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Biomedical subjects

J M Keller

Publications and source records attributed to J M Keller.

At least 19 recordsLinked to original sources
PubMed2001

In vivo protein expression from mRNA delivered into adult rat brain.

The expression of proteins after local mRNA delivery has a great potential for analysis of protein function in vivo. To explore the feasibility of such a technique within the central nervous system (CNS), we delivered luciferase-encoding mRNA into the rat brain. The tissue distribution and stability of injected mRNA were analyzed using in situ detection and Northern hybridization, while luciferase expression was measured by enzymatic assay. Following intracerebral injection of lipofectin-complexed mRNA, expression of luciferase was detectable as early as 1 h, was maximal at 2-3 h, but was below the level of detection by 24 h. The extent of luciferase expression correlated with the amount of mRNA delivered. Luciferase expression was higher when lipofectin-complexed rather than naked mRNA was injected. In addition, the luciferase expression increased significantly by adding a 50 nt-long poly(A) tail to the 3'-end of the mRNA. Delivering mRNA to the cerebral cortex or hippocampus resulted in measurable luciferase activity at the injection sites but not in adjacent areas. Accordingly, the luciferase mRNA was also localized to the injection site, and the amount of intact transcript was significantly higher at 3 h compared to 24 h after injection. These results demonstrate that in vivo mRNA delivery is a feasible technique for immediate, transient overexpression of desired proteins in the CNS and, therefore, can serve as a model system to study the neurobiological effects of specific proteins.

Animals↗
PubMed2001

Transcriptional state and chromatin structure of the murine entactin and laminin gamma1 genes.

The positions of nucleosomes in the proximal 5' regions of the coordinately regulated murine entactin/nidogen and laminin gamma1 genes have been identified in four different transcriptional states--constitutively off, basal, induced, and constitutively induced. In the entactin gene a 450 base pair (bp) region of open chromatin is present between three positioned nucleosomes and the transcriptional start site in the basal, induced, and constitutively induced states. Additionally there is a 200 bp open chromatin region at approximately -2.1 kbp that is only present in the induced and constitutively induced states. In the laminin gamma1 gene, a 650 bp region of nucleosome-free chromatin is present between nucleosomes positioned at approximately -750 and +120 in all transcriptionally active states. These results suggest that basal co-expression of these genes requires sites present in these near upstream regions. The induction to high levels appears to involve additional sites and possibly the production of new and/or the modification of existing trans-acting factors.

Animals↗
PubMed2000

Evidence for the presence of peroxisome proliferator-activated receptor (PPAR) alpha and gamma and retinoid Z receptor in cartilage. PPARgamma activation modulates the effects of interleukin-1beta on rat chondrocytes.

Peroxisome proliferator-activated receptor (PPAR) alpha, PPARgamma, and retinoid acid receptor-related orphan receptor (ROR) alpha are members of the nuclear receptor superfamily of ligand-activated transcription factors. Although they play a key role in adipocyte differentiation, lipid metabolism, or glucose homeostasis regulation, recent studies suggested that they might be involved in the inflammation control and especially in the modulation of the cytokine production. This strongly suggests that these transcriptional factors could modulate the deleterious effects of interleukin-1 (IL-1) on cartilage. However, to date, their presence in cartilage has never been investigated. By quantitative reverse transcription-polymerase chain reaction, Western blot, and immunocytochemistry analysis, we demonstrated, for the first time, the presence of PPARalpha, PPARgamma, and RORalpha in rat cartilage, at both mRNA and protein levels. Comparatively, the PPARalpha mRNA content in cartilage was much lower than in the liver but not significantly different to that of the adipose tissue. PPARgamma mRNA expression in cartilage was weak, when compared with adipose tissue, but similar to that found in the liver. RORalpha mRNA levels were similar in the three tissues. mRNA expression of the three nuclear receptors was very differently modulated by IL-1 or mono-iodoacetate treatments. This indicates that they should be unequally involved in the effects of IL-1 on chondrocyte, which is in accordance with results obtained in other cell types. Indeed, we showed that 15d-PGJ2 mainly, but also the drug troglitazone, that are ligands of PPARgamma could significantly counteract the decrease in proteoglycan synthesis and NO production induced by IL-1. By contrast, PPARalpha ligands such as Wy-14,643 or clofibrate had no effect on this process. Therefore, the presence of PPARgamma in chondrocytes opens up new perspectives to modulate the effects of cytokines on cartilage by the use of specific ligands. The function of the two other transcription factors, PPARalpha and RORalpha identified in chondrocytes remains to be explored.

