Stressed Jerusalem artichoke tubers (Helianthus tuberosus L.) excrete a protein fraction with specific cytotoxicity on plant and animal tumour cell

Wounds from Jerusalem artichoke (Helianthus tuberosus L.) tubers excrete bioactive metabolites from a variety of structural classes, including proteins. Here we describe a protein specifically active against tumour cells arising either from human, animal or plant tissues. The non-tumour animal cells or the plant callus cells are not sensitive to these excreta. The active product was only obtained after a wound-drought stress of plant tubers. The cytotoxicity varies according to the tumour cell type. For instance, some human tumour cell lines and especially the human mammary tumour cells MDA-MB-231 were shown to be very susceptible to the active product. The active agent is shown to contain an 18-kDa polypeptide with homology to a superoxide dismutase (SOD). A 28-kDa polypeptide, related to an alkaline phosphatase (AP), was shown to be tightly linked to this 18-kDa polypeptide. The excreted 28-kDa polypeptide also displayed a consensus sequence similar to the group of DING proteins, but with a smaller molecular weight. The superoxide dismutase polypeptide was shown to be involved in the antitumour activity, but the presence of smaller factors (MW<10 kDa), such as salicylic acid, can enhance this activity.     

Accumulation of a lectin-like breakdown product of β-conglutin catabolism in cotyledons of germinating Lupinus albus L. seeds

During germination of Lupinus albus seeds, a 20-kDa polypeptide accumulates in the cotyledons of 4-d-old plants (Ferreira et al., 1995b, J Exp Bot 46: 211–219). Immunological, polypeptide cleavage with cyanogen bromide and amino acid sequencing experiments indicate that the 20-kDa polypeptide and ubiquitin are structurally unrelated. However there is a strong sequence homology between the 20-kDa polypeptide and the vicilin-like storage proteins from pea and soybean. Our results indicate that the 20-kDa polypeptide is an intermediate breakdown product of β-conglutin catabolism, the vicilin-like storage protein from L. albus, and that its interaction with anti-ubiquitin antibodies results from the recognition of the antibodies by the 20-kDa polypeptide rather than by the opposite. Besides rabbit anti-ubiquitin antibodies, the 20-kDa polypeptide interacts with a variety of glycoproteins, including immunoglobulin G from several animal species, peroxidase and alkaline phosphatase, suggesting that it possesses a lectin-type activity. Its activity is resistant to sodium dodecyl sulfate or methanol treatments, boiling and autoclaving. Purification of the 20-kDa polypeptide and immunological studies with anti-20-kDa-polypeptide antibodies showed that the non-glycosylated polypeptide is part of a glycoprotein with an estimated molecular mass of 210 kDa, composed of several types of structurally related subunit with molecular masses ranging from 14 to 50 kDa. Purified native protein containing the 20-kDa polypeptide self-aggregates in a calcium-dependent manner as reported for some glycosylated lectins. The possible physiological function of the 20-kDa polypeptide is discussed. Received: 28 June 1996 / Accepted: 7 February 1997     

Tobacco nectarin I. Purification and characterization as a germin-like, manganese superoxide dismutase implicated in the defensE of floral reproductive tissues

Nectarin I, a protein that accumulates in the nectar of Nicotiana sp. , was determined to contain superoxide dismutase activity by colorimetric and in-gel assays. This activity was found to be remarkably thermostable. Extended incubations at temperatures up to 90 degrees C did not diminish the superoxide dismutase activity of nectarin I. This attribute allowed nectarin I to be purified to homogeneity by heat denaturation of the other nectar proteins. By SDS-polyacrylamide gel electrophoresis, nectarin I appeared as a 29-kDa monomer. If the protein sample was not boiled prior to loading the gel, then nectarin I migrated as 165-kDa oligomeric protein. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the protomer subunit was found to be a 22.5-kDa protein. Purified nectarin I contained 0.5 atoms of manganese/monomer, and the superoxide dismutase activity of nectarin I was not inhibited by either H(2)O(2) or NaCN. Following denaturation, the superoxide dismutase activity was restored after Mn(2+) addition. Addition of Fe(2+), Cu(2+), Zn(2+), and Cu(2+)/Zn(2+) did not restore superoxide dismutase activity. The quaternary structure of the reconstituted enzyme was examined, and only tetrameric and pentameric aggregates were enzymatically active. The reconstituted enzyme was also shown to generate H(2)O(2). Putative nectarin I homologues were found in the nectars of several other plant species.     

