Biomedical subjects

J R Vanfleteren

Publications and source records attributed to J R Vanfleteren.

At least 19 recordsLinked to original sources

PubMed1997

Modulation of kinase activities in dauers and in long-lived mutants of Caenorhabditis elegans.

Mutant alleles of the genes age-1 and daf-2 that lengthen life span (Age phenotype) of Caenorhabditis elegans cause higher protein kinase (PKA, PKC, PTK) activity levels in senescing worms relative to wild-type. Elevated levels of PKA and PTK were also present in dauer larvae, developmentally arrested juveniles specialized for long-term survival, relative to L3 larvae, the alternative developmental stage. PKC activity was downregulated in dauers of a non-Age control strain and in age-1 mutant dauers, compared to L3 larvae, but similar activities were measured in dauers and L3 larvae of a daf-2 mutant strain. Thus, age-1 and daf-2 mutant worms may express distinct elements of a dauer-specific survival program during adult life.

Alleles↗

PubMed1996

Rate of aerobic metabolism and superoxide production rate potential in the nematode Caenorhabditis elegans.

We have monitored oxygen consumption as a measure of the rate of aerobic metabolism during the lifetime of Caenorhabditis elegans. We have also developed a chemiluminescent technique which measures exogenous NADPH-stimulated superoxide anion production by freeze-thawed worms. In this assay light production depends on the combined activities of all of the enzymes involved in superoxide production, both directly and indirectly, thus reflecting their activity levels immediately prior to freeze fixation. We have designated this parameter the superoxide production rate potential. The superoxide production rate potential is controlled by the longevity determining gene age-1 and varies in a life cycle-dependent fashion. The metabolic rate generally follows these fluctuations, but additionally shows specific alterations as a response to environmental factors. Metabolic rate and superoxide production rate potential increase by 1.3- and 3-fold, respectively, in reproducing adults. This increase is not due to the contribution of embryonating eggs, however. Culture conditions have a large effect on metabolic rate, but not on the superoxide production rate potential. The energetic cost of movement, measured as consumed oxygen, is low relative to the costs of maintenance and reproduction. Identical superoxide production rate potentials are scored in paralyzed and motile worms, as would be expected.

Acridines↗

PubMed1995

The gerontogenes age-1 and daf-2 determine metabolic rate potential in aging Caenorhabditis elegans.

Mutations in the genes age-1 and daf-2 extend life span of Caenorhabditis elegans by 100 and 200%, respectively, in axenic culture. Adult worms that are mutant in either of these genes have higher metabolic capacities, called metabolic rate potentials, at all ages and the extension of their life expectancies are positively correlated with the increases of metabolic rate potential. The activities of catalase, superoxide dismutase, isocitrate dehydrogenase, isocitrate lyase, and malate synthase are all higher relative to those in worms that are wild type for these genes, but acid phosphatase is down-regulated and alkaline phosphatase activity is lowered to 10% of the activity measured in age-1(+) and daf-2(+) worms. These results suggest that genes that regulate metabolic activity may play central roles in longevity and senescence.

Acid Phosphatase↗

PubMed1994

Structural characterization of an Ascaris myoglobin.

Globin was purified from the body wall of adults of the parasitic nematode Ascaris suum. Internal peptide fragments were sequenced and cDNAs encoding a polypeptide of 154 amino acids isolated by polymerase chain reaction. The polypeptide lacks a signal sequence, identifying it as a cytosolic myoglobin-like species. The native protein is a dimer. The predicted amino acid sequence shares several unusual substitutions with other nematode globins. Like the abundant pseudocoelomic A. suum hemoglobin it has a Tyr at B10 and a Gln at E7, substitutions thought to be determinants of high affinity. However, the 10-fold lower oxygen affinity of body wall globin suggests that in this molecule Tyr(B10) does not form an additional hydrogen bond with the heme bound oxygen. Evolutionary analysis of the nematode globins suggests that the monodomain myoglobin-like molecules and the two-domain hemoglobin-like molecules diverged about 500 million years ago, well before the divergence of the ascarid genera Ascaris and Pseudoterranova. The absence of introns in the A. suum myoglobin, in contrast to other nematode globin genes, is consistent with the hypothesis that during evolution intron elimination was the predominant event.

