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Biomedical subjects

D R Crawford

Publications and source records attributed to D R Crawford.

35 records · Page 2Linked to original sources
PubMed1994

Metabolic regulation of gene transcription.

The impact of nutrients on gene expression has become an area of considerable interest as the number of genes coding for key regulatory proteins in metabolic pathways are studied in detail. This has been greatly aided by a number of new techniques developed to study gene transcription in animals. We will use as an example studies on the regulation of transcription of the gene coding for P-enolpyruvate carboxykinase, a key enzyme in hepatic and renal gluconeogenesis. The promoter for P-enolpyruvate carboxykinase contains a number of regulatory elements within 500 bp of the start-site of gene transcription that are required for the response of the gene to metabolic signals. These elements bind tissue-specific transcription factors in complex patterns of interactions, which result in the coordinate control of P-enolpyruvate carboxykinase gene expression. An analysis of the regulation of transcription of this gene involves the use of a number of techniques ranging from gene transfection into cells in culture to the introduction of chimeric genes containing the P-enolpyruvate carboxykinase promoter into transgenic mice. This review presents a progress report on the current status of research on the nutritional and hormonal regulation of transcription of the P-enolpyruvate carboxykinase gene.

Activating Transcription Factor 2↗
PubMed1994

Adaptive response and oxidative stress.

The ability of a cell, tissue, or organism to better resist stress damage by prior exposure to a lesser amount of stress is known as adaptive response. It is observed in all organisms in response to a number of different cytotoxic agents. One of these agents, oxidative stress, is known to induce an adaptive response in bacteria that is accompanied by the induction of many proteins. De novo protein synthesis is required for adaptive response to oxidative and other types of stress, indicating that newly synthesized protective proteins are necessary for adaptation. Adaptive response to oxidative stress also has been observed in mammalian cells. Several studies suggest it is necessary to first preexpose mammalian cells to a somewhat toxic oxidative stress in order to observe significant resistance to a subsequent highly lethal dose of oxidant. Cross-resistance of oxidatively stressed cells to other toxic agents including gamma- and X-irradiation, heat shock, aldehydes, heavy metals, MNNG, N-ethylmaleimide, and heme also has been reported. Understanding oxidant adaptive response in more detail and identifying the protective proteins involved may prove to be of clinical benefit.

Adaptation, Physiological↗
PubMed1993

Regulation of the phosphoenolpyruvate carboxykinase/human factor IX gene introduced into the livers of adult rats by receptor-mediated gene transfer.

Gene transfer systems targeting the asialoglycoprotein receptor have been developed to introduce functional genes into cells in culture and livers of intact animals. A synthetic neoglycoprotein carrier was constructed and complexed to a chimeric gene containing the cDNA for human factor IX ligated to the promoter-regulatory region of the gene for phosphoenolpyruvate carboxykinase from the rat. The complex was used to transfect human hepatoma cells that express the asialoglycoprotein receptor. Human factor IX DNA sequences were found in cells 10 days after treatment. A 1.4 kB mRNA transcript was detected by Northern blot hybridization, which was inducible by treatment with dexamethasone or cAMP with theophylline. Western blot hybridization of proteins secreted into the culture medium detected human factor IX. The chimeric gene was also transferred into livers of rats using the neoglycoprotein carrier system after partial hepatectomy. Although the results were variable, the exogenous gene was transcribed in livers of several animals, and maximal levels of expression of the fully processed human factor IX were detected 30 days after introduction. The concentration of factor IX in the blood returned to control levels 60 days after transfection. Factor IX production was induced as late as 96 days after treatment by feeding transfected animals a diet high in protein but devoid of carbohydrates. This DNA carrier system can be used to introduce functional genes into the livers of rats, and may be a useful technique for gene therapy targeting the liver.

Animals↗
PubMed1991

Active oxygen transforms murine myeloid progenitor cells in vitro.

