Showing papers by "Steven Clarke published in 1995"

Exceptional seed longevity and robust growth: ancient Sacred Lotus from China

Abstract: A 1,288 + 271-yr-old (1,350 ? 220 yr BP, radiocarbon age) seed of Sacred Lotus (Nelumbo nucifera Gaertn.) from an ancient lake bed at Pulantien, Liaoning Province, China, has been germinated and subsequently radiocarbon dated. This is the oldest demonstrably viable and directly dated seed ever reported, the preserved relict of one of the early crops of lotus cultivated by Buddhists at Pulantien after introduction of the religion into the region prior to 372 A.D. A small portion of the dry pericarp of a second lotus fruit from the same locale has been dated as being 332 + 135-yr-old (270 ? 60 yr BP, radiocarbon age) by accelerator mass spectroscopy at the Lawrence Livermore National Laboratory. This polycentenarian seed not only germinated but is still growing (since March, 1994). Of six old lotus fruits tested, two-thirds germinated, almost all in fewer than 4 d, as rapidly as fruits harvested from the progeny of Pulantien Sacred Lotus plants (under cultivation by the National Park Service in Washington, DC), and more rapidly than fresh fruits of Yellow Lotus [N. lutea (Willd.) Pers.]. Growth of the old lotus is robust: rhizome formation and leaf emergence at rhizome nodes are more rapid than those of the Pulantien progeny, although the leaf width is smaller. Activity of the protein-repair enzyme L-isoaspartyl methyltransferase in the old lotus seed is persistent during germination and is as robust as that in the progeny, and the degree of aspartyl racemization in proteins of the two groups of plants is minimal and essentially identical. The six dated ancient Sacred Lotus fruits range in age from 95 to 1,288 yr (with a mean age of 595 ? 380 yr), evidently reflecting their production, deposition, and preservation at varying times during the intervening millennium.

Repair, refold, recycle: how bacteria can deal with spontaneous and environmental damage to proteins

Abstract: Summary Proteins, like DNA, are subject to various forms of damage that can render them non-functional. Conformational changes and covalent chemical alterations occur spontaneously, and the rates of these reactions can be increased by environmental stresses such as heat, oxidative agents, or changes in pH or osmotic conditions. Although affected proteins can be replaced by de novo biosynthesis, cells – especially those subjected to stress or nutrient limitation – have developed mechanisms which can either restore damaged polypeptides to an active state or remove them. Such mechanisms can spare the biosynthetic capacity of the cell and ensure that the presence of non-functional molecules does not disrupt cell physiology. Three major mechanisms, which operate in bacteria as well as eukaryotic organisms, have been described. First, chaperones not only assist in proper de novo folding of proteins but also provide an important means of restoring activity to conformationally damaged proteins. Second, enzymatic‘repair’systems exist to directly reverse certain forms of protein damage, including proline isomerization, methionine oxidation and the formation of isoaspartyl residues. Finally, proteolysis provides a‘last-resort’means of dealing with abnormal proteins which cannot be repaired. Protein maintenance and repair may be of special importance for bacteria preparing to survive extended periods in stationary phase: both constitutive and induced mechanisms are utilized to permit survival despite greatly reduced protein synthesis.

Protein L-isoaspartyl methyltransferase from the nematode Caenorhabditis elegans: genomic structure and substrate specificity.

Abstract: We identified a protein L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77) in the nematode worm Caenorhabditis elegans. The methylation of abnormal L-isoaspartyl residues by this enzyme can lead to their conversion to L-aspartyl residues and represents a protein repair step for polypeptides damaged by spontaneous reactions during the aging process. We show that the levels of this enzyme increase 2-fold in C. elegans in the dauer larval form, a developmental stage where the organism can survive for extended periods of time. Utilizing degenerate oligonucleotide primers derived from conserved amino acid sequences of mammalian, plant, and bacterial L-isoaspartyl methyltransferases and PCR amplification, we made DNA probes that allowed us to obtain cDNA and genomic DNA clones encoding this enzyme in the nematode. The deduced amino acid sequence is 53% identical to the human enzyme and 29% identical to the Escherichia coli enzyme. Overexpression of the cDNA for the C. elegans enzyme in E. coli gave an active product with micromolar Km values for L-isoaspartyl-containing peptide substrates and for the methyl donor S-adenosyl-L-methionine. No methylation of D-aspartyl-containing peptides was detected under conditions where the human enzyme catalyzed this reaction, suggesting that the ability to methylate D-aspartyl residues in addition to L-isoaspartyl residues was a later evolutionary adaptation of this enzyme. The C. elegans gene for the methyltransferase, designated pcm-1, was mapped to a single site in a 31 kb region in the central portion of chromosome V. The gene is 3.2 kb in length and includes six introns. Although much smaller, its genomic organization is similar to that of the corresponding mouse gene, with identically positioned intron--exon splice junctions at five of seven sites. We propose that this gene plays an important role in facilitating the long term survival of this organisms.

Production and use of human and plant methyltransferases

Abstract: An isolated recombinant human L-isoaspartyl/D-aspartyl protein methyltransferase is obtained by overexpression of cDNA coding for isozyme II in an E. coli strain, and a cDNA clone of the wheat enzyme and a purified enzyme from wheat are obtained. These enzymes are useful in treatment of medical conditions and diagnosis of disease associated with an increase in L-isoaspartyl/D-aspartyl residues of polypeptides in a tissue.