Small, novel proteins from the mistletoe Phoradendron tomentosum exhibit highly selective cytotoxicity to human breast cancer cells.

Four novel proteins (phoratoxins C-F) have been isolated from the North American mistletoe Phoradendron tomentosum. The amino acid sequences of these phoratoxins were determined unambiguously using a combination of Edman degradation and trypsin enzymatic digestion, and by electrospray ionization tandem mass spectrometry sequencing. Phoratoxins C, E and F consist of 46 amino acid residues; and phoratoxin D of 41. All proteins had six cysteines, similar to the earlier described phoratoxins A and B, which are thionins. The cytotoxicity of each protein was evaluated in a human cell line panel that represented several cytotoxic drug-resistance mechanisms. For the half-maximal inhibitory concentrations (IC50 values) of the different cell lines in the panel, correlation with those of standard drugs was low. The most potent cytotoxic phoratoxin C was further tested on primary cultures of human tumor cells from patients. The solid tumor samples from breast cancer cells were 18 times more sensitive to phoratoxin C than the tested hematological tumor samples.

Plant low-molecular-weight phospholipase A2S (PLA2s) are structurally related to the animal secretory PLA2s and are present as a family of isoforms in rice (Oryza sativa).

Recently, we purified to homogeneity and characterized a low-molecular-weight calcium-dependent phospholipase A2 (PLA2) from developing elm seed endosperm. This represented the first purified and characterized PLA2 from a plant tissue. The full sequences of two distinct but homologous rice (Oryza sativa) cDNAs are given here. These encode mature proteins of 1 19 amino acids (PLA2-I, preceded by a 19 amino acid signal peptide) and 128 amino acids (PLA2-II. preceded by a 25 amino acid signal peptide), and were derived from four expressed sequence tag (EST) clones. Both proteins were homologous to the N-terminal amino acid sequence of the elm PLA2. They contained twelve conserved cysteine residues and sequences that are likely to represent the Ca(2+)-binding loop and active-site motif, which are characteristic of animal secretory PLA2s. A soluble PLA2s activity was purified 145 000-fold from green rice shoots. This had the same biochemical characteristics as the elm and animal secretory PLA2s. The purified rice PLA2 consisted of two proteins, with a molecular weight of 12 440 and 12 920, that had identical N-terminal amino acid sequences. This sequence was different from but homologous to the PLA2-I and PLA2-II sequences. Taken together, the results suggest that at least three different low-molecular-weight PLA2s are expressed in green rice shoots. Southern blot analysis suggested that multiple copies of such genes are likely to occur in the rice and in other plant genomes.

Molecular cloning and characterization of starch-branching enzyme II from potato.

Full-length cDNA for starch branching enzyme (SBE) II of potato was isolated and sequenced. In potato, similarly to most other investigated plants, the SBE-II isoform differs from SBE-I by having an acidic amino-terminal extension and a shorter carboxyterminus. Two forms of SBE-II, migrating as 98 and 95 kDa proteins in 6% SDS-polyacrylamide gels, were associated to tuber starch. The latter form was 16 amino acids shorter in the amino terminus. Transcript of SBE-II was present in leaf tissue, whereas significant expression was not seen in tubers. On the other hand, a significant amount of SBE-I transcript was detected in tuber tissue but not in leaves.

The multiple forms of starch-branching enzyme I in Solanum tuberosum.

Western blot analysis showed the presence of three forms of starch-branching enzyme (SBE), with apparent molecular masses of 103, 97 and 80 kDa, in extracts of leaves and stored tubers of Solanum tuberosum. The 80-kDa form was absent in extracts of fresh tuber. Active 80-kDa enzyme was partially purified from stored tubers and sequence analysis showed that it, similar to the two larger enzyme forms, was an SBE-I isoform. Limited proteolysis of isolated 103-kDa SBE-I under native conditions removed approximately 200 amino acid residues from the carboxy terminus. A stable intermediate with an apparent molecular mass of approximately 80 kDa was formed. Since the 80-kDa form displayed full enzymatic activity and its circular-dichroism spectrum did not differ significantly from that of the 103-kDa enzyme, the carboxy-terminal portion of the enzyme was suggested to have an extended, unordered structure and therefore to be easily accessible to proteolysis. A cDNA sequence encoding a mature SBE-I was amplified from tuber mRNA of S. tuberosum by means of PCR. The 3' end of this sequence differed significantly from that of previously published data. PCR amplification and DNA sequencing of the 3' ends of the sbeI gene showed that four sbeI alleles exist in the cultivar studied. Two of these four alleles, sbeia and sbeIb, had slightly longer 3' ends compared with the other two, sbeIc and sbeId. The difference between the two groups of alleles was due to a partial deletion in sbeIc and sbeId of a segment duplicated in all alleles. All four alleles were expressed in leaf and tuber.

