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1.
Neurogastroenterol Motil ; 30(6): e13299, 2018 06.
Article in English | MEDLINE | ID: mdl-29383802

ABSTRACT

BACKGROUND: Irritable bowel syndrome (IBS) is a common gastrointestinal disorder associated with altered gastrointestinal microflora and increased nociception to colonic distension. This visceral hypersensitivity can be reversed in our rat maternal separation model by fungicides. Menthacarin® is a proprietary combination of essential oils from Mentha x piperita L. and Carum carvi. Because these oils exhibit antifungal and antibacterial properties, we investigated whether Menthacarin® can reverse existing visceral hypersensitivity in maternally separated rats. METHODS: In non-handled and maternally separated rats, we used the visceromotor responses to colorectal distension as measure for visceral sensitivity. We evaluated this response before and 24 hours after water-avoidance stress and after 7 days treatment with Menthacarin® or control. The pre- and post-treatment mycobiome and microbiome were characterized by sequencing of fungal internal transcribed spacer 1 (ITS-1) and bacterial 16s rDNA regions. In vitro antifungal and antimicrobial properties of Menthacarin® were studied with radial diffusion assay. KEY RESULTS: Menthacarin® inhibited in vitro growth of yeast and bacteria. Water-avoidance caused visceral hypersensitivity in maternally separated rats, and this was reversed by treatment. Multivariate analyses of ITS-1 and 16S high throughput data showed that maternal separation, induced changes in the myco- and microbiome. Menthacarin® treatment of non-handled and maternally separated rats shifted the mycobiomes to more similar compositions. CONCLUSIONS & INFERENCES: The development of visceral hypersensitivity in maternally separated rats and the Menthacarin® -mediated reversal of hypersensitivity is associated with changes in the mycobiome. Therefore, Menthacarin® may be a safe and effective treatment option that should be tested for IBS.


Subject(s)
Hyperalgesia/drug therapy , Mycobiome/drug effects , Oils, Volatile/administration & dosage , Plant Oils/administration & dosage , Visceral Pain/drug therapy , Animals , Animals, Newborn , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/isolation & purification , Antifungal Agents/administration & dosage , Antifungal Agents/isolation & purification , Drug Combinations , Hyperalgesia/microbiology , Hyperalgesia/psychology , Male , Maternal Deprivation , Mentha piperita , Mycobiome/physiology , Oils, Volatile/isolation & purification , Plant Oils/isolation & purification , Rats , Rats, Long-Evans , Visceral Pain/microbiology , Visceral Pain/psychology
2.
J Crohns Colitis ; 11(7): 831-839, 2017 Jul 01.
Article in English | MEDLINE | ID: mdl-28158397

ABSTRACT

BACKGROUND AND AIM: T cells are key players in the chronic intestinal inflammation that characterises Crohn's disease. Here we aim to map the intestinal T-cell receptor [TCR] repertoire in patients with Crohn's disease, using next-generation sequencing technology to examine the clonality of the T-cell compartment in relation to mucosal inflammation and response to therapy. METHODS: Biopsies were taken from endoscopically inflamed and uninflamed ileum and colon of 19 patients with Crohn's disease. From this cohort, additional biopsies were taken after 8 weeks of remission induction therapy from eight responders and eight non-responders. Control biopsies from 11 patients without inflammatory bowel disease [IBD] were included. The TCRß repertoire was analysed by next-generation sequencing of biopsy RNA. RESULTS: Both in Crohn's disease patients and in non-IBD controls, a broad intestinal T-cell repertoire was found, with a considerable part consisting of expanded clones. Clones in Crohn's disease were more expanded [p = 0.008], with the largest clones representing up to as much as 58% of the total repertoire. There was a substantial overlap of the repertoire between inflamed and uninflamed tissue and between ileum and colon. Following therapy, responders showed larger changes in the T-cell repertoire than non-responders, although a considerable part of the repertoire remained unchanged in both groups. CONCLUSIONS: The intestinal T-cell repertoire distribution in Crohn's disease is different from that in the normal gut, containing profoundly expanded T-cell clones that take up a large part of the repertoire. The T-cell repertoire is fairly stable regardless of endoscopic mucosal inflammation or response to therapy.