Superoxide Dismutase, Catalase, and alpha-Tocopherol Content of Stored Potato Tubers

Activated oxygen or oxygen free radical mediated damage to plants has been established or implicated in many plant stress situations. The extent of activated oxygen damage to potato (Solanum tuberosum L.) tubers during low temperature storage and long-term storage is not known. Quantitation of oxygen free radical mediated damage in plant tissues is difficult. However, it is comparatively easy to quantitate endogenous antioxidants, which detoxify potentially damaging forms of activated oxygen. Three tuber antioxidants, superoxide dismutase, catalase, and α-tocopherol were assayed from four potato cultivars stored at 3°C and 9°C for 40 weeks. Tubers stored at 3°C demonstrated increased superoxide dismutase activities (up to 72%) compared to tubers stored at 9°C. Time dependent increases in the levels of superoxide dismutase, catalase, and α-tocopherol occurred during the course of the 40 week storage. The possible relationship between these increases in antioxidants and the rate of activated oxygen production in the tubers is discussed.     

Studies on the Rabies Virus RNA Polymerase: 2. Possible Relationships between the Two Forms of the Non-Catalytic Subunit (P Protein)

We investigated the relationship between the two forms of rabies virus P protein, a non-catalytic subunit of rabies virus RNA polymerase. The two displayed different electrophoretic mobilities as 37- and 40-kDa polypeptides, hence termed as p37 and p40, respectively. Double labeling experiments with [³H]leucine and [³²P]orthophosphate demonstrated that p40 was much more phosphorylated than p37. Treatment of the virion proteins with alkaline phosphatase eliminated only p40, and not 37-kDa polypeptide. The p37 was a major product of the P gene, and was accumulated in the infected cell and incorporated into the virion. On the other hand, p40 was apparently detected only in the virion, and little detected in the cells. Treatment of infected cells with okadaic acid, however, resulted in significant accumulation of p40 in the cell, suggesting that p40 was continuously produced in the cell but dephosphorylated quickly. We detected both 37- and 40-kDa products in P cDNA-transfected animal cells, while only a 37-kDa product was produced in Escherichia coli. Incubation of 37-kDa products from E. coli with the lysates of animal cells in vitro resulted in the production of a 40-kDa product, which was also shown to be suppressed by the heparin. From these results, it is suggested that p40 is produced by the hyperphosphorylation of a 37-kDa polypeptide, which depends on certain heparin-sensitive cellular enzyme(s) and occurs even in the absence of the other viral gene products, and that p40 is reverted quickly to p37 in the infected cells, probably being dependent on some virus-induced factor(s).     

Serine and tyrosine phosphorylation of 28- and 35-kDa proteins of human T lymphocytes stimulated by streptococcal M protein

Purified polypeptide fragments of certain surface M proteins of group A streptococci stimulate blastogenesis and the differentiation of cytotoxic T lymphocytes of normal human lymphocytes. The biochemical basis of lymphocyte stimulation by a type M5 protein polypeptide fragment (pep M5) was investigated. Optimal blastogenic doses of pep M5 or phytohemagglutinin stimulated the phosphorylation of several cellular proteins. However, pep M5 but not phytohemagglutinin induced the phosphorylation of 28- and 35-kDa proteins. The 28-kDa protein was shown to be phosphorylated only at serine residues, whereas the 35-kDa protein was phosphorylated only at tyrosine residues. Stimulation of peripheral blood lymphocytes with pep M5 caused a two-fold increase in the CD8+ and CD4+ 4B4+ subpopulations of T lymphocytes. The phosphorylation of the 28-kDa protein appeared to be confined to the CD4+ T cell subpopulation.     

