Identification and characterization of a recombinant metallothionein protein from a marine alga, Fucus vesiculosus.
A cDNA library was constructed from macroalgae adapted to prolonged elevated environmental copper levels. To investigate the possible existence of a metallothionein (MT) gene, the library was screened with degenerate probes designed using plant MT cysteine-rich motifs. A gene was identified (1229 bp) with a putative open reading frame (204 bp) encoding a 67-amino-acid protein exhibiting several characteristic features of MT proteins, including 16 cysteine residues (24%) and only one aromatic residue. Although the protein sequence showed high identity with plant and invertebrate MTs, it contained a unique 'linker' region (14 amino acid residues) between the two putative metal-binding domains which contained no cysteine residues. This extended linker is larger than the tripeptide found in archetypal vertebrate MTs, but does not conform either with the 40-amino-acid linkers commonly found in plant MT sequences. An S-peptide Fucus MT fusion protein expressed in Escherichia coli exhibited a relative molecular mass of approximately 14 kDa. The recombinant fusion bound seven Cd ions, of which 50% were dissociated at pH 4.1. Under anaerobic conditions, the Cd ions were displaced by Cu(I), which associated with the protein at a ratio of 13:1. Laboratory exposure of F. vesiculosus to elevated copper resulted in induction of the MT gene. Thus this paper describes, for the first time, an MT gene identified from macroalgae which is induced by copper exposure and whose encoded protein product binds cadmium and copper. (+info)
Interactions of membrane potential and cations in regulation of ciliary activity in Paramecium.
Ciliary activity in Paramecium was investigated in different external solutions using techniques of voltage clamp and high frequency cinematography. An increase in the external concentration of K, Ca or Mg ions decreased the resting potential. It had no effect on ciliary activity. When the membrane potential was fixed, an increase in external Ca or Mg and, to a lesser extent, an increase in K concentration, raised the frequency of normal beating or decreased the frequency of reversed beating of the cilia. Similar effects resulted from membrane hyperpolarization with constant ionic conditions. Increase in concentration of Ca, but not of Mg or K, enhanced hyperpolarization-induced augmentation of ciliary frequency. Increase in Ca concentration also specifically augmented the delayed increase in inward current during rapid hyperpolarizing clamp. The results support the view that [Ca]i regulates the frequency and direction of ciliary beating. It is suggested that the insensitivity of the ciliary motor system to elevations of the external concentrations of ions results from compensation of their effects on [Ca]i. Depolarization itself appears to increase [Ca]i while elevation of the external ion concentrations at a fixed membrane potential appears to decrease [Ca]i. (+info)
Purification and properties of a low-molecular-weight, high-alkaline pectate lyase from an alkaliphilic strain of Bacillus.
A low-molecular-weight, high-alkaline pectate lyase (pectate transeliminase, EC 22.214.171.124) was found in an alkaline culture of Bacillus sp. strain KSM-P15, purified to homogeneity, and crystallized. The enzyme had a relative molecular weight of approximately 20,300 as measured by sedimentation equilibrium, with a sedimentation coefficient (s20,w0) of 1.73 S. It was a basic protein with an isoelectric point of pH 10.3, and the alpha-helical content was only 6.6%. In the presence of Ca2+ ions, the enzyme degraded polygalacturonic acid in a random manner to yield 4,5-unsaturated oligo-galacturonides and had its optimal activity around pH 10.5 and 50-55 degrees C. It also had a protopectinase-like activity on cotton fibers. The N-terminal amino acid sequences of the intact protein (28 amino acids) and its two lysyl endopeptidase-cleaved peptide fragments (8 and 12 amino acids) had very low sequence similarity with pectate lyases reported to date. These results strongly suggest that the pectate lyase of Bacillus sp. strain KSM-P15 may be a novel enzyme and belongs in a new family. (+info)
Purification and characterization of Aspergillus ficuum endoinulinase.
Endoinulinase from Aspergillus ficuum, which catalyzes the hydrolysis of inulin via an endo-cleavage mode, was purified by chromatography from Novozym 230 as a starting commercial enzyme mixture on CM-Sephadex and DEAE-Sepharose, and by preparative electrophoresis under native conditions. The enzyme was estimated to be pure on the basis of its I/S ratio, whose value was infinite in our assay conditions. Two forms separated by using this method. SDS gel electrophoresis showed the two purified forms to respectively exhibit molecular weights of 64,000 +/- 500 and 66,000 +/- 1,000. The results of deglycosylation indicated that the two forms were originally the same protein but with different sugar contents. A molecular weight of 54,800 +/- 1,500 was found by gel filtration of the native enzyme, indicating the native functional protein to be a monomer. The enzyme showed nearly absolute substrate specificity towards inulin and inulooligosaccharides, and acted via an endo-attack to produce mainly inulotriose during the late stage of the reaction. The apparent Km and Vmax values for inulin hydrolysis were 8.1 +/- 1.0 mM and 773 +/- 60 U/mg, respectively. The internal peptides of the enzyme showed sequence homology to the endoinulinase of Penicillium purpurogenum. (+info)
Analysis of 118 second-generation metal-on-metal retrieved hip implants.