Subject(s)
Crohn Disease/immunology , Crohn Disease/pathology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , Adalimumab/therapeutic use , Adult , Anti-Inflammatory Agents/therapeutic use , Biopsy , Budesonide/therapeutic use , C-Reactive Protein/metabolism , Case-Control Studies , Clone Cells/drug effects , Colon/pathology , Crohn Disease/drug therapy , Female , Gastrointestinal Agents/therapeutic use , Humans , Ileum/pathology , Inflammation/immunology , Inflammation/pathology , Infliximab/therapeutic use , Male , Middle Aged , Severity of Illness Index , T-Lymphocytes/drug effects , Young Adult
3.
Biochem Biophys Res Commun ; 482(4): 1013-1018, 2017 Jan 22.
Article in English | MEDLINE | ID: mdl-27908722

ABSTRACT

Maternal diabetes is one of major causes of congenital malformations in offspring, but the underlying mechanism is still unclear. MiRNAs play an important role in transcriptional and post-transcriptional regulation of gene expression. However, no miRNA expression profiling of hyperglycemic offspring are thus far available. Female mice were made diabetic with streptozotocin, treated with slow-release insulin tablets, and mated. MiRNA expression profiling with Next Generation Sequencing on the SOLiD5 platform was performed on 8 control and 5 hyperglycemic embryonic day (ED)8.5 and 9 control and 6 hyperglycemic ED9.5 embryos. Differential expression was analyzed with the Wald test. On ED8.5, the abundance of expressed miRNAs was similar in control and hyperglycemic ED8.5 embryos. The spectrum of expressed miRNAs had not changed in ED9.5 embryos, but the abundance of most miRNAs increased ∼5-fold in control embryos. However, hyperglycemic D9.5 embryos were unable to mount this increase in prevalence. Only 3 miRNAs were differentially expressed in control and hyperglycemic ED9.5 embryos, but their putative target genes were underrepresented in the Jackson database of genes causing cardiovascular or neural malformations.


Subject(s)
Gene Expression Regulation, Developmental , MicroRNAs/genetics , Pregnancy in Diabetics , Somites/embryology , Animals , Embryo, Mammalian/metabolism , Female , Hyperglycemia/etiology , Hyperglycemia/genetics , Male , Mice , Pregnancy , Pregnancy in Diabetics/etiology , Somites/metabolism , Transcriptome
4.
Genes Brain Behav ; 8(3): 290-5, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19170755

ABSTRACT

Glutamine synthetase (GS) is a pivotal glial enzyme in the glutamate-glutamine cycle. GS is important in maintaining low extracellular glutamate concentrations and is downregulated in the hippocampus of temporal lobe epilepsy patients with mesial-temporal sclerosis, an epilepsy syndrome that is frequently associated with early life febrile seizures (FS). Human congenital loss of GS activity has been shown to result in brain malformations, seizures and death within days after birth. Recently, we showed that GS knockout mice die during embryonic development and that haploinsufficient GS mice have no obvious abnormalities or behavioral seizures. In the present study, we investigated whether reduced expression/activity of GS in haploinsufficient GS mice increased the susceptibility to experimentally induced FS. FS were elicited by warm-air-induced hyperthermia in 14-day-old mice and resulted in seizures in most animals. FS susceptibility was measured as latencies to four behavioral FS characteristics. Our phenotypic data show that haploinsufficient mice are more susceptible to experimentally induced FS (P < 0.005) than littermate controls. Haploinsufficient animals did not differ from controls in hippocampal amino acid content, structure (Nissl and calbindin), glial properties (glial fibrillary acidic protein and vimentin) or expression of other components of the glutamate-glutamine cycle (excitatory amino acid transporter-2 and vesicular glutamate transporter-1). Thus, we identified GS as a FS susceptibility gene. GS activity-disrupting mutations have been described in the human population, but heterozygote mutations were not clearly associated with seizures or epilepsy. Our results indicate that individuals with reduced GS activity may have reduced FS seizure thresholds. Genetic association studies will be required to test this hypothesis.


Subject(s)
Genetic Predisposition to Disease/genetics , Glutamate-Ammonia Ligase/genetics , Glutamic Acid/metabolism , Haplotypes/genetics , Seizures, Febrile/genetics , Animals , Biomarkers/analysis , Biomarkers/metabolism , Brain/enzymology , Brain/physiopathology , Brain Chemistry/genetics , Disease Models, Animal , Down-Regulation/genetics , Excitatory Amino Acid Transporter 2/analysis , Excitatory Amino Acid Transporter 2/metabolism , Glial Fibrillary Acidic Protein/analysis , Glial Fibrillary Acidic Protein/metabolism , Mice , Mice, Knockout , Reaction Time/genetics , Seizures, Febrile/enzymology , Seizures, Febrile/physiopathology , Vesicular Glutamate Transport Protein 1/analysis , Vesicular Glutamate Transport Protein 1/metabolism , Vimentin/analysis , Vimentin/metabolism
5.
Biochimie ; 88(9): 1255-64, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16839656

