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Biomedical subjects

T L Woods

Publications and source records attributed to T L Woods.

10 recordsLinked to original sources
PubMed2001

S100A2 coding sequence polymorphism: characterization and lack of association with psoriasis.

Psoriasis is a chronic inflammatory skin disease with a strong genetic component. Linkage studies have identified several susceptibility loci for psoriasis including a region on chromosome 1q21 termed the 'epidermal differentiation complex'. At least 20 genes involved in epidermal differentiation and proliferation have been mapped to this region including S100A2, a gene known to be over-expressed in psoriasis lesions. In the course of cloning and sequencing several S100A2 cDNAs, we identified an A/G (Asn62Ser) polymorphism at nucleotide 185 of the S100A2 coding region. To determine whether this polymorphism is associated with psoriasis, we tested DNA from 38 unrelated normal and 40 unrelated psoriatic individuals. The 185G allele was present in 148 of the 156 chromosomes analysed, giving an allele frequency of 94.9%. All of the remaining chromosomes carried 185A. There was no significant difference in the allele distribution between normal and psoriatic individuals (normals 72G, 4A; psoriatics 76G, 4A; P = 1.00 by Fisher's exact test). Our data explain conflicting results in the literature regarding the sequence of S100A2 but provide no support for a direct causal role for S100A2 in psoriasis.

Adolescent↗
PubMed2000

Biochemical characterization of S100A2 in human keratinocytes: subcellular localization, dimerization, and oxidative cross-linking.

S100A2 is a calmodulin-like protein of unknown function, whose transcription is positively regulated in response to ErbB and p53 signaling. Expression of S100A2 is markedly increased in the context of ErbB-driven reactive epidermal hyperplasia, and decreased in the context of hypofunctional p53 mutations in carcinoma cell lines and tumors. This bimodal pattern of regulation suggests an important function for S100A2 in keratinocyte differentiation and carcinogenesis. Taking the biochemical approach to the determination of S100A2 function, we have characterized its physical state and subcellular localization in normal human keratinocytes. S100A2 in hypotonic lysates remained soluble after centrifugation at 100 000 x g, indicating that it is not associated with cell membranes. Permeabilization experiments confirmed the lack of membrane association and revealed a digitonin-insoluble nuclear fraction of S100A2, which was confirmed by immunofluorescence microscopy. Pulldown assays of epitope-tagged S100A2 and yeast two-hybrid screening revealed that S100A2 displays a strong propensity to homodimerize. Naturally expressed S100A2 dimers in normal human keratinocytes readily underwent intermolecular disulfide cross-linking unless a strong denaturant was present during cell lysis. Treatment of intact normal human keratinocytes with hydrogen peroxide strongly promoted S100A2 cross-linking. These results demonstrate that native S100A2 is a homodimer that does not depend on disulfide cross-linking for stability, but undergoes intermolecular cross-linking at cysteine residues in response to oxidative stress. Based on these findings, we propose that S100A2 may protect normal keratinocytes against carcinogens by participating in the cellular proof-reading response to oxidative stress.

Antibodies, Monoclonal↗
PubMed1999

Effects of IGF-I, IGF-II, bFGF and PDGF on the initiation of mRNA translation in C2C12 myoblasts and differentiating myoblasts.

