Isolation of Proteins Related to the β-Lectin from Potato which Bind to Hyphal Wall Components of Phytophthora infestans

Proteins from potato cells which recognize fungal cell wall components of Phytophthora infestans were isolated after passage of a potato homogenate through an affinity column which contained bound fungal cell wall components. Bound potato proteins were eluted with NN′-diacetylchitobiose, and fractionated by SDS-polyaciylamide gel electrophoresis. Eluted proteins from cv. Yukijiro (R₁-gene) and cv. Irish Cobbler (r-gene) had similar profiles and the same apparent Mr, 66, 58 and 41.5 kD and other proteins with lower molecular weight. Only the 41.5 kD and lower molecular weight proteins reaeted with polyclonal antibodies against the β-lectin from the R₁ cultivar, Rishiri. The surfaces of protoplasts from cvs Yukijiro and Irish Cobbler also reacted with the antibodies to the lectin. Treatment of potato protoplasts with hyphal cell wall components of P. infestans caused cytoplasmic aggregation, a response characteristic of the hypersensitive reaction. Oligomers of N-acetyl glucosamine reduced the ability of fungal cell wall components to cause this cytoplasmic aggregation. These results suggest that binding between fungal cell wall eomponents and certain potato proteins is sensitive to N-acetylglucosamine, and may play a role in the hypersensitive reaction.     

Calcium-dependent Protein Kinases are Involved in Potato Signal Transduction in Response to Elicitors from the Oomycete Phytophthora infestans

Plant response to pathogens involves an intricate network of signal transduction pathways. Here, potato cell cultures were used to study signal transduction in response to elicitors from Phytophthora infestans. Pretreatment of cells with Ser/Thr protein kinase inhibitors, EGTA, calmodulin antagonists or a channel blocker abolished the induction of two enzymes involved in defence responses, phenylalanine ammonia‐lyase (PAL) and peroxidase. Phosphatase inhibitors caused an increase of these activities in the absence of elicitors. Hyphal cell wall components (HWC) from an incompatible race (HWC 0) produced a rapid and transient increment of histone phosphorylation, whereas induction by HWC from a compatible race (HWC C) was less pronounced and more sustained. As activities were calcium‐dependent, a fraction enriched in calcium‐dependent protein kinases (CDPKs) was obtained by DEAE chromatography. Fractions from HWC 0‐ and HWC C‐treated cells presented higher kinase activity than that from untreated cells. Moreover, total activity was higher in the incompatible than in the compatible interaction. Activity was calcium‐dependent, partially inhibited by calmodulin antagonists and able to phosphorylate syntide‐2, a specific substrate of CDPKs. An in‐gel kinase assay showed the presence of a band of approximately 50 kDa whose activity was higher in HWC 0‐ than in HWC C‐treated cells and was not detected in control extracts. This report presents evidences of the differential activation of CDPKs in response to elicitors from different races of P. infestans, revealing that these protein kinases participate in the defence response to oomycete.     

Biochemical, Immunochemical and Immunohistochemical Identification of Myosin Heavy Chains in Cultured Cells of Catharanthus roseus

In the pollen tubes of the lily Lilium longiflorum, myosin,composed of 170-kDa heavy chains is responsible for the intracellulartransport of organelles [Yokota and Shimmen (1994) Protoplasma177: 153]. Polypeptides of 170 kDa with similar antigenicityto this pollen-tube myosin have also been found in other angiospermcells [Yokota et al. (1995) Protoplasma 185: 178]. To clarifythe role of this type of myosin in cytoplasmic streaming, weprepared partially purified myosin fraction from cultured cellsof Catharanthus roseus by co-precipitation with F-actin. Ina motility assay in vitro with this fraction, rhodamine-phalloidin-labeledF-actin moved with an average velocity of 10.7 µm s^-1.This sliding velocity was similar to that of the cytoplasmicstreaming observed in intact cultured cells. Antibodies raisedagainst the 170-kDa heavy chain of pollen-tube myosin recognizedonly a single polypeptide of 170 kDa in this partially purifiedfraction. The same polypeptide was also identified by theseantibodies in a crude extract of proteins from cultured cells.The myosin-specific fluorescence was concentrated around thenuclei and was associated with particles of various sizes. Duallocalization using antibodies against myosin and against actinrevealed that these particles were preferentially co-localizedwith actin filaments. On the other hand, no component of thecrude extract or of the partially purified myosin fraction cross-reactedwith antibodies against heavy chains of myosin II from animalcells. These results suggest that the 170-kDa polypeptide is the myosinheavy chain and that this myosin generates the motive forcefor cytoplasmic streaming in cultured cells of Catharanthusroseus. (Received March 28, 1995; Accepted September 14, 1995)     