Role of Protease Inhibitors in Potato Protection

Mechanical wounding or infection of potatoes with Phytophthora infestans caused an accumulation of only serine protease inhibitors in exudates of potato tubers. Among them, proteins prevailed that are structurally similar to those present in healthy tubers: a 22-kDa trypsin inhibitor, a 21-kDa serine protease inhibitor consisting of two polypeptide chains, and a 8-kDa potato chymotrypsin I inhibitor produced de novo. The accumulated proteins inhibited the growth of hyphae and germination of zoospores of P. infestans. Treatment with elicitors, jasmonic and arachidonic acids, intensified the accumulation of these inhibitors in tubers in response to the wound stress, whereas salicylic acid blocked this process. These results suggest that lipoxygenase metabolism plays a substantial role in signal transduction of the protective system of resting potato tubers.     

GM2 gangliosidoses in Spain: Analysis of the HEXA and HEXB genes in 34 Tay-Sachs and 14 Sandhoff patients

Acid α-glucosidase (GAA) is a lysosomal enzyme that hydrolyzes glycogen to glucose. Deficiency of GAA causes Pompe disease. Mammalian GAA is synthesized as a precursor of ~ 110,000 Da that is N-glycosylated and targeted to the lysosome via the M6P receptors. In the lysosome, human GAA is sequentially processed by proteases to polypeptides of 76-, 19.4-, and 3.9-kDa that remain associated. Further cleavage between R²⁰⁰ and A²⁰⁴ inefficiently converts the 76-kDa polypeptide to the mature 70-kDa form with an additional 10.4-kDa polypeptide. GAA maturation increases its affinity for glycogen by 7-10 fold. In contrast to human GAA, processing of bovine and hamster GAA to the 70-kDa form is more rapid. A comparison of sequences surrounding the cleavage site revealed human GAA contains histidine at 201 while other species contain hydrophobic amino acids at position 201 in the otherwise conserved sequence. Recombinant human GAA (rhGAA) containing the H201L substitution was expressed in 293 T cells by transfection. Pulse chase experiments in 293 T cells expressing rhGAA with or without the H201L substitution revealed rapid processing of rhGAA^H201L but not rhGAA^WT to the 70-kDa form. Similarly, when GAA precursor was endocytosed by human Pompe fibroblasts rhGAA^H201L but not rhGAA^WT was rapidly converted to the 70-kDa mature GAA. These studies indicate that the amino acid at position 201 influences the rate of conversion of 76-kDa GAA to 70-kDa GAA. The GAA sequence rather than the lysosomal protease environment explains the predominance of the 76-kDa form in human tissues.     

Biosynthesis and secretion of an osteopontin-related 20-kDa polypeptide in the Madin-Darby canine kidney cell line

We describe a 20-kDa phosphorylated polypeptide, which is secreted constitutively at the apical surface of the kidney-derived Madin-Darby canine kidney cell line. Using polyclonal antibodies raised against this protein, we show that it is generated from a 60-kDa O-glycosylated, sulfated, and phosphorylated precursor protein by an intracellular proteolytic maturation step, which is pH-sensitive. Amino acid sequence analysis of the 20-kDa secreted polypeptide demonstrated that it displays 70% identity with the carboxyl-terminal amino acids of human osteopontin. The amino-terminal amino acid of the 20-kDa polypeptide corresponds to amino acid 213 of human osteopontin. Thrombin has been shown to cleave rat osteopontin in vivo and in vitro at amino acid 153, yielding two fragments of 28 and 26 kDa. A similar cleavage product can be detected by thrombin treatment of the 60-kDa precursor, suggesting that the precursor is identical or closely related to osteopontin. In the rat nephron, the protein has been localized along the luminal surfaces of the proximal and distal tubule and the collecting duct cells. These results show that in the kidney-derived cell line Madin-Darby canine kidney osteopontin or a closely related protein is proteolytically processed to a 20-kDa polypeptide, raising the possibility that diverse functions of osteopontin in various tissues might be attributed to specific processing to distinct polypeptides.     