Active oxygen (AO) is ubiquitous in nature and its many forms can act as natural carcinogens. Their effect on the transformation of a mouse myeloid progenitor cell line was studied using anchorage-independent colony formation in methylcellulose as the primary assay. Both cytotoxic and non-toxic concentrations of t-butylhydroperoxide, hydrogen peroxide and menadione were examined. At non-cytotoxic concentrations, no AO transformation of these cells from interleukin-3 dependence to factor independence (FI) was observed, even after as many as 25 treatments. At cytotoxic concentrations, however, all 3 classes of AO transformed the cells to FI growth. The most potent agent was t-butyl hydroperoxide (43-fold induction), followed by hydrogen peroxide and then menadione. As little as one exposure to cytotoxic levels of these oxidants induced significant transformation, with relative potencies the same as those observed for multiple exposures. These inductions were not due to general cytotoxic effects, since sodium fluoride and heat-shock treatment gave minimal inductions. AO-induced colonies in methylcellulose that were removed, examined and then injected into pre-irradiated mice uniformly produced tumors. Control, non-treated cells did not form tumors. Tumorigenic cells did not form colonies in methylcellulose at lower plating densities. Furthermore, low numbers of transformed cells supplemented to high density with normal cells showed a small but insufficient increase in colony number as compared with high-density cultures of transformed cells. Our results suggest that the transformants depend upon a paracrine mechanism of growth that is mediated by the transformed cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Thrombospondin: biosynthesis, distribution, and changes associated with wound repair in corneal endothelium.

Thrombospondin is a cell adhesion molecule which interacts via specific domains with a wide array of extracellular matrix components, including fibrinogen, fibrin, fibronectin, collagen, and heparan sulfate proteoglycan. Although this protein has been localized in several human tissues, its presence in corneal tissues had not been previously established. In the present study, we have demonstrated that cultured bovine corneal endothelial cells synthesize thrombospondin and incorporate it into their extracellular matrix. We have also shown immunofluorescently the presence and distribution of thrombospondin in these cultured cells and in the noninjured and injured corneal endothelium in situ. Ultrastructural immunoperoxidase cytochemistry revealed that thrombospondin could be displaced from the cell surface by heparin, but not by keratan sulfate. Confluent cultures of corneal endothelium synthesize and secrete the three cell adhesion proteins laminin, thrombospondin, and fibronectin in the ratios 1:8.2:51.8. Only the laminin B chains were detected in immunoprecipitates. Immunofluorescent studies of these cultured cells, using a polyclonal antiserum raised against purified thrombospondin, revealed a low level of fluorescence associated with the cell layer but a punctate fluorescent pattern at the level of the extracellular matrix. Noninjured corneal endothelium in situ also demonstrated a low level of fluorescence throughout the cell layer. However, this dramatically changed after a circular freeze injury to the tissue. By 24 h after wounding, cells surrounding the injury zone displayed a prominent fluorescence that was still observed at 48 h post-injury. In addition to its increased intracellular fluorescence, thrombospondin was also localized as migration tracks, oriented in the direction of cellular migration into the wound site. Thus, in corneal endothelium, thrombospondin appears to play a major role in injury-induced cell migration in situ along a natural basement membrane.

Animals↗
PubMed1989

Constitutive and phorbol-myristate-acetate regulated antioxidant defense of mouse epidermal JB6 cells.