Amino Acid Sequence↗

PubMed1994

Analysis of the proteins of aging Caenorhabditis elegans by high resolution two-dimensional gel electrophoresis.

The metabolic rate decreases dramatically as a function of age in the nematode worm Caenorhabditis elegans. Superoxide anion production, which is tightly linked to oxygen consumption, and thus to metabolic rate, drops to a 20-fold lower level in 10-day-old, senescent worms, as compared to 4-day-old young adults. In a long-lived mutant strain of the same species metabolic activity is much better preserved. High resolution two-dimensional gel electrophoresis was employed to study alterations in the protein profile, correlating with changes of metabolic activity. Surprisingly, few proteins show age- or age- and strain-specific variations of spot intensity. The abundance of the huge majority of proteins displayed on these gels remains unaltered, irrespective of age and strain differences. These results imply that there are no major age-related alterations of proteins due to faulty protein synthesis or free radical attack, and that age-related changes in the rate of protein synthesis and breakdown must be strictly coordinated throughout the aging process.

Aging↗

PubMed1994

Molecular genealogy of some nematode taxa as based on cytochrome c and globin amino acid sequences.

We have begun to reconstruct the ancient history of the nematode phylum based on cytochrome c and globin amino acid sequences. The data suggest that the nematode ancestor diverged from a line leading to mammals about 1 billion years ago and that the most recent common ancestor of the extant species Caenorhabditis elegans, Trichostrongylus colubriformis, Nippostrongylus brasiliensis, Ascaris suum, and Pseudoterranova decipiens lived about 550 MY ago. The rhabditids and strongylids emerged as one offshoot of this ancestor, the ascarids as another. Rhabditids and strongylids diverged some 400 MY ago, whereas the genera Trichostrongylus and Nippostrongylus diverged slightly over 200 MY ago. A gene duplication event in the strongylid branch is predicted to have occurred around 250-335 MY ago. There are two globin genes in Nippostrongylus, expressed in anatomically distinct compartments (body and cuticle), and the single sequence from Trichostrongylus is most like the Nippostrongylus body globin gene. A strikingly different duplication event occurred within the same period in the line leading to the extant ascarid genera, creating a single polypeptide containing two globin domains. The genera Ascaris and Pseudoterranova diverged some 150-250 MY ago. Interestingly, the second globin repeat evolved at a faster rate in both species examined. This is possibly related to the acquisition of an unusual carboxyterminal extension, composed of alternating positively and negatively charged residues, that is necessary for the assembly of several monomers into the native polymeric molecules.

Amino Acid Sequence↗

PubMed1993

Oxidative stress and ageing in Caenorhabditis elegans.

Mutations in the age-1 gene double both the mean and maximum life span of Caenorhabditis elegans. They also result in an age-specific increase of catalase and Cu/Zn superoxide dismutase activity levels. The higher superoxide dismutase activity levels in age-1 mutants confer hyperresistance to the superoxide-anion-generating drug paraquat. The rate of superoxide anion production by microsome fractions declines linearly with age in age-1(+) worms, but, after an initial decline, is stabilized at a higher level in senescent age-1 mutant nematodes. These results clearly show that oxidative stress resistance and potential life span are correlated in this organism, and they suggest that the natural product of age-1 either directly or indirectly downregulates the activities of several other genes as a function of age.

Aging↗

PubMed1992

Peptide mapping and microsequencing of proteins separated by SDS-PAGE after limited in situ acid hydrolysis.

A method is described for the isolation of peptide fragments from proteins separated by polyacrylamide gel electrophoresis. After completion of the electrophoresis step, gels are stained with Ponceau S or Coomassie Blue. Gel portions containing protein stained with Ponceau S are excised and transferred to borosilicate glass digestion tubes containing 0.9 ml of 1 mM NaOH or 5 mM Na2HPO4. After complete dissociation of the dye from the protein, 0.1 ml of 20% formic acid is added and the protein is hydrolyzed in situ at 112 degrees C for four hours. Subsequently the acid solution is made 10% in acetonitrile and chromatographed as such on a C18 (C4) reversed-phase column using an appropriate large-volume sample loading syringe and injection loop. Proteins stained with Coomassie Blue can be hydrolyzed in situ after complete removal of the dye with an aqueous solution containing 40% acetone, 10% triethylamine and 5% acetic acid. The gel slices are next washed with HPLC-grade water and protein is hydrolyzed in 2% formic acid under standard conditions. Gel-related contaminants do not interfere with the peptide separation under the proper conditions of HPLC analysis.

