Promoter analysis and immunolocalisation show that puroindoline genes are exclusively expressed in starchy endosperm cells of wheat grain

The purolindolines are small cysteine-rich proteins which are present in the grain of wheat. They have a major impact on the utilisation of the grain as they are the major determinants of grain texture, which affects both milling and baking properties. Bread and durum wheats were transformed with constructs comprising the promoter regions of the Puroindoline a (Pina) and Puroindoline b (Pinb) genes fused to the uidA (GUS) reporter gene. Nine lines showing 3:1 segregation for the transgene and comprising all transgene/species combinations were selected for detailed analysis of transgene expression during grain development. This showed that transgene expression occurred only in the starchy endosperm cells and was not observed in any other seed or vegetative tissues. The location of the puroindoline proteins in these cells was confirmed by tissue printing of developing grain, using a highly specific monoclonal antibody for detection and an antibody to the aleurone-localised 8S globulin as a control. This provides clear evidence that puroindolines are only synthesised and accumulated in the starchy endosperm cells of the wheat grain.

Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel alpha-amylase inhibitory properties

Lipid transfer proteins (LTPs) were thus named because they facilitate the transfer of lipids between membranes in vitro. This study was triggered by the characterization of a 9-kDa LTP from Capsicum annuum seeds that we call Ca-LTP(1). Ca-LTP(1) was repurified, and in the last chromatographic purification step, propanol was used as the solvent in place of acetonitrile to maintain the protein`s biological activity. Bidimensional electrophoresis of the 9-kDa band, which corresponds to the purified Ca-LTP(1), showed the presence of three isoforms with isoelectric points (pIs) of 6.0, 8.5 and 9.5. Circular dichroism (CD) analysis suggested a predominance of alpha-helices, as expected for the structure of an LTP family member. LTPs immunorelated to Ca-LTP(1) from C. annuum were also detected by western blotting in exudates released from C. annuum seeds and also in other Capsicum species. The tissue and subcellular localization of Ca-LTP(1) indicated that it was mainly localized within dense vesicles. In addition, isolated Ca-LTP(1) exhibited antifungal activity against Colletotrichum lindemunthianum, and especially against Candida tropicalis, causing several morphological changes to the cells including the formation of pseudohyphae. Ca-LTP(1) also caused the yeast plasma membrane to be permeable to the dye SYTOX green, as verified by fluorescence microscopy. We also found that Ca-LTP(1) is able to inhibit mammalian alpha-amylase activity in vitro.

Immunolocalisation of sodium/proton exchanger-like proteins in the gills of elasmobranchs

Na⁺/H⁺ exchangers are integral membrane proteins that exchange Na⁺ and H⁺ across cell membranes. The Na⁺/H⁺ exchangers 2 and 3 are epithelial isoforms in mammals and contribute to acid–base homeostasis. The gills of fishes, including elasmobranchs, are also associated with acid/base balance, and are probably the primary acid/base regulatory organ. This study examines the presence of Na⁺/H⁺ exchangers 2 and 3 using immunohistochemistry and immunoblotting in the gills of four species of elasmobranchs, the banjo ray (Trygonorrhina fasciata), southern eagle ray (Myliobatis australis), the gummy shark (Mustelus antarcticus) and the Australian angel shark (Squatina australis) using heterologous antibodies. Na⁺/H⁺ exchanger 2-like immunoreactivity was observed in the gills of the banjo ray, eagle ray and angel shark. In the banjo and eagle rays, this Na⁺/H⁺ exchanger-like immunoreactivity co-localised with immunoreactivity to Na⁺/K⁺-ATPase, a marker for the mitochondrial-rich cells of fishes. Na⁺/H⁺ exchanger 3-like immunoreactivity was only observed in the gills of the angel and gummy sharks, some Na⁺/H⁺ exchanger 3-like cells also showed Na⁺/K⁺-ATPase immunoreactivity. However, immunoblotting of banjo and eagle ray gill membranes demonstrated Na⁺/H⁺ exchanger 3-like immunoreactivity, which was not consistent with the immunohistochemical results. These data demonstrate the presence of epithelial Na⁺/H⁺ exchangers 2 and 3 in the gills of elasmobranchs and a link with acid/base regulation is suggested.