Cytoskeletal changes in platelets induced by thrombin and phorbol myristate acetate (PMA).

Changes in shape, and aggregation that accompanies platelet activation, are dependent on the assembly and reorganization of the cytoskeleton. To assess the changes in cytoskeleton induced by thrombin and PMA, suspensions of aspirin-treated,32P-prelabeled, washed pig platelets in Hepes buffer containing ADP scavengers were activated with thrombin, and with PMA, an activator of protein kinase C. The cytoskeletal fraction was prepared by adding Triton extraction buffer. The Triton-insoluble (cytoskeletal) fraction isolated by centrifugation was analysed by SDS-PAGE and autoradiography. Incorporation of actin into the Triton-insoluble fraction was used to quantify the formation of F-actin. Thrombin-stimulated platelet cytoskeletal composition was different from PMA-stimulated cytoskeletal composition. Thrombin-stimulated platelets contained not only the three major proteins: actin (43 kDa), myosin (200 kDa) and an actin-binding protein (250 kDa), but three additional proteins of Mr56 kDa, 80 kDa and 85 kDa in the cytoskeleton, which were induced in by thrombin dose-response relationship. In contrast, PMA-stimulated platelets only induced actin assembly, and the 56 kDa, 80 kDa and 85 kDa proteins were not found in the cytoskeletal fraction. Exposure of platelets to thrombin or PMA induced phosphorylation of pleckstrin parallel to actin assembly. Staurosporine, an inhibitor of protein kinase C, inhibited actin assembly and platelet aggregation induced by thrombin or PMA, but did not inhibit the incorporation of 56 kDa, 80 kDa and 85 kDa into the cytoskeletal fraction induced by thrombin. These three extra proteins seem to be unrelated to the induction of protein kinase C. We conclude that actin polymerization and platelet aggregation were induced by a mechanism dependent on protein kinase C, and suggest that thrombin-activated platelets aggregation could involve additional cytoskeletal components (56 kDa, 80 kDa, 85 kDa) of the cytoskeleton, which made stronger actin polymerization and platelet aggregation more.     

Systemic Induction of Resistance in Potato Plants Against Phytophthora infestans by Local Treatment with Hyphal Wall Components of the Fungus

Potato plants (cv.‘Irish Cobbler’ with no major resistance genes to Phytophthora infestans), the lower or upper leaves of which were previously treated with hyphal wall components (HWC) of the fungus by rubbing with carborundum, acquired an induced resistance in other untreated leaves against cultivar-pathogenic races of P. infestans when challenged by spraying with a zoospore suspension. Such induced resistance was significantly shown to exist from at least 1 to 20 days after induction treatment with HWC. Thus, the treated plants were protected from severe late blight disease while non-induced control plants finally died of the disease. The induced resistance was due to a reduction of the number of successfully germinating zoospores, and subsequent penetration and then occurrence of hypersensitive-like cell response to the penetrating organisms. These results suggested that local leaf tissues of potato plants reacting to HWC may provide some systemic information that activates or enhances some resistance to P. infestans.     