Osteolysis is due to particulate wear debris and is responsible for the long-term failure of total hip replacements. It has stimulated the development of alternative joint surfaces such as metal-on-metal or ceramic-on-ceramic implants. Since 1988 the second-generation metal-on-metal implant Metasul has been used in over 60 000 hips. Analysis of 118 retrieved specimens of the head or cup showed rates of wear of approximately 25 microm for the whole articulation per year in the first year, decreasing to about 5 microm per year after the third. Metal surfaces have a 'self-polishing' capacity. Scratches are worn out by further joint movement. Volumetric wear was decreased some 60-fold compared with that of metal-on-polyethylene implants, suggesting that second-generation metal-on-metal prostheses may considerably reduce osteolysis. (+info)
Adaptation of bulk constitutive equations to insoluble monolayer collapse at the air-water interface.
A constitutive equation based on stress-strain models of bulk solids was adapted to relate the surface pressure, compression rate, and temperature of an insoluble monolayer of monodendrons during collapse at the air-water interface. A power law relation between compression rate and surface pressure and an Arrhenius temperature dependence of the steady-state creep rate were observed in data from compression rate and creep experiments in the collapse region. These relations were combined into a single constitutive equation to calculate the temperature dependence of the collapse pressure with a maximum error of 5 percent for temperatures ranging from 10 degrees to 25 degrees C. (+info)
SAG, a novel zinc RING finger protein that protects cells from apoptosis induced by redox agents.
SAG (sensitive to apoptosis gene) was cloned as an inducible gene by 1,10-phenanthroline (OP), a redox-sensitive compound and an apoptosis inducer. SAG encodes a novel zinc RING finger protein that consists of 113 amino acids with a calculated molecular mass of 12.6 kDa. SAG is highly conserved during evolution, with identities of 70% between human and Caenorhabditis elegans sequences and 55% between human and yeast sequences. In human tissues, SAG is ubiquitously expressed at high levels in skeletal muscles, heart, and testis. SAG is localized in both the cytoplasm and the nucleus of cells, and its gene was mapped to chromosome 3q22-24. Bacterially expressed and purified human SAG binds to zinc and copper metal ions and prevents lipid peroxidation induced by copper or a free radical generator. When overexpressed in several human cell lines, SAG protects cells from apoptosis induced by redox agents (the metal chelator OP and zinc or copper metal ions). Mechanistically, SAG appears to inhibit and/or delay metal ion-induced cytochrome c release and caspase activation. Thus, SAG is a cellular protective molecule that appears to act as an antioxidant to inhibit apoptosis induced by metal ions and reactive oxygen species. (+info)
Amyloid beta peptides do not form peptide-derived free radicals spontaneously, but can enhance metal-catalyzed oxidation of hydroxylamines to nitroxides.
Amyloid beta (Abeta) peptides play an important role in the pathogenesis of Alzheimer's disease. Free radical generation by Abeta peptides was suggested to be a key mechanism of their neurotoxicity. Reports that neurotoxic free radicals derived from Abeta-(1-40) and Abeta-(25-35) peptides react with the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) to form a PBN/.Abeta peptide radical adduct with a specific triplet ESR signal assert that the peptide itself was the source of free radicals. We now report that three Abeta peptides, Abeta-(1-40), Abeta-(25-35), and Abeta-(40-1), do not yield radical adducts with PBN from the Oklahoma Medical Research Foundation (OMRF). In contrast to OMRF PBN, incubation of Sigma PBN in phosphate buffer without Abeta peptides produced a three-line ESR spectrum. It was shown that this nitroxide is di-tert-butylnitroxide and is formed in the Sigma PBN solution as a result of transition metal-catalyzed auto-oxidation of the respective hydroxylamine present as an impurity in the Sigma PBN. Under some conditions, incubation of PBN from Sigma with Abeta-(1-40) or Abeta-(25-35) can stimulate the formation of di-tert-butylnitroxide. It was shown that Abeta peptides enhanced oxidation of cyclic hydroxylamine 1-hydroxy-4-oxo-2,2,6, 6-tetramethylpiperidine (TEMPONE-H), which was strongly inhibited by the treatment of phosphate buffer with Chelex-100. It was shown that ferric and cupric ions are effective oxidants of TEMPONE-H. The data obtained allow us to conclude that under some conditions toxic Abeta peptides Abeta-(1-40) and Abeta-(25-35) enhance metal-catalyzed oxidation of hydroxylamine derivatives, but do not spontaneously form peptide-derived free radicals. (+info)