Lignin biosynthesis in Norway spruce: from a model system to the tree

Lignin is a hydrophobic polymer that is synthesised in the secondary cell walls of all vascular plants. It enables water conduction through the stem, supports the upright growth habit and protects against invading pathogens. In addition, lignin hinders the utilisation of the cellulosic cell walls of plants in pulp and paper industry and as forage. Lignin precursors are synthesised in the cytoplasm through the phenylpropanoid pathway, transported into the cell wall and oxidised by peroxidases or laccases to phenoxy radicals that couple to form the lignin polymer. This study was conducted to characterise the lignin biosynthetic pathway in Norway spruce (Picea abies (L.) Karst.). We focused on the less well-known polymerisation stage, to identify the enzymes and the regulatory mechanisms that are involved. Available data for lignin biosynthesis in gymnosperms is scarce and, for example, the latest improvements in precursor biosynthesis have only been verified in herbaceous plants. Therefore, we also wanted to study in detail the roles of individual gene family members during developmental and stress-induced lignification, using EST sequencing and real-time RT-PCR. We used, as a model, a Norway spruce tissue culture line that produces extracellular lignin into the culture medium, and showed that lignin polymerisation in the tissue culture depends on peroxidase activity. We identified in the culture medium a significant NADH oxidase activity that could generate H2O2 for peroxidases. Two basic culture medium peroxidases were shown to have high affinity to coniferyl alcohol. Conservation of the putative substrate-binding amino acids was observed when the spruce peroxidase sequences were compared with other peroxidases with high affinity to coniferyl alcohol. We also used different peroxidase fractions to produce synthetic in vitro lignins from coniferyl alcohol; however, the linkage pattern of the suspension culture lignin could not be reproduced in vitro with the purified peroxidases, nor with the full complement of culture medium proteins. This emphasised the importance of the precursor radical concentration in the reaction zone, which is controlled by the cells through the secretion of both the lignin precursors and the oxidative enzymes to the apoplast. In addition, we identified basic peroxidases that were reversibly bound to the lignin precipitate. They could be involved, for example, in the oxidation of polymeric lignin, which is required for polymer growth. The dibenzodioxocin substructure was used as a marker for polymer oxidation in the in vitro polymerisation studies, as it is a typical substructure in wood lignin and in the suspension culture lignin. Using immunolocalisation, we found the structure mainly in the S2+S3 layers of the secondary cell walls of Norway spruce tracheids. The structure was primarily formed during the late phases of lignification. Contrary to the earlier assumptions, it appears to be a terminal structure in the lignin macromolecule. Most lignin biosynthetic enzymes are encoded for by several genes, all of which may not participate in lignin biosynthesis. In order to identify the gene family members that are responsible for developmental lignification, ESTs were sequenced from the lignin-forming tissue culture and developing xylem of spruce. Expression of the identified lignin biosynthetic genes was studied using real-time RT-PCR. Candidate genes for developmental lignification were identified by a coordinated, high expression of certain genes within the gene families in all lignin-forming tissues. However, such coordinated expression was not found for peroxidase genes. We also studied stress-induced lignification either during compression wood formation by bending the stems or after Heterobasidion annosum infection. Based on gene expression profiles, stress-induced monolignol biosynthesis appeared similar to the developmental process, and only single PAL and C3H genes were specifically up-regulated by stress. On the contrary, the up-regulated peroxidase genes differed between developmental and stress-induced lignification, indicating specific responses.

Distribution of the Receptor for Advanced Glycation End Products (RAGE) in the Human Male Reproductive Tract. Prevalence in Men with Diabetes Mellitus.

BACKGROUND: Diabetics have a significantly higher percentage of sperm with nuclear DNA (nDNA) fragmentation and increased levels of advanced glycation end products (AGEs), in their testis, epididymis and sperm. As the receptor for AGEs (RAGE) is important to oxidative stress and cell dysfunction, we hypothesise, that it may be involved in sperm nDNA damage. METHODS: Immunohistochemistry was performed to determine the presence of RAGE in the human testis and epididymis. A comparison of the receptor's incidence and localisation on sperm from 10 diabetic and 11 non-diabetic men was conducted by blind semi-quantitative assessment of the immunostaining. ELISA analysis ascertained RAGE levels in seminal plasma and sperm from 21 diabetic and 31 non-diabetic subjects. Dual labelling immunolocalisation was employed to evaluate RAGE's precise location on the sperm head. RESULTS: RAGE was found throughout the testis, caput epididymis, particularly the principle cells apical region, and on sperm acrosomes. The number of sperm displaying RAGE and the overall protein amount found in sperm and seminal plasma were significantly higher in samples from diabetic men (p

Fasciola hepatica cathepsin L-like proteases: biology, function and potential in the development of first generation liver fluke vaccines.

Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are members of a lineage that gave rise to the human cathepsin Ls, Ks and Ss, but while they exhibit similarities in their substrate specificities to these enzymes they differ in having a wider pH range for activity and an enhanced stability at neutral pH. There are presently 13 Fasciola cathepsin L cDNAs deposited in the public databases representing a gene family of at least seven distinct members, although the temporal and spatial expression of each of these members in the developmental stage of F. hepatica remains unclear. Immunolocalisation and in situ hybridisation studies, using antibody and DNA probes, respectively, show that the vast majority of cathepsin L gene expression is carried out in the epithelial cells lining the parasite gut. Within these cells the enzyme is packaged into secretory vesicles that release their contents into the gut lumen for the purpose of degrading ingested host tissue and blood. Liver flukes also express a novel multi-domain cystatin that may be involved in the regulation of cathepsin L activity. Vaccine trials in both sheep and cattle with purified native FheCL1 and FheCL2 have shown that these enzymes can induce protection, ranging from 33 to 79%, to experimental challenge with metacercariae of F. hepatica, and very potent anti-embryonation/hatch rate effects that would block parasite transmission. In this article we review the vaccine trials carried out over the past 8 years, the role of antibody and T cell responses in mediating protection and discuss the prospects of the cathepsin Ls in the development of first generation recombinant liver fluke vaccines. Author Keywords: Helminths; Trematodes; Parasites; Cathepsins; Proteases; Vaccines; Immunology; Biochemistry

Leucine aminopeptidase of the human blood flukes, Schistosoma mansoni and Schistosoma japonicum

An array of schistosome endoproteases involved in the digestion of host hemoglobin to absorbable peptides has been described, but the exoprotease responsible for catabolising these peptides to amino acids has yet to be identified. By searching the public databases we found that Schistosoma mansoni and Schistosoma japonicum express a gene encoding a member of the M17 family of leucine aminopeptidases (LAPs). A functional recombinant S. mansoni LAP produced in insect cells shared biochemical properties, including pH optimum for activity, substrate specificity and reliance on metal cations for activity, with the major aminopeptidase activity in soluble extracts of adult worms. The pH range in which the enzyme functions and the lack of a signal peptide indicate that the enzyme functions intracellularly. Immunolocalisation studies showed that the S. mansoni LAP is synthesised in the gastrodermal cells surrounding the gut lumen. Accordingly, we propose that peptides generated in the lumen of the schistosome gut are absorbed into the gastrodermal cells and are cleaved by LAP to free amino acids before being distributed to the internal tissues of the parasite. Since LAP was also localised to the surface tegument it may play an additional role in surface membrane re-modelling. (C) 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Cofilin immunolabelling correlates with depth of invasion in gastrointestinal endocrine cell tumors

Gastrointestinal endocrine cell tumors are a heterogeneous population of lesions believed to arise from neuroendocrine cells of the gastrointestinal mucosa. The current classification of these tumors is based on tumor size, microscopic features and clinical evidence of metastasis. Although diagnostic categories generally correlate with prognosis, molecular prognostic markers will be clinically useful adjuncts. Cofilin has been implicated in tumor invasion, and its immunolocalisation was studied in gastrointestinal endocrine cell tumors. The immunolocalisation of cofilin was studied by immunohistochemistry in 34 formalin-fixed, paraffin wax-embedded gastrointestinal endocrine cell tumors using a tissue microarray platform. A significant correlation was found between high cofilin immunolabelling and the depth of invasion (p

A novel calmodulin-like protein from the liver fluke, Fasciola hepatica

An 18.2 kDa protein from the liver fluke, Fasciola hepatica has been identified and characterised. The protein shows strongest sequence similarity to egg antigen proteins from Schistosoma mansoni, Schistosoma japonicum and Clonorchis sinensis. The protein is predicted to adopt a calmodulin-like fold; it thus represents the third calmodulin-like protein to be characterised in F. hepatica and has been named FhCaM3. Compared to the classical calmodulin structure there are some variations. Most noticeably, the central, linker helix is disrupted by a cysteine residue. Alkaline native gel electrophoresis showed that FhCaM3 binds calcium ions. This binding event increases the ability of the protein to bind the hydrophobic fluorescent probe 8-anilinonaphthalene-1-sulphonate, consistent with an increase in surface hydrophobicity as seen in other calmodulins. FhCaM3 binds to the calmodulin antagonists trifluoperazine and W7, but not to the myosin regulatory light chain binding compound praziquantel. Immunolocalisation demonstrated that the protein is found in eggs and vitelline cells. Given the critical role of calcium ions in egg formation and hatching this suggests that FhCaM3 may play a role in calcium signalling in these processes. Consequently the antagonism of FhCaM3 may, potentially, offer a method for inhibiting egg production and thus reducing the spread of infection.