Alginates↗
PubMed2000

Differential expression of peroxisome proliferator-activated receptors (PPARs) in the developing human fetal digestive tract.

We investigated the spatiotemporal distributions of the different peroxisome proliferator-activated receptor (PPAR) isotypes (alpha, beta, and gamma) during development (Week 7 to Week 22 of gestation) of the human fetal digestive tract by immunohistochemistry using specific polyclonal antibodies. The PPAR subtypes, including PPARgamma, are expressed as early as 7 weeks of development in cell types of endodermal and mesodermal origin. The presence of PPARgamma was also found by Western blotting and nuclease-S1 protection assay, confirming that this subtype is not adipocyte-specific. PPARalpha, PPARbeta, and PPARgamma exhibit different patterns of expression during morphogenesis of the digestive tract. Whatever the stage and the gut region (except the stomach) examined, PPARgamma is expressed at a high level, suggesting some fundamental role for this receptor in development and/or physiology of the human digestive tract.

Antibody Specificity↗
PubMed2000

Use of a two-step Percoll gradient for separation of loggerhead sea turtle peripheral blood mononuclear cells.

In order to determine a suitable procedure for isolating peripheral blood mononuclear cells (PBMCs) from loggerhead sea turtles (Caretta caretta), blood was collected using three different anticoagulants (sodium heparin, sodium citrate or potassium EDTA) and separated using a single step commercially-prepared arabinogalactan gradient of 1.077 g/ml density or multiple step Percoll gradients between 1.053 and 1.076 g/ml density (40-60% stock isotonic Percoll suspension). Heparinized blood centrifuged over a two-step 45/55% (1.059/1.070 g/ml) Percoll gradient yielded 99 to 100% mononuclear cells at the 45/55% interface. Mononuclear cell viability ranged from 85 to 97% with cell yields up to 9.2 x 10(6) cells/mL. An unexpected finding was a population of low density granulocytes migrating to 40% (1.053 g/ml) and 45% Percoll layers in the multiple step gradients. These granulocytes could be eliminated from the PBMC preparation by use of the two-step 45/55% Percoll gradient. Isolated PBMCs can be used for cellular immunology and toxicology studies on these threatened marine organisms for which other tissues can usually be obtained only sporadically from post-mortem specimens.

Animals↗
PubMed2000

Implications of peroxisome proliferator-activated receptors (PPARS) in development, cell life status and disease.

The past several years have seen an increasing interest in the peroxisome proliferator-activated receptors (PPARs). These transcriptional factors belong to the superfamily of the steroid/thyroid/retinoid receptors. They are activated by fatty acids or their metabolites as well as by different xenobiotic peroxisome proliferators. These receptors are expressed in both the embryo and the adult organism. They have been implicated in cell proliferation, differentiation and apoptosis. In this review, we will attempt to point out some of the more salient features of this expression pattern during development and the different steps of cell life. The current understanding of how PPARs are involved in some human diseases will also be described.

Animals↗
PubMed1999

Effects of the peroxisome proliferator clofibric acid on superoxide dismutase expression in the human HepG2 hepatoma cell line.