Effect of Mastoparan on Phospholipase A2 Activity in Potato Tubers Treated with Fungal Elicitor

Participation of phospholipase (PL) A₂ in signal trans-ductionhas been reported to elicitate a resistance reaction in potatocells by inoculation of an incompatible race of Phytophthorainfestans, the late blight fungus, or by treatment with fungalelicitor hyphal wall components (HWC). Mastoparan, a genericG protein activator, has been shown to activate PLA in a G protein-dependentmanner in animal cells. We analyzed the effects of mastoparanand the inactive analog Mas-17 on PLA₂ activity in potato tubers.In healthy potato tubers, the activation of PLA₂ by mastoparanwas detected in the soluble fraction, but not micro-somal fraction.However, in potato tubers treated with HWC, PLA₂ activity wasstimulated by mastoparan in both soluble and microsomal fractions.Pretreatment of the microsomal fraction with neomycin, a PLCinhibitor, and staurosporine, a protein kinase inhibitor, inhibitedthe mastoparan-induced activation of PLA₂. This suggested thatthe PLA₂ activation in potato tubers by mastoparan was mediatedby the PLC pathway and protein phospho-rylation. We also examinedthe accumulation of potato phytoalexin rishitin. Mastoparanstimulated rishitin accumulation induced by HWC, but did notinduce the accumulation. This indicated that mastoparan mightactivate the signal transduction pathway in the resistance reactionsinduced in potato tubers. (Received March 12, 1998; Accepted August 6, 1998)     

Spectrin-like proteins in green algae (Desmidiaceae)

Immunochemical detection of actin as well as spectrin-like proteins have been carried out in the green algae Micrasterias denticulata, Closterium lunula, and Euastrum oblongum. In these algae, actin is detected on Western blots at 43 kDa with antibodies to actin from higher plant and animal origin. By use of antibodies to human and chicken erythrocyte spectrin a cross-reactivity with desmid proteins is found at about the molecular mass of 220 kDa, where also human erythrocyte spectrin is detected. Additional bands are present at 120 kDa and 70 kDa, which are probably breakdown products. An antibody against chicken alpha-actinin, a small protein of the spectrin superfamily, recognizes bands at 90 kDa, where it is expected, and 70 kDa, probably the same breakdown product as mentioned for spectrin. Isoelectric focusing provides staining at pI 4.6 with antibodies against spectrin. Immunogold labelling of spectrin and alpha-actinin antigens on high-pressure frozen, freeze-substituted Micrasterias denticulata cells with the same antibodies exhibits staining, especially at membranes of different populations of secretory vesicles, at dictyosomes, and the plasma membrane. However, no clear correlation to the growth pattern of the cell could be observed. Taken together, our results demonstrate the presence of spectrin-like proteins in desmid cells which are probably functional in exocytosis.     

Role of c-Src in cellular events associated with colony-stimulating factor-1-induced spreading in osteoclasts