Role of proteinase inhibitors in potato protection

Mechanical damage or infection of potatoes with Phytophthora infestans caused an accumulation of only serine protease inhibitors in exudates of potato tubers. Among them, proteins prevailed that are structurally similar to those present in healthy tubers: a 22-kDa trypsin inhibitor, a 21-kDa serine protease inhibitor consisting of two polypeptide chains, and a 8-kDa potato chymotrypsin I inhibitor produced de novo. The accumulated proteins inhibited the growth of hyphae and germination of zoospores of P. infestans. Treatment with elicitors, jasmonic and arachidonic acids, intensified the accumulation of these inhibitors in tubers in response to the wound stress, whereas salicylic acid blocked this process. These results suggest that the lipoxygenase metabolism plays a substantial role in signal transduction of the protective system of resting potato tubers.     

Purification and characterization of adult diarrhea rotavirus: identification of viral structural proteins.

Adult diarrhea rotavirus (ADRV) is a newly identified strain of noncultivable human group B rotavirus that has been epidemic in the People's Republic of China since 1982. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western (immuno-) blot analysis to examine the viral proteins present in the outer and inner capsids of ADRV and compared these with the proteins of a group A rotavirus, SA11. EDTA treatment of double-shelled virions removed the outer capsid and resulted in the loss of three polypeptides of 64, 61, and 41, kilodaltons (kDa). Endo-beta-N-acetylglucosaminidase H digestion of double-shelled virions identified the 41-kDa polypeptide as a glycoprotein. CaCl2 treatment of single-shelled particles removed the inner capsid and resulted in the loss of one polypeptide with a molecular mass of 47 kDa. The remaining core particle had two major structural proteins of 136 and 113 kDa. All of the proteins visualized on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were antigenic by Western blot analysis when probed with convalescent-phase human and animal antisera. A 47-kDa polypeptide was most abundant and was strongly immunoreactive with human sera, animal sera raised against ADRV and against other group B animal rotaviruses (infectious diarrhea of infant rat virus, bovine and porcine group B rotavirus, and bovine enteric syncytial virus) and a monoclonal antibody prepared against infectious diarrhea of infant rat virus. This 47-kDa inner capsid polypeptide contains a common group B antigen and is similar to the VP6 of the group A rotaviruses. Human convalescent-phase sera also responded to a 41-kDa polypeptide of the outer capsid that seems similar to the VP7 of group A rotavirus. Other polypeptides have been given tentative designations on the basis of similarities to the control preparation of SA11, including a 136-kDa polypeptide designated VP1, a 113-kDa polypeptide designated VP2, 64- and 61-kDa polypeptides designated VP5 and VP5a, and several proteins in the 110- to 72-kDa range that may be VP3, VP4, or related proteins. The lack of cross-reactivity on Western blots between antisera to group A versus group B rotaviruses confirmed that these viruses are antigenically quite distinct.     

Protective effect of Operculina turpethum against 7,12-dimethyl benz(a)anthracene induced oxidative stress with reference to breast cancer in experimental rats

Reactive oxygen species (ROS) directly or indirectly involves in multistage process of carcinogenesis. Antioxidant activity of methanolic extract of Operculina turpethum stems (MEOT) on 7,12 dimethylbenz(a)anthracene (DMBA) induced breast cancer was investigated in female Sprague-Dawley rats. Changes in the levels of lipid peroxidation and antioxidants system was evaluated in addition to tumour development. Twenty four female rats were divided into four groups: control, DMBA, DMBA + MEOT and MEOT. In the DMBA group, rats were intragastrically administered with 20 mg of DMBA using corn oil as vehicle. Animals of DMBA + MEOT group received a single dose of 20 mg of DMBA dissolved in corn oil intragastrically followed by O. turpethum extract (100 mg/kg body weight), while MEOT group received O. turpethum extract (100 mg/kg body weight) intragastrically daily for a period of 45 days. After the experimental period of 45 days, oxidative stress parameters were assessed in serum, liver and breast of both control and experimental groups. In addition to this, tumour weight of breast was also assessed. A significant increase in lipid peroxidation levels were observed in the tested samples of cancer induced rats while the activities of enzymic antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and non-enzymic antioxidants like glutathione (GSH), ascorbic acid (Vitamin C) and α-tocopherol (Vitamin E) were decreased in cancer-bearing animals when compared to control animals. A significant (P < 0.05) increase in the tumour weight was observed in the breast of DMBA group and the breast tumour weight decreased significantly (P < 0.05) in the DMBA + MEOT groups. Oral administration of MEOT remarkably reduced the lipid peroxidation activity and increased the antioxidants level in drug treated animals and decreased the tumour weight significantly (P < 0.05). This result suggests that MEOT shows antioxidant activity and play a protective role against DMBA induced breast cancer.     