We examined the effects of clofibric acid, a peroxisome proliferator, on the production of superoxide radicals, on the levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), and on the expression of superoxide dismutases (SODs) in the human HepG2 hepatoma cell line. To this end, HepG2 cells were treated for 1 or 5 days with 0.25, 0.50, or 0.75 mM clofibric acid. The production of superoxide radicals was only enhanced in HepG2 cells exposed for 5 days to the different clofibric acid concentrations. However, this overproduction of superoxide radicals was not accompanied by increased rates of lipid peroxidation, as the MDA and 4-HNE levels did not change significantly. Manganese (Mn) SOD activity was increased when HepG2 cells were treated for 1 day with 0.50 or 0.75 mM clofibric acid. For this duration of treatment, no change was observed in total SOD and copper/zinc (Cu/Zn) SOD activities. For a 5-day treatment, total SOD and MnSOD activities as well as the enzyme apoprotein and MnSOD mRNA levels increased whatever the clofibric acid concentration used. This transcriptional induction of the MnSOD gene was correlated with an activation of the activator protein-1 transcription factor for 1 and 5 days of treatment, but was independent of nuclear factor-kappa B and of peroxisome proliferator-activated receptor. On the other hand, the PP exerted very little effect if any on Cu,ZnSOD expression. In contrast to rodent data, PP treatment of human hepatoma cells induces MnSOD expression.

Apoproteins↗
PubMed1998

Effect of aluminum on superoxide dismutase activity in the adult rat brain.

Male rats were treated daily with an intraperitoneal injection of 15 mg aluminum (Al chloride)/kg body weight for 17 d, in order to study the effects on superoxide dismutase (SOD) activities in the brain (cortex). No significant difference between control and treated animals was registered in the Cu/Zn and Mn SOD activities in the gray matter of the cortex. High Al levels were found in the plasma, the spleen, and the liver of the treated animals in comparison to the controls, but not in the cortex homogenates (gray matter). In addition, Al induced a significant decrease in food ingestion and weight gain.

Aluminum Chloride↗
PubMed1998

Data-driven homologue matching for chromosome identification.

Karyotyping involves the visualization and classification of chromosomes into standard classes. In "normal" human metaphase spreads, chromosomes occur in homologous pairs for the autosomal classes 1-22, and X chromosome for females. Many existing approaches for performing automated human chromosome image analysis presuppose cell normalcy, containing 46 chromosomes within a metaphase spread with two chromosomes per class. This is an acceptable assumption for routine automated chromosome image analysis. However, many genetic abnormalities are directly linked to structural or numerical aberrations of chromosomes within the metaphase spread. Thus, two chromosomes per class cannot be assumed for anomaly analysis. This paper presents the development of image analysis techniques which are extendible to detecting numerical aberrations evolving from structural abnormalities. Specifically, an approach to identifying "normal" chromosomes from selected class(es) within a metaphase spread is presented. Chromosome assignment to a specific class is initially based on neural networks, followed by banding pattern and centromeric index criteria checking, and concluding with homologue matching. Experimental results are presented comparing neural networks as the sole classifier to our homologue matcher for identifying class 17 within normal and abnormal metaphase spreads.

Chromosome Banding↗
PubMed1996

Peroxisomes and peroxisomal enzymes in the human fetal small intestine.

The appearance and development of peroxisomes and the expression of their enzymes in the human fetal intestine have been investigated between 11 and 22 weeks of gestation. In the youngest samples (11-16 weeks of age), cytochemistry at the ultrastructural level revealed the presence of rare, mostly circular peroxisomes. From 16 weeks of gestation onwards, an increase was noted in the number of peroxisomes. Two peroxisomal types were distinguished: round to oval forms and elongated and/or tailed organelles. Biochemical assays revealed that total and specific intestinal catalase activities increased gradually between 11 and 20 weeks of gestation. The activity of fatty acylCoA oxidase, the first enzyme of the peroxisomal beta-oxidation system, was detectable as early as 11 weeks of gestation. Thereafter, total and specific activities of the enzyme increased steadily. Activities of other peroxisomal oxidases (D-amino acid oxidase, L-alpha-hydroxyacid oxidase) appeared more slowly in the fetal intestine during the period studied. This investigation establishes the presence and the morphological changes that occur in intestinal peroxisomes during human fetal development as well as the developmental patterns of associated enzymes.

Acyl-CoA Dehydrogenase↗
PubMed1995

A 43-kDa protein related to c-Erb A alpha 1 is located in the mitochondrial matrix of rat liver.