We and others have observed that in response to treatment with Colony Stimulating Factor-1 (CSF-1) neonatal rat osteoclasts demonstrate rapid cytoplasmic spreading. The receptor for CSF-1, c-Fms, is expressed in osteoclasts, possesses intrinsic tyrosine-kinase activity, and signals via rapid phosphorylation of selected proteins. It has been reported previously that c-Src becomes tyrosine phosphorylated following CSF-1 treatment of fibroblasts overexpressing c-Fms. We therefore examined the cellular events associated with CSF-1-induced spreading in osteoclasts and what role, if any, c-Src played in these processes. Confocal microscopic studies using phosphotyrosine (P-tyr) monoclonal antibodies demonstrated that CSF-1 induced a significant dose- and time-dependent increase in P-tyr labeling of neonatal rat osteoclasts. Phalloidin staining was consistent with partial to complete disassembly of the actin attachment ring with redistribution of actin to the spreading cytoplasmic edge of the cell. Quantitation of cellular F-actin using NBD-phallicidin confirmed a decrease in polymerized actin following exposure to CSF-1. In contrast, CSF-1 failed to induce any cytoplasmic spreading in osteoclasts isolated from mice with targeted disruption of the src gene. Further, in src osteoclasts no well defined attachment ring could be identified. To investigate cell-signaling events associated with osteoclast spreading, detergent lysates were made from purified multinucleated osteoclast-like cells (OCLs) obtained by coculturing murine bone marrow and osteoblasts with calcitriol. Western blot analyses of lysates from control and CSF-1-treated normal cells indicated that several proteins were specifically phosphorylated in response to CSF-1, most notably proteins of 165, 60, and 85–90 kDa. Immunoprecipitation studies revealed that the 165 and 60 kDa proteins were, respectively, c-Fms and c-Src. The c-Src kinase activity was increased 2.9-fold following CSF-1 treatment. The 85–90 kDa protein is as yet unidentified. Since activated receptor tyrosine kinases may induce spreading in part by reducing phosphoinositol 4,5-bisphosphate (PIP₂) binding to actin-associated proteins, a monoclonal antibody to PIP₂ was used to assess the nature of PIP₂ binding proteins in OCLs. Proteins of 85–90 kDa, 43 kDa, and 30 kDa were consistently demonstrated to bind PIP₂. Further, the PIP₂ content of the 85–90 kDa protein appeared to decrease with CSF-1 treatment. Whether this protein represents the phosphoprotein of the same M.W. is unclear. We also examined the effect of CSF-1 on the PIP₂ content of α-actinin. Alpha -actinin showed low-level PIP₂ binding, which was demonstrable only after immunoprecipitation and did not change with CSF-1 treatment. However, CSF-1 did cause a significant decline in the phosphotyrosine content of α-actinin. In contrast, in src OCLs, CSF-1 induced more prolonged phosphorylation of c-Fms, and the 85–90 kDa protein was markedly hypophosphorylated. Further, α-actinin did not dephosphorylate in src cells. We conclude that CSF-1-induced osteoclast spreading is accompanied by rapid reorganization of the actin cytoskeleton and phosphorylation of several cellular substrates, including c-Fms and c-Src. PIP₂ binding to at least one protein appears to decrease with CSF-1 treatment, which may favor actin depolymerization. The reduced tyrosine phosphorylation of α-actinin could effect its ability to bind to actin. Thus c-Src may play an important role in these cellular events since in its absence, osteoclasts do not spread and signaling events downstream are altered. Whether these changes relate in part to the basal abnormalities in the cytoskeletal organization of src osteoclasts remains to be determined. Mol Reprod Dev 46:104–108, 1997. © 1997 Wiley-Liss, Inc.     

The 92-kDa chitinase from Streptomyces olivaceoviridis contains a lysine-C endoproteinase at its N-terminus

Abstract Serine proteinases of 42, 22 and 14 kDa were purified from the culture fluid of Streptomyces olivaceoviridis by FPLC. The first 14 amino acids at their N-termini were identical and coincide with the N-terminal amino acid sequence of 92-kDa chitinase, which was found to hydrolyse casein. The four proteins hydrolyse synthetic substrates at the carboxyl group of lysine and (more slowly) arginine. The 14-kDa endoproteinase releases only two fragments of 42 and 43 kDa from β-galactosidase. When the pure 92-kDa chitinase was incubated at 37°C in Tris·HCl buffer, it was cleaved into a 70-kDa chitinase and a 22-kDa proteinase which in its part is rapidly degraded to a 14-kDa proteinase.     

Actin is ADP-ribosylated by the Salmonella enterica virulence-associated protein SpvB

The Salmonella enterica virulence-associated protein SpvB was recently shown to contain a carboxy-terminal mono(ADP-ribosyl)transferase domain. We demonstrate here that the catalytic domain of SpvB as well bacterial extracts containing full-length SpvB modifies a 43 kDa protein from macrophage-like J774-A.1 and epithelial MDCK cells as shown by label transfer from [32P]-nicotinamide adenine dinucleotide (NAD) to the 43 kDa protein. When analysed by two-dimensional gel electrophoresis, the same protein was modified in cells infected with S. enterica serovariant Dublin strain SH9325, whereas infection with an isogenic spvB mutant strain did not result in modification. Immunoprecipitation and immunoblotting experiments using SH9325-infected cells identified the modified protein as actin. The isolated catalytic domain of SpvB mediated transfer of 32P from [32P]-NAD to actins from various sources in vitro, whereas isolated eukaryotic control proteins or bacterial proteins were not modified. In an in vitro actin polymerization assay, the isolated catalytic SpvB domain prevented the conversion of G actin into F actin. Microscopic examination of MDCK cells infected with SH9325 revealed morphological changes and loss of filamentous actin content, whereas cells infected with the spvB mutant remained virtually unaffected. We conclude that actin is a target for an SpvB-mediated modification, most probably ADP-ribosylation, and that the modification of G actin interferes with actin polymerization.     