In order to study the mechanisms by which growth factors stimulate protein synthesis, C2C12 myogenic cells were treated with a variety of growth factors and the recruitment of free ribosomes to polysomes was quantified. All experiments were conducted on C2C12 myoblasts (24 h prior to induction of fusion) and differentiating myoblasts (24 h after induction of fusion). After the 2 h incubation, cells were rinsed with phosphate buffered saline and quickly frozen at -80 degrees C. Cell lysates were fractionated on 15-60% sucrose gradients by centrifugation at 200,000 x g for 1 h. Absorbance at 254 nm was recorded continuously across the gradient. The response to each of the four growth factors, IGF-I and-II, basic fibroblast growth factor (FGF), and platelet-derived growth factor was a decrease (P < 0.05) in monosome peak height and a increase (P < 0.05) in polysome percentage (P < 0.05). All responses were linear, except IGF-I, and the monosome peak height response to FGF which were quadratic (P < 0.05). None of the growth factors had a significant effect (P > 0.05) on RNA concentrations over the 2-h incubation. Protein content did not vary due to growth factor or level of treatment. This corroborates the hypothesis that the acute increase of protein synthesis exhibited by growth factor treated cells is due to an increase in the activity of existing ribosomes rather than an increase in ribosome synthesis. These results suggest that we can study the mechanisms regulating protein synthesis in muscle cells effectively by studying shifts in ribosomal activity. This method gave more consistent results than the H3-tyrosine incorporation and has the added benefit of not requiring the use of radioactivity. The strong correlation between monosome peak heights and percentage polysomes will allow researchers to measure total protein synthetic activity in a culture from the free or cytoplasmic fraction and to reserve the polysomes for other uses. The similarity of response among the various growth factors may indicate a common mechanism for increasing the initiation of protein synthesis.

Animals↗
PubMed1999

Effects of serum deprivation, insulin and dexamethasone on polysome percentages in C2C12 myoblasts and differentiating myoblasts.

An increase in the rate of protein synthesis in living cells can be achieved by regulating the quantity of mRNA, ribosomes, and enzymes available for translation or by regulating the efficiency at which existing components are used. Efficiency can be measured by comparing the number of ribosomes actively engaged in the synthesis of protein (polysomes) to the pool of free ribosomes. The objective of this study was to determine the percentage of ribosomes found as polysomes in C2C12 cells deprived of serum or exposed to insulin or dexamethasone 24 h before and after being stimulated to differentiate. Individual 60 mm culture dishes were exposed to serum-free control medium, medium containing serum, insulin, or dexamethasone for a period of 1 h or 2 h and then quickly frozen. The ribosomes and polysomes from these cells were separated by ultracentrifugation on 15 to 60% sucrose gradients and the absorbance across the gradient at 254 nm was recorded. Polysome percentages were determined as the area under the polysome peak divided by the total area under the curve. Serum deprivation caused a 12% decline in the percentage of ribosomes found as polysomes (P < 0.01). Dexamethasone caused a quadratic decline (P < 0.05) in polysome percentage, while insulin yielded a quadratic increase (P < 0.05). Protein synthesis assays measuring 3H-tyrosine uptake showed similar responses. These changes occurred in the absence of any differences in total RNA concentration. It was concluded that differentiation and the absence of serum in the media reduced the rate of recruitment of ribosomes for protein synthesis. Insulin increased ribosome recruitment which was also observed by a similar increase in incorporation of radio-labeled tyrosine.

Animals↗
PubMed1997

Conditions for the culture of bovine embryonic myogenic cells.

The objective of this experiment was to determine the growth characteristics of bovine embryonic muscle cells and to optimize the growth conditions for these cells using commercially-prepared media and sera. In the first study, the growth of muscle cells isolated from the hindlimb was determined by measuring DNA content. The DNA concentration was lowest (P < 0.001) at 24 h post-plating and increased to a maximum at approximately 60 h. The slopes of creatine kinase activity and fusion index curves were similar to the DNA; however, the creatine kinase activity achieved a maximum at 140 h post-plating, while the fusion index reached maximum at 120 h. In the second study, cells were cultured on different substrata, either plastic, gelatin, or collagen. There were no differences (P > 0.05) in the cell growth rates for any of the three substrata. In the third study, cells were grown in 10% fetal bovine serum (FBS) and either a balanced salt solution (BSS; 30 mM Hepes, 10 mM glucose, 120 mM NaCl, 2.5 mM Na2HPO4, and 3 mM KCl), McCoy's 5A, Dulbecco's Minimal Essential Medium/Ham's F12 (DMEM/F12), or 70% DMEM/20% M-199. Cell numbers adhering to the plate at 26 h post-plating were different (P > 0.001) between each medium (DMEM/M-199 > McCoy's 5A > DMEM/F12 > BSS). Cell proliferation rates for each treatment medium were greatest for DMEM/M-199, followed by McCoy's 5A, DMEM/F12, and BSS. Cell differentiation was highest (P < 0.05) in the DMEM/F12, followed by McCoy's 5A, DMEM/M-199, and BSS. In the final study, the cells were treated with different sources of serum added at 10% to DMEM/M-199. The sera consisted of FBS, newborn calf serum (NCS), horse serum (HS) and iron-supplemented calf serum (Fe(2+)-CS). The cells were added to each well at 10(4) cells. At 24 h post-plating, the serum-free, NCS, and FBS-treated cell numbers were greater (P < 0.05) than the cells treated with HS or Fe(2+)-CS, which may reflect the efficient adherence to the surface or faster adaptation to the serum by the cells. The proliferation rate was greatest (P < 0.001) for the cells treated with Fe(2+)-CS, followed by FBS = NCS, HS, and no serum. Therefore, the muscle cells obtained from bovine embryos grow and differentiate similar to muscle cells from other species. The optimal growth medium for growing these cells in vitro is DMEM/M-199 plus 10% Fe(2+)-CS, while the optimal differentiation medium is McCoy's 5A.