In order to characterize Sterling's triiodothyronine (T3) mitochondrial receptor using photoaffinity labeling, we observed two specific T3-binding proteins in the inner membrane (28 kDa) and in the matrix (43 kDa) of rat liver mitochondria. Western blots and immunoprecipitation using antibodies raised against the T3-binding domain of the T3 nuclear receptor c-Erb A alpha 1 indicated that at least the 43-kDa protein was c-Erb A alpha 1-related. In addition, gel mobility shift assays demonstrated the occurrence of a c-Erb A alpha 1-related mitochondrial protein that specifically binds to a natural or a palindromic thyroid-responsive element. Moreover, this protein specifically binds to a direct repeat 2 sequence located in the D-loop of the mitochondrial genome. Furthermore, electron microscopy studies allowed the direct observation of a c-Erb A-related protein in mitochondria. Lastly, the relative amounts of the 43-kDa protein related to c-Erb A alpha 1 were in good correlation with the known mitochondrial mass in three typical tissues. Interestingly, expression of a truncated form of the c-Erb A alpha 1 nuclear receptor in CV1 cells was associated with a mitochondrial localization and a stimulation of mitochondrial activity. These results supply evidence of the localization of a member of the nuclear receptor superfamily in the mitochondrial matrix involved in the regulation of mitochondrial activity that could act as a mitochondrial T3-dependent transcription factor.

Affinity Labels↗
PubMed1995

Escherichia coli O157:H7 restriction pattern recognition by artificial neural network.

An artificial neural network model for the recognition of Escherichia coli O157:H7 restriction patterns was designed. In the training phase, images of two classes of E. coli isolates (O157:H7 and non-O157:H7) were digitized and transmitted to the neural network. The system was then tested for recognition of images not included in the training set. Promising results were achieved with the designed network configuration, providing a basis for further study. This application of a new generation of computation technology serves as an example of its usefulness in microbiology.

Animals↗
PubMed1995

Comparative effects of clofibrate on peroxisomal enzymes of human (Hep EBNA2) and rat (FaO) hepatoma cell lines.

We compared the responses of the human Hep EBNA2 and rat FaO hepatoma lines to the peroxisome proliferator, clofibrate. Using spectrophotometrical assays performed with peroxisome-enriched fractions, the dose- and time-dependent increase of catalase and acyl-CoA oxidase activities were determined. For catalase activity a maximum stimulation of 1.2-fold for Hep EBNA2 and 1.7-fold for FaO lines was obtained. This increase was neither dose- nor time-dependent. The activity of the initial enzyme of the peroxisomal beta-oxidation system, acyl-CoA oxidase, was tested using two different biochemical assays. The maximum stimulation of acyl-CoA oxidase was 2.4 to 3-fold for human Hep EBNA2 and 6 to 11-fold for rat FaO lines. The specific activity of acyl-CoA oxidase increased with the concentration of clofibrate and with the length of the treatment. Dot blot analyses carried out using mRNAs from FaO and Hep EBNA2 cells treated with 0.5 mM clofibrate for 5 days and from control cells, confirmed the increase in the level of acyl-CoA oxidase mRNAs from the clofibrate-treated cells. In the human cell line, the level of mRNA encoding for the peroxisomal bifunctional enzyme which is involved in the second and the third step of the beta-oxidation system, was also increased by clofibrate treatment.

Acyl-CoA Oxidase↗
PubMed1994

Studies on peroxisomes of colonic mucosa in Crohn's disease.

The etiology and pathogenesis of Crohn's disease, a chronic inflammatory bowel pathology, have not been elucidated yet. In particular, the behavior of peroxisomes in inflamed colonic mucosa has not been investigated despite their important role in cellular oxidative metabolism. Using cytochemistry at the ultrastructural level, we have observed these catalase-positive organelles. In addition, biochemical analyses have revealed the specific activities of catalase and cyanide-insensitive acyl-CoA oxidase. Mucosal biopsy specimens from inflamed and noninflamed areas of Crohn's patients were compared to control biopsies. We found that Crohn's disease was marked by an important diminution in the peroxisomal frequency per cell unit area. If catalase activity was not affected by this pathology, cyanide-insensitive acyl-CoA oxidase, an enzyme of the peroxisomal beta-oxidation system, was found diminished in inflamed and in noninflamed areas. In conclusion, our results showed that Crohn's disease is accompanied by peroxisomal modifications but the number and the enzyme activities of colonic peroxisomes are less deeply altered in Crohn's disease than during neoplasia. This fact suggests that a relation may exist between the degree of peroxisomal deficiency and the clinical severity of colonic disease.