Because oxidative processes can participate in tumor promotion, it is likely that the cellular antioxidant defense also plays a role. We have compared the levels of the three major antioxidant enzymes, Cu,Zn-superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx), in promotable mouse epidermal JB6 cells clone 41 and nonpromotable cells, clone 30. We found that the constitutive activities of SOD and catalase were approximately twice as high in clone 41 as in clone 30 while the GPx activities were comparable. Correspondingly, catalase protein concentrations were higher in clone 41, according to immunoblots. Northern blot analysis indicated that the steady-state mRNA concentrations for SOD and catalase, but not for GPx, were considerably higher in clone 41 than in clone 30. Southern blot analysis showed no difference between the two clones in their complements of the SOD and catalase genes. Clone 41 also contained slightly higher constitutive levels of glutathione. The higher antioxidant capacity of promotable clone 41 may protect it from excessive toxicity of oxidant promoters and allow growth stimulation. Certain tumor promoters that lack oxidizing properties may generate a cellular prooxidant state by a variety of mechanisms (e.g., it had been reported that the phorbol ester PMA decreases the activities of catalase and SOD in mouse skin). We found for JB6 cells that this loss of enzyme activity was due to a decrease in the steady-state concentrations of catalase and SOD mRNA. No significant changes in the rates of transcription were detected in nuclear run-off experiments. The observed decreases in catalase and SOD can be considered as part of the complex reprogramming of gene expression that is set in motion by phorbol-12-myristate-13-acetate.

Animals↗
PubMed1988

Molecular defect in human acatalasia fibroblasts.

The human hereditary disease Acatalasia (AC) is characterized by low or no catalase activity in all body tissues. We have studied the molecular basis of AC. In order to assess their antioxidant defense status we measured the enzyme activities, protein levels and m-RNA concentrations of catalase, superoxide dismutase and glutathione peroxidase in fibroblasts from a Japanese (AC65) and a Swiss (AC64) patient and several normal individuals. Our results point to genetic heterogeneity. While strain AC64 contained normal levels of catalase mRNA and -protein, strain AC65 was completely devoid of both. A structural mutation in the catalase gene is probably responsible for the inactivation of the enzyme in AC64. Since AC65 contains at least a major portion of the catalase gene it may represent a regulatory mutation in which the gene is not transcribed.

Acatalasia↗
PubMed1987

An RNA-binding protein from Xenopus oocytes is associated with specific message sequences.

Monoclonal antibodies directed against an RNA-binding protein from Xenopus oocytes were used to immunoselect messenger ribonucleoprotein (mRNP) particles. RNA was extracted from both the immunoselected and nonselected fractions and was used to direct the synthesis of oligo (dT)-primed 32P-cDNA. These two cDNA preparations were then used to probe Xenopus stage-1 oocyte cDNA libraries to identify sequences that had been specifically coimmunoselected by the antibodies. Three cDNA clones were shown to be derived specifically from the antibody-selected mRNPs. During very early oogenesis (stage 1-2), the RNA-binding protein and the three coselected mRNAs sediment in the nontranslating mRNP region of a sucrose gradient. By oocyte stage 6, the binding protein concentration decreases by as much as 22-fold relative to polyadenylated RNA. At this stage of development, the three mRNAs are found predominantly in the polysome region of a sucrose gradient. These data demonstrate that Xenopus oocytes contain an RNA-binding protein which binds specific message sequences and may regulate their expression.

Animals↗
PubMed1985

Standard threshold shift criteria. An investigation of the most reliable indicator of noise-induced hearing loss.

Definition of a standard threshold shift (STS) and its use as an early indicator to identify those employees with deteriorating hearing are necessary aspects of hearing conservation programs in industry. STS criteria were evaluated using techniques borrowed from decision theory and signal detection theory to separate audiometric threshold shifts into true-positive and false-positive shifts. The current Occupational Safety and Health Administration definition of a "change in hearing threshold relative to the baseline audiogram of an average of 10 dB or more at 2, 3 or 4 kHz in either ear" and the American Academy of Otolaryngology-Head and Neck Surgery recommendation of a "10 dB or greater change for the worse for either the 0.5, 1, 2 kHz pure tone average or the 3, 4, 6 kHz pure tone average, in either ear" were supported. Revision of baselines after an STS occurs is recommended.

Audiometry↗
PubMed1984

Expression of type I and III collagen genes during differentiation of embryonic chicken myoblasts in culture.