Amino Acid Sequence↗

PubMed1990

The primary structure of cytochrome c from the nematode Caenorhabditis elegans.

The complete amino acid sequence of cytochrome c from the nematode Caenorhabditis elegans was determined. The native protein displays the same spectral properties in the oxidized and reduced states as horse heart cytochrome c. The apoprotein consists of 110 amino acid residues and differs from human cytochrome c by 44 substitutions, one internal deletion, five N-terminal additions and two C-terminal additions. One of the substitutions is the replacement of an 'invariant' phenylalanine residue at position 15 by tyrosine. The N-terminal sequence extension contains a short peptide motif, which is highly homologous with a peptide fragment present at the N-terminus of annelid and insect cytochrome c sequences. From the number of amino acid changes and the evolutionary rate of cytochrome c it would appear that nematodes diverged from a line leading to man about 1.4 billion years ago. When similar data based on the amino acid sequences of the histones H1, H2A, H2B and H3 are taken into account, the average estimate is 1.1 +/- 0.1 billion years.

Amino Acid Sequence↗

PubMed1990

The primary structure of a minor isoform (H1.2) of histone H1 from the nematode Caenorhabditis elegans.

The complete amino acid sequence of a minor isoform (H1.2) of histone H1 from the nematode Caenorhabditis elegans was determined. The amino acid chain consists of 190 residues and has a blocked N-terminus. Histone subtype H1.2 is 17 residues shorter than the major isoform H1.1, mainly as the result of deletions of short peptide fragments. Considerable divergence from isoform H1.1 has occurred in the N-terminal domain and the very C-terminus of the molecule, but the central globular domain and most of the C-terminal domain, including two potential phosphorylation sites, have been well conserved. Secondary-structure predictions for both H1 isoforms reveal a high potential for helix formation in the N-terminal region 1-33 of isoform H1.1 whereas the corresponding region in isoform H1.2 has low probability of being found in alpha-helix. No major differences in secondary structure are predicted for other parts of both H1 subtypes. The aberrant conformation of isoform H1.2 may be indicative of a significantly different function.

Amino Acid Sequence↗

PubMed1990

Chromatographic recovery of polypeptides from copper-stained sodium dodecyl sulfate polyacrylamide gels.

A procedure of preparative electrophoresis is described in which proteins separated on sodium dodecyl sulfate gels, stained with copper and eluted by simple diffusion, are highly concentrated on a fluorocarbon packing and freed of small molecular weight substances, including sodium dodecyl sulfate and buffer components and gel-related substances. This method can be used for microscale preparations or it can be scaled up to recover milligram amounts of protein. The purified polypeptides, however denatured, are suitable for amino acid sequencing.

Chromatography, High Pressure Liquid↗

PubMed1989

The histones of Caenorhabditis elegans: no evidence of stage-specific isoforms. An overview.

The nematode Caenorhabditis elegans expresses one species of H2A and one species of H4 molecules, at least two species of H1 (H1.1, H1.2), two species of H2B (H2B.1, H2B.2) and 2-4 species of H3 (H3.1 and H3.3 and an unassigned Ile/Leu microheterogeneity in H3). The study of their primary structures has been completed now and all of them, with the exception of the Ile/Leu microheterogeneity in H3, have been assigned to protein spots on two-dimensional gels. One spot, previously designated H3.2, probably represents C-terminally cleaved H3.1. The relative abundance of the isohistones was essentially the same when derived from either eggs, gravid adults or postreproductive, senescent worms. The degree of post-translational modification, however, particularly acetylation of H2A, H2B and H3 histone species, was reduced at old age.

Acetylation↗

PubMed1989

Sequential sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase chromatography of unfolded proteins.