Animals↗
PubMed1996

Effects of dexamethasone and anabolic agents on proliferation and protein synthesis and degradation in C2C12 myogenic cells.

The objective of this experiment was to determine the dose response of dexamethasone (DEX) on C2C12 myogenic cells and to examine the effects of the anabolic compounds estradiol (E), testosterone (T), and dihydrotestosterone (D) alone and in combination with DEX on proliferation and protein turnover in cultured C2C12 myogenic cells. In the first study, cells were treated with seven concentrations (0, 25, 50, 75, 100, 150, or 200 nM) of DEX in medium with or without 5% horse serum (HS) for the determination of protein synthesis and degradation, and six concentrations (0, 50, 100, 150, 200, or 250 nM) of DEX in medium with 5% fetal bovine serum for cell proliferation measurements. Proliferation of myoblasts decreased (P < .05) with DEX. As DEX concentration increased, protein degradation in myotubes increased (P < .05) up to 100 nM, then declined. Protein synthesis decreased linearly (P < .01) as DEX concentration increased. The presence of HS in the medium decreased (P < .01) protein degradation by 32% as compared with no HS and increased (P < .05) protein synthesis. In the second study, cells were treated with E, T, or D at four concentrations (0, 100, 500, or 1,000 nM) in medium containing 0 or 100 nM DEX. Cells were assayed for protein synthesis or protein degradation. Synthesis decreased (P < .01) and degradation increased (P < .01) with DEX. No differences (P > .05) were found between E, T, or D hormone treatments or concentrations. To measure proliferation, myoblasts were treated 1 d after plating with the same anabolic hormone treatments in medium containing 0 to 100 nM DEX. Cells were grown to confluence and assayed for proliferation. Proliferation decreased (P < .01) in the presence of DEX in each treatment compared with controls. Cells treated with E had significantly lower (P < .05) proliferation rates than cells treated with T and D. The presence of concentrations of DEX at 100 nM inhibited proliferation and protein synthesis and increased protein degradation. Anabolic agents at pharmacological doses do not inhibit the DEX effects on C2C12 myogenic cells, nor do they directly affect proliferation or protein turnover.

Animals↗
PubMedDate not supplied

Determination of 3-methylhistidine by capillary electrophoresis.

The post-translational methylation of histidine to form 3-methylhistidine (3MH) is a modification principally found in contractile proteins, and thus, the level of free 3MH has been used to monitor muscle protein turnover. This work describes procedures for the capillary electrophoretic separation and determination of the phenylthiohydantoin (PTH) derivative of 3MH using uncoated fused-silica capillaries. The procedure described here utilized UV detection and resulted in a linear standard curve in the range of 2-15 pmole, which is more sensitive than previously reported HPLC methods using fluorescent detection. In addition, good agreement for theoretical amounts of 3MH in hydrolyzed rabbit skeletal muscle actin and myofibril preparations from bovine skeletal muscle cells was found.

Actins↗