Garlic (Allium sativum) chitinases: characterization and molecular cloning

Leaves and bulbs of garlic ( Allium sativum L.) contain a chitinase which can be separated into three different isoforms with similar molecular structure and N- terminal amino acid sequence. SDS-PAGE of the alkylated chitinase revealed two distinct polypeptides of 32 and 33 kDa. Induction studies of the chitinase in leaves of garlic plants indicated that not only treatment with ethephon or salicylate and wounding but also a temperature shock strongly increased the enzyme level.
cDNA libraries constructed from poly(A)-rich RNA isolated from young garlic shoots and bulbs were screened for chitinase clones using the cDNA clone CCH4 encoding a basic potato chitinase as a probe. Two different cDNA clones (designated CHITAS 1 and CHITAS 2)of ca 1 000 bp were isolated and their sequences analyzed. The amino acid sequences deduced from both cDNA clones were homologous though not identical to the N-terminal sequences of the mature chitinases. Although both clones encode highly homologous chitinases their sequences definitely differ in that they have different signal peptides and one of them contains a glycine-rich domain. The garlic chitinases are apparently translated from an mRNA of 1200 nucleotides which encodes a proprotein of approximately 32 or 33 kDa for CHITAS 1 and CHITAS 2, respectively. Co-translational removal of the signal peptide will result in a 30 (for CHITAS 1) or 31 kDa (for CHITAS 2) protein with an isoelectric point of 4. 94 (for CHITAS 1) or 6. 12 (for CHITAS 2). Garlic chitinases are encoded by a small gene family as shown by Southern blot analysis of genomic DNA isolated from garlic.
The garlic chitinases show a high degree of sequence homology to the previously isolated chitinases from dicotyledonous as well as monocotyledonous species, indicating that these proteins have been conserved from an evolutionary point of view.     

A 51 kDa Protein Kinase of Potato Activated with Hyphal Wall Components from Phytophthora infestans

In potato tuber tissue, treatment of fungal elicitor, hyphalwall components (HWC) induces various plant defense reactions.As treatment of protein kinase inhibitor prior to HWC treatmentblocks some of defense reactions induced by HWC, involvementof protein kinases in plant defense induction is proposed. Here,we demonstrate HWC-induced activation of a 51-kDa protein kinase(abbreviated p51-PK) using myelin basic protein as a substratein potato tuber discs. The activity of p51-PK was not detectedin the absence of phosphatase inhibitor, NaF, and p51-PK wasimmuroprecipitated with antibody against phosphotyrosine. Pretreatmentof phospholipase C inhibitor, neomycin, and GTP-binding proteinactivator, mastoparan, partially inhibited the HWC-induced activationof p51-PK, suggesting possible involvement of phospholipaseC and GTP-binding protein in the activation of p51-PK. Exogenouslysupplied elicitors, salicylic acid and arachidonic acid, whichare known to induce various defense responses in potato plants,also activated the protein kinase showing the same migrationas p51-PK on SDS-PAGE and different activation patterns. Theseresults implied that p51-PK might be involved in several signaltransduction pathways leading to plant defense responses initiatedby different stimuli. (Received January 11, 1999; Accepted May 25, 1999)     

Induction of pathogenesis-related proteins in sugarcane leaves and cell-cultures by a glycoprotein elicitor isolated from <Emphasis Type="Italic">Colletotrichum falcatum</Emphasis>