Sequential sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography on a fluorocarbon packing (poly F column, DuPont) provide fully denatured but highly purified protein, which is free of low-molecular-weight substances and directly amenable to structural analysis. Conditions for the preparative elution of four test proteins (bovine serum albumin, carbonic anhydrase, myoglobin, cytochrome c) blotted to Immobilon membranes have been optimized. Phenol saturated with 50 mM Tris-HCl (pH 8.25) and containing 2% SDS and 1% 2-mercaptoethanol is able to elute proteins that have been blotted to Immobilon membranes, stained with Coomassie R-250, and stored as dry sheets, largely irrespective of their molecular mass. Polypeptides that are not degraded by exposure to low pH can also be efficiently extracted directly from stained gels with 70% formic acid. If further separation of polypeptides is not needed, a simple run of a protein sample dissolved in 1-2% SDS on the poly F column will efficiently remove low-molecular-weight substances, including SDS.

Blotting, Western↗

PubMed1989

Sequential two-dimensional and acetic acid/urea/Triton X-100 gel electrophoresis of proteins.

A method is described which combines the resolving power of two-dimensional gel electrophoresis with that of acetic acid/urea/Triton X-100 gel electrophoresis, avoiding the necessity of eluting protein from the gels at any step of the procedure. The combination of electrophoretic separation on the basis of charge, mass, and hydrophobic properties of the proteins has the potential of resolving modified forms and isoforms present in very complex protein populations. The technique can be used for analytical purposes, or it may be scaled up to yield microgram amounts of highly purified proteins. The resolution obtained by tandem application of nonequilibrium pH gradient electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and polyacrylamide gel electrophoresis in the presence of nonionic detergent was evaluated using crude nuclear proteins of the nematode Caenorhabditis elegans.

Acetates↗

PubMed1988

The primary structure of the major isoform (H1.1) of histone H1 from the nematode Caenorhabditis elegans.

The complete primary structure of the major isoform (H1.1) of histone H1 from the nematode Caenorhabditis elegans was determined. The amino acid chain consists of 207 amino acids and has a blocked N-terminus. The nematode histone shows rather little sequence identity when compared with proteins of the H1 family derived from other organisms. However, the main characteristic features of H1 molecules have been well conserved: a tripartite domain structure consisting of a central hydrophobic core of about 80 residues, flanked by an N-terminal domain which is somewhat acidic at the very N-terminus, but very basic further on, and a long C-terminal domain very rich in lysine, alanine and proline. Several repeat structures, including a twice (with modification)-repeated and well-conserved phosphorylation site, can be recognized in this region. The presence of O-phosphoserine at these sites could not be demonstrated, however.

Amino Acid Sequence↗

PubMed1987

Age-specific nuclear proteins in the nematode worm Caenorhabditis elegans.

The nematode worm Caenorhabditis elegans is known to undergo characteristic morphological as well as physiological signs of senescence. Two-dimensional gel electrophoresis shows that alterations also occur in the pattern of the nuclear proteins as a function of age. Non-histone proteins whose level exhibits a steep fall with age are egg-specific and not involved in senescence. However, a distinct set of non-histones accumulates with age and can be considered as senescence markers. Some of these are glycoproteins, as shown by their concanavalin A-binding properties. One age-specific polypeptide, called 'protein S-28', was further characterized by peptide mapping and determination of its N-terminal amino acid sequence.

Aging↗

PubMed1987

The primary structure of histone H2A from the nematode Caenorhabditis elegans.

The complete primary structure of histone H2A from the nematode Caenorhabditis elegans was determined. The amino acid chain consists of 126 amino acid residues and has a blocked N-terminus. By comparison with calf thymus histone H2A, the nematode protein shows five deletions, two insertions and 16 substitutions. Most of the changes occur in the N- and C-terminal regions of the molecule, whereas the central part covering the residues 21-120 is quite well conserved. The lysine residues 5, 8 and 10 were found to be partially acetylated.

Amino Acid Sequence↗

PubMed1987

The primary structure of histone H3 from the nematode Caenorhabditis elegans.

The complete amino acid sequence of histone H3 (135 residues) from the nematode Caenorhabditis elegans has been established. Microheterogeneity occurs at positions 96 and 100 of the chain. The sequences of the nematode H3 isoforms are very similar to the major chain of calf thymus H3 with which they show 4 substitutions in total. The major variant has cysteine in position 96. This is the first report of cysteine in this position in H3 from non-mammalian tissue. An exceptional methylation site has been detected at position 79. Various other sites of secondary modification are of a conservative nature.

Amino Acid Sequence↗