Expression of type I and III procollagen genes was studied in embryonic chicken myoblast cell cultures, obtained from thigh muscles of 11-day-old embryos. Differentiation initiated by the addition of ovotransferrin (30 micrograms/ml) was followed visually by phase-contrast microscopy. Myoblast fusion and myotube formation were detected by day 3 and appeared to be complete by day 7. The synthesis of procollagens was monitored by labeling cell cultures for 1 h with [3H]proline and determining the radioactivity in procollagen chains by scanning densitometry of the fluorograms of the sodium dodecyl sulfate-polyacrylamide gels. A 10- to 20-fold increase in the rate of pro alpha-1(I), pro alpha-2(I), and pro alpha-1(III) collagen synthesis was observed, with the greatest increase occurring between days 3 and 9. Collagen mRNA levels in the myoblast cultures were examined by Northern blot and dot blot hybridization assays. The 10- to 20-fold increased rate of protein synthesis was accompanied by a 15-fold increase in the steady-state levels of pro alpha-1(I) and pro alpha-2(I) mRNAs and a 10-fold increase in the steady-state levels of pro alpha-1(III). As a correlate to the studies of collagen expression during myoblast differentiation, the expression of actin mRNAs was examined. Although alpha actin could be detected by day 4, a complete switch from lambda and beta to alpha actin was not observed in the time periods examined. Similar results were obtained in the analysis of RNA extracted from embryonic legs at days 12 and 17 of gestation. Myoblast differentiation is manifested by the accumulation of both muscle-specific mRNAs, such as actin, and type I and III procollagen mRNAs.

Animals↗
PubMed1981

Isolation of plasma membrane from human neutrophils and determination of cytochrome b and quinone content.

Analyses of plasma membrane and other subcellular fractions indicate that the primary location of cytochrome b in human neutrophils is not the plasma membrane. The procedure developed for the purification of plasma membrane from fresh human neutrophils yielded a 14-fold enrichment in the marker enzyme 5'-nucleotidase and a 10-fold enrichment in ouabain-sensitive ATPase. On sucrose density gradients, the peak density of 5'-nucleotidase activity was 1.12 g/ml, and was shifted after digitonin addition to 1.15 g/ml. Protein in the plasma membrane equalled approximately 8 percent of the whole cell protein. A b-type cytochrome was found to be present in the plasma membrane fraction at a concentration of 205 pmol/mg of protein, which is three times greater than that in the neutrophil overall. Although this cytochrome has been reported previously in the neutrophil, this is the first determination for purified plasma membrane and may indicate that b-type cytochrome has a dual localization in the human neutrophil. Differential centrifugation results suggest that the primary location is in the granules, probably specific granules. Quinone content in the plasma membrane was found to be 740 pmol/mg of protein, a concentration two times greater than in the whole cell. Such a small enhancement of quinone indicates that quinone also is not primarily located in the plasma membrane.

Cell Membrane↗
PubMed1978

Effects of in vivo and in vitro dialysis on plasma transaminase activity.

The effects of dialysis on plasma glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) were investigated. GOT was measured using Autoanalyzer (SMA) and kinetic (Karmen) methods. Hemodialysis of uremic subjects was associated with a significant increase of GOT (SMA) and GPT (SMA). In contrast, hemodialysis had no effect on GOT (Karmen). However, the SMA method is influenced by substances affecting the blank value. Therefore, the results suggest that the increase in transaminase activities measured by the SMA method are not due to true increases in enzyme activities. Plasma from dialysis patients, obtained prior to hemodialysis, was also dialyzed in vitro. In vitro dialysis of uremic plasma significantly increased GOT (SMA), GOT (Karmen) and GPT vitro dialysis of uremic plasma significantly increased GOT (SMA), OGT (Karmen) and GPT (SMA). The results suggest that an inhibitor of transaminase activity may accumulate in renal failure. In vitro dialysis may remove this inhibitor and thus increase true transaminase activity.

Alanine Transaminase↗