Rhizobium meliloti nodulation genes: identification of nodDABC gene products, purification of nodA protein, and expression of nodA in Rhizobium meliloti.

A set of conserved, or common, bacterial nodulation (nod) loci is required for host plant infection by Rhizobium meliloti and other Rhizobium species. Four such genes, nodDABC, have been indicated in R. meliloti 1021 by genetic analysis and DNA sequencing. An essential step toward understanding the function of these genes is to characterize their protein products. We used in vitro and maxicell Escherichia coli expression systems, together with gel electrophoresis and autoradiography, to detect proteins encoded by nodDABC. We facilitated expression of genes on these DNA fragments by inserting them downstream of the Salmonella typhimurium trp promoter, both in colE1 and incP plasmid-based vectors. Use of the incP trp promoter plasmid allowed overexpression of a nodABC gene fragment in R. meliloti. We found that nodA encodes a protein of 21 kilodaltons (kDa), and nodB encodes one of 28 kDa; the nodC product appears as two polypeptide bands at 44 and 45 kDa. Expression of the divergently read nodD yields a single polypeptide of 33 kDa. Whether these represent true Rhizobium gene products must be demonstrated by correlating these proteins with genetically defined Rhizobium loci. We purified the 21-kDa putative nodA protein product by gel electrophoresis, selective precipitation, and ion-exchange chromatography and generated antiserum to the purified gene product. This permitted the immunological demonstration that the 21-kDa protein is present in wild-type cells and in nodB- or nodC-defective strains, but is absent from nodA::Tn5 mutants, which confirms that the product expressed in E. coli is identical to that produced by R. meliloti nodA. Using antisera detection, we found that the level of nodA protein is increased by exposure of R. meliloti cells to plant exudate, indicating regulation of the bacterial nod genes by the plant host.     

Conformational stability of recombinant manganese superoxide dismutase from the filamentous fungus Trichoderma reesei

Superoxide dismutases (SODs; EC 1.15.1.1) are part of the antioxidant system of aerobic organisms and are used as a defense against oxidative injury caused by reactive oxygen species (ROS). The cloning and sequencing of the 788-bp genomic DNA from Trichoderma reesei strain QM9414 (anamorph of Hypocrea jecorina) revealed an open reading frame encoding a protein of 212 amino acid residues, with 65-90% similarity to manganese superoxide dismutase from other filamentous fungi. The TrMnSOD was purified and shown to be stable from 20 to 90 °C for 1 h at pH from 8 to 11.5, while maintaining its biological activity.     

Expression of transfected human CuZn superoxide dismutase gene in mouse L cells and NS20Y neuroblastoma cells induces enhancement of glutathione peroxidase activity

The human CuZn superoxide dismutase (superoxide dismutase 1) a key enzyme in the metabolism of oxygen free-radicals, is encoded by a gene located on chromosome 21 in the region 21 q 22.1 known to be involved in Down's syndrome. A gene dosage effect for this enzyme has been reported in trisomy 21. To assess the biological consequences of superoxide dismutase 1 overproduction within cells, the human superoxide dismutase 1 gene and a human superoxide dismutase 1 cDNA were introduced into mouse L cells and NS20Y neuroblastoma cells. Both cell types expressed elevated levels (up to 3-fold) of enzymatically active human superoxide dismutase 1. These human superoxide dismutase 1 overproducers, especially neuronal cell lines, showed an increased activity in the selenodependent glutathione peroxidase. These data are consistent with the possibility that gene dosage of superoxide dismutase 1 contributes to oxygen metabolism modifications previously described in Down's syndrome.     

