data. In addition, the observed reciprocal relationship of intensity and saturation may serve as a standardized criterion for bleaching. Finally, this study demonstrates that GIS has broad interdisciplinary applications for spatial and spectral analyses from the individual colony to reef scale assessments.     

Investigation of the steps involved in the difference of susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Bacillus thuringiensis Vip3Aa16 toxin

BUPM95 is a Bacillusthuringiensis subsp. kurstaki strain producing the Vip3Aa16 toxin with an interesting insecticidal activity against the Lepidopteran larvae Ephestia kuehniella. Study of different steps in the mode of action of this Vegetative Insecticidal Protein on the Mediterranean flour moth (E. kuehniella) was carried out in the aim to investigate the origin of the higher susceptibility of this insect to Vip3Aa16 toxin compared to that of the Egyptian cotton leaf worm Spodoptera littoralis. Using E. kuehniella gut juice, protoxin proteolysis generated a major band corresponding to the active toxin and another band of about 22 kDa, whereas the activation of Vip3Aa16 by S. littoralis gut juice proteases generated less amount of the 62 kDa active form and three other proteolysis products. As demonstrated by zymogram analysis, the difference in proteolysis products was due to the variability of proteases in the two gut juices larvae. The study of the interaction of E. kuehniella BBMV with biotinylated Vip3Aa16 showed that this toxin bound to a putative receptor of 65 kDa compared to the 55 and 100 kDa receptors recognized in S. littoralis BBMV. The histopathological observations demonstrated similar damage caused by the toxin in the two larvae midguts. These results demonstrate that the step of activation, mainly, is at the origin of the difference of susceptibility of these two larvae towards B. thuringiensis Vip3Aa16 toxin.