A plasma membrane Ca2+-ATPase (PMCA) from the liver fluke, Fasciola hepatica

Fasciolosis is a parasitic infection by the liver fluke Fasciola hepatica, which costs the global agricultural community over US $2 billion per year. Its prevalence is rising due to factors such as climate change and drug resistance. ATP-dependent membrane transporters are considered good potential drug targets as they are essential for cellular processes and are in an exposed, accessible position in the cell. Immunolocalisation studies demonstrated that a plasma membrane calcium ATPase (PMCA) was localised to the parenchymal tissue in F. hepatica. The coding sequence for a F. hepatica PMCA (FhPMCA) has been obtained. This sequence encodes a 1,163 amino acid protein which contains motifs which are commonly conserved in PMCAs. Molecular modelling predicted that the protein has 10 transmembrane segments which include a potential calcium ion binding site and phosphorylation motif. FhPMCA interacts with the calmodulin-like protein FhCaM1, but not the related proteins FhCaM2 or FhCaM3, in a calcium-ion dependent manner. This interaction occurs through a region in the C-terminal region of FhPMCA which most likely adopts an a-helical conformation. When FhPMCA was heterologously expressed in a budding yeast strain deleted for its PMCA (Pmc1p), it restored viability. Microsomes prepared from these yeast cells had calcium ion stimulated ATPase activity which was inhibited by the known PMCA inhibitors, bisphenol and eosin. The potential of FhPMCA as a new drug target is discussed.

Immunological homologues of the Arabidopsis thaliana beta 1 tubulin are polyglutamylated in Nicotiana tabacum

Peptide-specific antibody AABI, raised to the C-terminal 13 amino acids of Arabidopsis thaliana beta 1 tubulin, identifies a single electrophoretically separable beta-tubulin on 2-D-gel Western blots of total protein extracts from A. thaliana seedlings. We show that AABI crossreacts with two of the eight polyglutamylated beta-tubulin isoforms present in purified Nicotiana tabacum tubulin fractionated by high-resolution isoelectric focussing. Immunolocalisation studies using AAB1 revealed that the two N. tabacum polyglutamylated beta 1-tubulin isoforms are utilised in all four plant microtubule arrays (the interphase cortical array, the preprophase band, the spindle and the phragmoplast) indicating that there is no apparent subcellular sorting of these isotypes.

Purification of a cathepsin L-like proteinase secreted by adult Fasciola hepatica

A cysteine proteinase released in vitro by Fasciola hepatica was purified to homogeneity by Sephacryl S-200 gel filtration chromatography followed by QAE-Sephadex chromatography. The purified enzyme resolves as a single band with an apparent molecular size of 27 kDa on reducing SDS-polyacrylamide gel electrophoresis; however, under non-reducing conditions it migrates as multiple bands, each with enzymatic activity, in the apparent molecular size range 60-90 kDa. The sequence of the first 20 N-terminal amino acids of the enzyme shows considerable homology with cathepsin L-like proteinases. Immunolocalisation studies revealed that the cathepsin L-like proteinase is concentrated within vesicles in the gut epithelial cells of liver fluke.

Differential expression of liver fluke β-tubulin isotypes at selected life cycle stages

We have shown that Fasciola hepatica expresses at least six ß-tubulins in the adult stage of its life cycle, designated F.hep-ß-tub1-6 (Ryan et al., 2008). Here we show that different complements of tubulin isotypes are expressed in different tissues and at different life cycle stages; this information may inform the search for novel anthelmintics. The predominant (as judged by quantitative PCR) isotype transcribed at the adult stage was F.hep-ß-tub1 and immunolocalisation studies revealed that this isotype occurred mainly in mature spermatozoa and vitelline follicles. Quantitative PCR indicated that changes occurred in the transcription levels of ß-tubulin isotypes at certain life cycle stages and may be of importance in the efficacy of benzimidazole-based anthelmintic drugs, but there were no significant differences between the triclabendazole (TCBZ)-susceptible Leon isolate and the TCBZ-resistant Oberon isolate in the transcription levels of each of the isotypes. When three well-characterised isolates with differing susceptibilities to TCBZ were compared, only one amino acid change resulting from a homozygous coding sequence difference (Gly269Ser) in isotype 4 was observed. However, this change was not predicted to alter the overall structure of the protein. In conclusion, these findings indicate that there is tissue-specific expression of tubulin isotypes in the liver fluke but the development of resistance to TCBZ is not associated with changes in its presumed target molecule.