The major integral proteins of spinach leaf plasma membranes are putative aquaporins and are phosphorylated in response to Ca2+ and apoplastic water potential.

We show that homologs of the major intrinsic protein (MIP) family are major integral proteins of the spinach leaf plasma membrane and constitute approximately 20% of integral plasma membrane protein. By using oligonucleotide primers based on partial amino acid sequences for polymerase chain reaction and screening of a spinach leaf cDNA library, we obtained two full-length clones of MIP homologs (pm28a and pm28b). One of these clones, pm28a, was sequenced, and it encodes a protein (PM28A) of 281 amino acids with a molecular mass of 29.9 kD. DNA gel blots indicated that PM28A is the product of a single gene, and RNA gel blots showed that pm28a is ubiquitously expressed in the plant. In vivo phosphorylation of the 28-kD polypeptide(s), corresponding to PM28A and PM28B, was dependent on apoplastic water potential, suggesting a role in regulation of cell turgor for these putative aquaporins. In vitro, only one of the homologs, PM28A, was phosphorylated. Phosphorylation of PM28A occurred on Ser-274, seven amino acids from the C terminus of the protein, within a consensus phosphorylation site (Ser-X-Arg) for vertebrate protein kinase C. In vitro phosphorylation of PM28A was due to a plasma membrane-associated protein kinase and was strictly dependent on submicromolar concentrations of Ca2+.

Cloning and sequencing of human gp330, a Ca(2+)-binding receptor with potential intracellular signaling properties.

We present here the complete primary structure of human gp330, the human variant of the principal kidney autoantigen causing Heymann membranous glomerulonephritis in rats. The deduced 4655 amino acid residues give a calculated molecular mass of 519636 Da for the mature protein and consists of a probable 25-amino-acid N-terminal signal peptide sequence, an extracellular region of 4398 amino acids, a single transmembrane-spanning domain of 23 amino acids, and an intracellular C-terminal region of 209 amino acid residues. Three types of cysteine-rich repeats characteristic of the low density lipoprotein receptor (LDLR) superfamily are present in human gp330. In the extracellular region, there are a total of 36 LDLR ligand-binding repeats, comprising four distinct domains, 16 growth factor repeats separated by eight YWTD spacer regions, and one epidermal growth factor-like repeat. No consensus cleavage sequence for the processing endoprotease furin is detected in human gp330. The intracellular tail contains not only two copies of the F(X)NPXY coated-pit mediated internalization signal characteristic of LDLR superfamily members, but also intriguing and potentially functional motifs including several Src-homology 3 recognition motifs, one Src-homology 2 recognition motif for the p85 regulatory subunit of phosphatidylinositol 3-kinase, and additional sites for protein kinase C, casein kinase II and cAMP-/cGMP-dependent protein kinase. There is approximately 77% amino acid identity between human and rat gp330 with minor differences between the extracellular and intracellular regions. Recently gp330 has been implicated in Ca2+ regulation in the parathyroid, the placenta, and the renal tubule, but its overall physiological and pathological role still remains uncertain.

Synthesis, antioxidant properties, biological activity and molecular modelling of a series of chalcogen analogues of the 5-lipoxygenase inhibitor DuP 654.

2-Phenylsulfenyl- (1b), 2-phenylselenenyl- (1c) and 2-phenyltellurenyl-1-naphthol (1d) were prepared and their antioxidative properties evaluated in comparison with 2-benzyl-1-naphthol (1a; DuP 654). 2-Phenyltellurenyl-1-naphthol had a significantly lower (1.00 V versus SCE) oxidation potential than the other three compounds (1.24, 1.27 and 1.25 V, respectively, versus SCE for compounds 1a, 1b and 1c) as determined by cyclic voltammetry. In contrast to the other materials, compound 1d was able to catalyze the reduction of hydrogen peroxide in the presence of thiols as stoichiometric reducing agents. The organotellurium compound was also the most efficient inhibitor of azo-initiated peroxidation of linoleic acid in a two-phase model system. Ab initio geometry optimization at the 3-21G(*) level revealed infinitesimal changes in the molecular conformations of the carbon, sulfur, selenium and tellurium analogues. As judged by their ability to inhibit stimulated LTB4 biosynthesis in human neutrophils, compounds 1a-1d all turned out to be highly potent 5-lipoxygenase inhibitors with IC50-values ranging from 0.40 microM for 2-benzyl-1-naphthol (1a) to 0.063 microM for 2-phenyltellurenyl-1-naphthol (1d).