The induction of pathogenesis-related (PR) proteins in sugarcane (Saccharum officinarum L.) leaves and suspension-cultured cells in response to treatment with a glycoprotein elicitor isolated from Colletotrichum falcatum (the red rot pathogen) was investigated. Treatment of leaves and cells with the elicitor resulted in a much marked increase in the activities of chitinase and β-1,3-glucanase in red rot resistant (BO 91) than susceptible (CoC 671) sugarcane cultivar. SDS-PAGE analysis revealed that C. falcatum elicitor induced the accumulation of several proteins in suspension-cultured cells of resistant cultivar (BO 91); among them the 35 kDa protein was predominant. Whereas, a 27 kDa protein was induced predominantly in the cells of susceptible cultivar upon treatment with the elicitor. When sugarcane leaves were treated with C. falcatum elicitor, two proteins with apparent molecular masses of 25 and 27 kDa were induced both in the resistant and susceptible cultivars. However, the induction was stronger in the resistant than the susceptible cultivar. Immunoblot analysis for chitinase indicated that a protein with an apparent molecular mass of 37 kDa cross-reacting with barley chitinase antiserum was strongly induced in the suspension cultured cells of both the cultivars. The induction of 37 kDa chitinase was more in the cells of resistant cultivar than in the susceptible cultivar. Western blot analysis revealed that a 25 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antiserum was strongly induced in leaves and cultured cells of both resistant and susceptible cultivars due to elicitor treatment.     

Induction of Chitinase and Phenylalanine Ammonia-Lyase in Cultured Carrot Cells Treated with Fungal Mycelial Walls

Addition of insoluble mycelial walls of a fungus, Chaetomiumglobosum, stimulated the induction of chitinase and phenylalanineammonia-lyase (PAL), as well as the accumulation of phenolicacids in cultured carrot cells. Mycelial wall fragments solubilizedby chitinase treatment also elicited accumulation of phenolicacids. The induction of chitinase and PAL were highly dependenton the age of the carrot cell cultures, as are other defenseresponses, including phytoalexin production. (Received April 2, 1986; Accepted August 22, 1986)     

Exoproteinases of the Oomycete Phytophthora infestans

When grown in a medium containing heat-stable potato tuber proteins, the oomycete Phytophthora infestans (Mont.) de Bary produces a set of exoproteinases active at neutral and mildly basic pH values. These extracellular proteinases have been shown (by SDS-PAGE in the presence of gelatin) to include at least six components differing in molecular weight. Inhibitory analysis and studies of the effects of the enzymes on various synthetic substrates show that the culture liquid of P. infestans contains mainly serine proteinases (specific for trypsin and subtilisin) and metalloproteinases. Their activity is suppressed by proteinase-inhibiting proteins from potato tubers. It is suggested that exoproteinases of P. infestans may be the metabolic target for natural proteinase inhibitors from potato.     

Pathogenesis-related proteins in lima bean leaves infected with tobacco ringspot virus and their distribution within and around local lesions

Tobacco ringspot virus (TRSV) induces circular, darkbrown local lesions on primary leaves of lima bean (Phaseolus lunatus cv Nemagreen) with a concomitant production of three basic and three acidic pathogenesisrelated (PR) proteins. The three basic proteins are: a 21 kDa protein related serologically to Pinto bean PR-4d and tobacco PR-5 proteins; a 36 kDa glucanase that is related to tobacco PR-2; and, a 31 kDa chitinase related serologically to ethylene-induced bean chitinase. The three acidic 18 kDa lima bean PR proteins are serologically similar and probably are charged isomers of the same protein. The 21 kDa basic protein and the 18 kDa acidic protein accumulated preferentially at the lesion center while the 31 kDa chitinase and TRSV were distributed evenly throughout the necrotic area. In green tissue immediately surrounding a lesion, the amounts of PR proteins were comparable to or lower than those in the necrotic area, and virions were not detected. This mode of spatial distribution indicates that lima bean PR proteins are not involved in TRSV localization, and is consistent with other observations that PR proteins play no direct role in restricting viral spread.     

Differential regulation of CD43 glycoforms on CD4+ and CD8+ T lymphocytes in graft-versus-host disease