Comparison of redox state of cells of tatar buckwheat morphogenic calluses and non-morphogenic calluses obtained from them

Intracellular content of hydrogen peroxide and of the product of lipid peroxidation malonic dialdehyde as well as activity of antioxidant enzymes catalase, ascorbate peroxidase, and superoxide dismutase were studied in cells of morphogenic and derived from them non-morphogenic calluses of tatar buckwheat Fagopyrum tataricum L. Non-morphogenic calluses were characterized by significantly higher content of hydrogen peroxide and malonic dialdehyde, low catalase activity, and high activity of superoxide dismutase compared to morphogenic cultures. The results may indicate that cells of non-morphogenic calluses are in the state of continuous oxidative stress. Nevertheless, proliferative activity of non-morphogenic cultures and the biomass increase significantly exceeded these parameters in morphogenic calluses. An analogy is drawn between animal cancer cells and non-morphogenic plant calluses.     

Analysis of Rotavirus Nonstructural Protein NSP5 Phosphorylation

The rotavirus nonstructural phosphoprotein NSP5 is encoded by a gene in RNA segment 11. Immunofluorescence analysis of fixed cells showed that NSP5 polypeptides remained confined to viroplasms even at a late stage when provirions migrated from these structures. When NSP5 was expressed in COS-7 cells in the absence of other viral proteins, it was uniformly distributed in the cytoplasm. Under these conditions, the 26-kDa polypeptide predominated. In the presence of the protein phosphatase inhibitor okadaic acid, the highly phosphorylated 28- and 32- to 35-kDa polypeptides were formed. Also, the fully phosphorylated protein had a homogeneous cytoplasmic distribution in transfected cells. In rotavirus SA11-infected cells, NSP5 synthesis was detectable at 2 h postinfection. However, the newly formed 26-kDa NSP5 was not converted to the 28- to 35-kDa forms until approximately 2 h later. Also, the protein kinase activity of isolated NSP5 was not detectable until the 28- and 30- to 35-kDa NSP5 forms had been formed. NSP5 immunoprecipitated from extracts of transfected COS-7 cells was active in autophosphorylation in vitro, demonstrating that other viral proteins were not required for this function. Treatment of NSP5-expressing cells with staurosporine, a broad-range protein kinase inhibitor, had only a limited negative effect on the phosphorylation of the viral polypeptide. Staurosporine did not inhibit autophosphorylation of NSP5 in vitro. Together, the data support the idea that NSP5 has an autophosphorylation activity that is positively regulated by addition of phosphate residues at some positions.     

Evolutionary conservation of a germ cell-specific lamin persisting through mammalian spermiogenesis.

We had identified earlier a germ cell-specific lamin of 60 kDa in rat which is related to somatic lamin B. This polypeptide was shown to be the only major component organizing the lamina structure of round spermatids. In the present study, we find that this 60-kDa polypeptide persists in the testicular and epididymal sperms of rat. We also show, by indirect immunofluorescence studies, that the 60-kDa protein is antigenically conserved in the germ cells of grasshopper, rooster, and frog and in plant meiocytes. The distribution of fluorescence among the various germ cell populations shows that the antigen is located around the nuclear cortex of pre- and postmeiotic germ cells, while it is distributed all over the pachytene nuclei. The anti-60-kDa polyclonal antibodies also reacted with a 60-kDa polypeptide in the Western blot analysis of nuclear matrix proteins of grasshopper germ cells. The similar fluorescent localization pattern of the antigen observed in various eukaryotic species strongly suggests that this germ cell-specific lamin may play a very crucial role during meiotic prophase, particularly during homologous chromosome pairing and recombination.     

Plant rac proteins induce superoxide production in mammalian cells

The small GTP-binding protein family including Rac proteins represents a paradigm for signaling molecules shared by animal and plants. In mammalian cells, Rac induces the activation of NADPH oxidase leading to superoxide production. In plants, evidence suggests that resistance to pathogens depends on superoxide that is generated via NADPH oxidase-like enzymes. We have identified four closely related Rho/Rac genes from Zea mays that exhibit a high degree of homology to the human Rac. We hypothesized that these plant Rac proteins could function as their mammalian counterpart and activate an enzymatic complex that leads to superoxide production. Here, we show that like human Rac1, activated Zea mays Rac genes can induce superoxide production, when expressed in a mammalian system: NIH 3T3 cells. Our results suggest that in plants, Rac proteins can function as activators of oxidative burst and indicate the remarkable functional and structural conservation of Rho/Rac proteins between plant and animal kingdoms during evolution.