Acyl-CoA Oxidase↗
PubMed1993

Peroxisome through cell differentiation and neoplasia.

Peroxisomes are essential in cellular metabolism as their dysgenesis or defects in single enzymes or impairment of multiple peroxisomal enzymatic functions have been found in several inherited metabolic diseases with serious clinical sequelae. The assembly and formation of these cytoplasmic organelles constitute a major and intriguing research topic. In the present study the biogenesis of peroxisomes and the developmental patterns of their enzymes have been reviewed during embryonic and/or post-embryonic ontogenesis of lower (amphibians) and higher (avians, mammals) vertebrates. In developing vertebrates, epithelial cell differentiation is accompanied by increases in frequency and size of peroxisomes. The tissue-specific expression of peroxisomal enzymes contributes substantially to the biochemical maturation of epithelial cells. The relationship between biogenesis of peroxisomes, expression of peroxisomal enzymes and structural and functional cellular phenotype has also been investigated in differentiating epithelial cells along the crypt-villus axis of the adult rat intestine. Cytochemical studies at the ultrastructural level have provided evidence that peroxisomes are already present in proliferating cells of the intestinal crypt region before they begin to differentiate. Migration and differentiation of intestinal epithelial cells from crypt to villus compartments are marked by significant increases in number and size of catalase-positive structures. Increasing activity gradients from crypt to surface areas are found for the peroxisomal oxidases examined (enzymes of the peroxisomal beta-oxidation system, D-amino acid oxidase and polyamine oxidase). Thus, peroxisomes are more and more involved in oxidative metabolic pathways as intestinal epithelial cells differentiate. Finally, we have analyzed the peroxisomal behaviour in human neoplastic epithelial cells. The presence of peroxisomes has been cytochemically revealed in human breast and colon carcinomas. Peroxisomal enzyme specific activities are significantly lower in human breast and colon carcinomas than in the adjacent healthy mucosa. Furthermore, a relationship is found between the specific activities of some peroxisomal enzymes and the histological tumour grades.

Amphibians↗
PubMed1993

Carboxy-terminal deletion analysis of oat phytochrome A reveals the presence of separate domains required for structure and biological activity.

A series of seven carboxy-terminal deletion mutants of oat phytochrome A were stably expressed in transgenic tobacco to localize phytochrome domains involved in chromophore attachment, spectral integrity, photoreversibility between the red light (Pr)- and far-red light (Pfr)-absorbing forms, dimerization, and biological activity. Amino acids necessary for chromophore attachment in vivo were localized to the amino-terminal 398 residues because mutant proteins this small had covalently bound chromophore. Deletion mutants from the carboxy terminus to residue 653 were spectrally indistinguishable from the full-length chromoprotein. In contrast, further truncation to residue 399 resulted in a chromoprotein with a bleached Pfr absorbance spectrum, Pr and Pfr absorbance maxima shifted toward shorter wavelengths, and reduced Pfr to Pr phototransformation efficiency. Thus, residues between 399 ad 652 are required for spectral integrity but are not essential for chromophore attachment. The sequence(s) between residues 919 and 1093 appears to be necessary for dimerization. Carboxy-terminal mutants containing this region behaved as dimers under nondenaturing conditions in vitro, whereas truncations without this region behaved as monomers. None of the plants expressing high levels of deletion mutants lacking the 35 carboxy-terminal amino acids displayed the light-exaggerated phenotype characteristic of plants expressing biologically active phytochrome A, even when the truncated phytochromes were expressed at levels 6- to 15-fold greater than that effective for the full-length chromoprotein. Collectively, these data show that the phytochrome protein contains several separable carboxy-terminal domains required for structure/function and identify a domain within 35 residues of the carboxy terminus that is critical for the biological activity of the photoreceptor in vivo.

Base Sequence↗