Two distinct T-cell glycoforms of CD43 result from differentialglycosylation of a single gene product in vivo. The 115 kDaglycoform carries mainly tetrasaccharides and is a pan T-cellmarker, whereas the 130 kDa glycoform carries mainly hexasaccharidesand is associated with T-cell activation. CD43 has been shownto play a role both in enhancing and inhibiting cell adhesion;however, the function of the individual glycoforms is unknown.We have examined the distribution and regulation of the CD43glycoforms in a murine model of acute graft-versus-host disease(GVHD) using monoclonal antibodies (mAbs) S7 and 1B11 specificfor the 115 and 130 kDa CD43 glycoforms, respectively. An increasein T-lymphocyte CD43 130 kDa expression occurred during GVHDfrom day 4 onwards and coincided with splenomegaly and upregulationof the ß1-6GlcNAc transferase (C2GnT), the key enzymeresponsible for the addition of complex O-glycan branching toCD43. When T-lymphocyte subsets were examined for CD43 expression,we found that in GVHD, both CD43 glycoforms were upregulatedon CD4⁺ T cells. However, in CD8⁺ T cells, CD43 115 kDa wasdownregulated while CD43 130 kDa was dramatically upregulated,such that two distinct CD8⁺1B11⁺ T-cell subsets were observed.These data demonstrate differential expression of the CD43 glycoformsin both resting and activated CD4⁺ and CD8⁺ T cells, and suggestthat glycosylation differences between the CD43 glycoforms mayreflect participation in the different functions of these T-cellsubsets in immune disorders in vivo. activation CD43 glycosyltransferases graft-versushost disease T lymphocytes     

Immunolocalization of an extracellular domain of connexin43 in rat heart gap junctions.

Antipeptide antibodies directed to residues 55 to 66 (NTQQPGCENVCY) of connexin43 (cx43) specifically recognize this protein on Western blots of intact and urea-split gap junctions isolated from rat heart. These antibodies detect a single protein of 43 kDa, corresponding to cx43, on Western blots of whole fractions of various vertebrate hearts. Immunogold labeling by electron microscopy shows that the epitopes recognized by these antibodies are not localized on the cytoplasmic surfaces of intact gap junctions but only at the edges of these junctions. In urea-split gap junctions the gold particles are seen in the junctional space, associated with the extracellular faces of junctional membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses of rat heart gap junctions treated with trypsin show that they are constituted with either two polypeptides of Mr 12,000 and 14,000 or a single polypeptide of Mr 22,000 according to whether the analyses are performed under reducing or non-reducing conditions, respectively. The antibodies directed to residues 55 to 66 of cx43 cross-react with both the 12 and 22 kDa polypeptides. These results suggest that the two protected domains of 12 and 14 kDa which contain the first extracellular loop and a putative second extracellular loop, respectively, are linked by disulfide bonds. In adult rat heart sections analyzed by indirect immunofluorescence the intercalated discs are labeled with antibodies directed to a cytoplasmic carboxy-terminal domain of cx43 (El Aoumari et al., J. Membr. Biol. 115, 229-240 (1990)). The same intercalated discs are also labeled in adjacent sections incubated with the antibodies directed to residues 55 to 66. Two hypotheses might explain these results: either the antibodies have access to the extracellular domain of cx43 molecules localized at the edges of the gap junctions, or cx43 molecules are present in the non-junctional membranes of the intercalated discs.     

Molecular characterization of four chitinase cDNAs obtained fromCladosporium fulvum-infected tomato

Complementary DNA clones encoding acidic and basic isoforms of tomato chitinases were isolated fromCladosporium fulvum-infected leaves. The clones were sequenced and found to encode the 30 kDa basic intracellular and the 26 and 27 kDa acidic extracellular tomato chitinases previously purified (M.H.A.J. Joostenet al., in preparation). A fourth truncated cDNA which appears to encode an extracellular chitinase with 82% amino acid similarity to the 30 kDa intracellular chitinase was also isolated. Characterization of the clones revealed that the 30 kDa basic intracellular protein is a class I chitinase and that the 26 and 27 kDa acidic extracellular proteins which have 85% peptide sequence similarity are class II chitinases. The characterized cDNA clones represent four from a family of at least six tomato chitinases. Southern blot analysis indicated that, with the exception of the 30 kDa basic intracellular chitinase, the tomato chitinases are encoded by one or two genes. Northern blot analysis showed that the mRNA encoding the 26 kDa acidic extracellular chitinase is induced more rapidly during an incompatibleC. fulvum-tomato interaction than during a compatible interaction. This difference in timing of mRNA induction was not observed for the 30 kDa basic intracellular chitinase.