cDNA cloning and sequencing of mouse mastocytoma glucosaminyl N-deacetylase/N-sulfotransferase, an enzyme involved in the biosynthesis of heparin.

A 110-kDa protein involved in heparin biosynthesis in mouse mastocytoma cells was previously shown to express both glucosaminyl N-deacetylase and N-sulfotransferase activity. In this study, the complete nucleotide sequence corresponding to this protein is reported. The mRNA, estimated to contain 3.9 kilobases encodes a protein with an M(r) of 101,092. The predicted domain structure of the protein resembles those of previously characterized Golgi proteins with an N-terminal cytoplasmic tail, a single membrane-spanning domain, and a large catalytic domain linked to the transmembrane domain through a "stem region." Comparison of the deduced amino acid sequence of the mouse mastocytoma protein and a previously cloned similar enzyme from rat liver demonstrated that while large portions of the proteins, corresponding essentially to the putative catalytic domains, were closely related, other portions, in particular in the N-terminal parts, were markedly different. The divergence was not due to species differences since two separate mouse transcripts could be identified that hybridized with probes specific for the two proteins. Also, functional differences were noted since the mastocytoma enzyme, contrary to the liver enzyme, requires a polycation cofactor for expression of N-deacetylase activity. The results are discussed in relation to the structural properties of heparin and heparan sulfate.

Reoxygenation-induced cell damage of isolated neonatal rat ventricular myocytes can be reduced by chain-breaking antioxidants.

To study the role of chain-breaking antioxidants on reperfusion injury in the ischemic heart, cultured ventricular heart cells (myocytes) were subjected to hypoxia and reoxygenation. The myocytes were prepared from neonatal rats and cultured in F10 medium that was supplemented with serum. As a marker for cell damage, lactate dehydrogenase was analyzed in the medium. Cells subjected to hypoxia for 5 h showed a 1.9 fold increase in lactate dehydrogenase (LD) leakage, while cells subjected to 1 h hypoxia followed by 4 h reoxygenation showed a 5-fold increase in LD intake. Alpha-tocopherol, beta-carotene, nordihydroguairetic acid (NDGA), butylated hydroxyltoluene (BHT), and ICI211965 were added to the cell medium every 24 h for 6 d prior to reoxygenation. All compounds protected against reoxygenation-induced cell damage. In the presence of the 5-lipoxygenase inhibitor ICI211965, protection against LD leakage was found only at high concentrations, which corresponded to the antioxidative effect of ICI211965, and not to inhibition of 5-lipoxygenase. We conclude that cultured ventricular myocytes can be used to evaluate the protective effect of antioxidants on reoxygenation-induced cell damage, and that chain-breaking antioxidants protected well against reoxygenation-induced cell damage.

Characterization of the 97 and 103 kDa forms of starch branching enzyme from potato tubers.

N-Terminal analysis, peptide mapping and partial peptide sequencing of the 97 and 103 kDa forms of starch branching enzyme from potato tubers showed that the two forms are highly related. A comparison with sequence data in the literature showed that these forms belong to the starch branching enzyme isoform I family. An internal cDNA fragment was obtained using PCR technology on potato tuber RNA with two oligonucleotide primers constructed from the peptide sequence data. Southern blot analysis using the PCR fragment as probe showed that there is only one gene locus encoding this isoform of the enzyme in Solanum tuberosum as well as in Solanum commersonii.

Effect of long term treatment with hydrophilic colloid on serum lipids.

Thirteen patients with essential hyperlipoproteinaemia were treated over periods of 2--29 months with hydrophilic colloid made from Psyllium. Reduction of the increased serum cholesterol levels averaged 16.9 per cent, and the corresponding figure for the increased triglyceride levels was 52.0 per cent. The reduction of cholesterol and triglyceride was statistically significant (p less than 0.0025 and p less than 0.0005 respectively). No significant change in normal blood lipid levels was observed on administration of the Psyllium colloid.