ABSTRACT

Glutamine synthetase (GS) is expressed at various levels in a wide range of tissues, suggesting that a complex network of modules regulates its expression. We explored the interactions between the upstream enhancer, regulatory regions in the first intron, and the 3'-untranslated region and immediate downstream genomic sequences of the GS gene (the GS "tail"), and compared the results with those obtained previously in conjunction with the bovine growth hormone (bGH) tail. The statistical analysis of these interactions revealed that the GS tail was required for full enhancer activity of the combination of the upstream enhancer and either the middle or the 3'-intron element. The GS tail also prevented a productive interaction between the upstream enhancer and the 5'-intron element, whereas the bGH tail did not, suggesting that the 5'-intron element is a regulatory element that needs to be silenced for full GS expression. Using the CMV promoter/enhancer and transfection experiments, we established that the 2.8 kb GS mRNA polyadenylation signal is approximately 10-fold more efficient than the 1.4 kb mRNA signal. Because the steady-state levels of both mRNAs are similar, the intervening conserved elements destabilize the long mRNA. Indeed, one but not all constructs containing these elements had a shorter half life in FTO-2B cells. A construct containing only 300 bases before and 100 bases after the 2.8 kb mRNA polyadenylation site sufficed for maximal expression. A stretch of 21 adenines inside this fragment conferred, in conjunction with the upstream enhancer and the 3'-part of the first intron, sensitivity of GS expression to ambient glutamine.


Subject(s)
3' Untranslated Regions/metabolism , Glutamate-Ammonia Ligase/genetics , Glutamine/metabolism , RNA Stability , RNA, Messenger/metabolism , Regulatory Sequences, Nucleic Acid , Animals , Base Sequence , Cattle , Gene Expression Regulation , Glutamate-Ammonia Ligase/drug effects , Glutamate-Ammonia Ligase/metabolism , Growth Hormone/metabolism , Molecular Sequence Data , RNA, Messenger/biosynthesis , Rats
6.
Burns ; 27(7): 717-22, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11600251

ABSTRACT

UNLABELLED: Increasing data suggest that the skin nerve system is involved in wound healing. The objective of this study was to investigate the outgrowth of nerve fibers during the burn wound remodeling process and to analyze possible differences between normotrophic and hypertrophic burn wounds. In a prospective study, biopsies were taken from 22 patients with spontaneously healed partial-thickness burns at 1, 4 and 7-month post-burn. Nerve outgrowth and the expression of the neuropeptides substance P, neurokinin A, calcitonin gene-related peptide, vasoactive intestinal peptide and neuropeptide Y was monitored using immunohistochemistry. Our results showed that the number of nerve fibers gradually increased in both the dermis and the epidermis, but that they did not reach the levels of expression present in matched unburned skin of the same patient. A significantly higher number of nerve fibers were observed in normotrophic scars compared with hypertrophic scars. The number of neuropeptides-containing nerves in normotrophic and hypertrophic scars were similar. IN CONCLUSION: 7 months after wound closure, burn wound scars contain less nerve fibers than unburned skin. The significantly higher number of nerve fibers in normotrophic, compared with hypertrophic scars suggests a regulatory role for the skin nerve system in the outcome of burn wound healing.


Subject(s)
Burns/physiopathology , Calcitonin Gene-Related Peptide/analysis , Cicatrix, Hypertrophic/physiopathology , Nerve Growth Factors/analysis , Neurokinin A/analysis , Wound Healing , Adult , Aged , Biomarkers/analysis , Burns/metabolism , Cicatrix/physiopathology , Cicatrix, Hypertrophic/metabolism , Follow-Up Studies , Gene Expression Regulation , Humans , Middle Aged , Prospective Studies , Skin/chemistry , Skin/physiopathology
7.
J Hepatol ; 34(5): 699-710, 2001 May.
Article in English | MEDLINE | ID: mdl-11434616

ABSTRACT

BACKGROUND/AIMS: In the embryo, rapidly proliferating hepatocytes migrate from the liver primordium into the surrounding mesenchyme, whereas foetal hepatocytes are mitotically quiescent and accumulate hepatocyte-specific enzymes. We investigated the timing and topography of this behavioural switch. METHODS: The expression of the c-met receptor and its ligand, hepatocyte growth factor (HGF), was investigated in prenatal rat liver by in situ hybridization, immunohistochemistry and western-blot analysis. RESULTS: c-Met was expressed by hepatocytes and HGF by non-parenchymal liver cells. Their mRNA levels peaked during embryonic day (ED) 11-13. c-Met protein was weakly expressed in the entire liver during ED 11 and 12, but more abundantly at ED 13, when its expression withdrew to the hepatic periphery. Simultaneously, the periportal hepatocellular marker carbamoylphosphate synthetase began to accumulate in the centre of the liver. Although the definitive vascular architecture develops simultaneously, the downstream, pericentral hepatocytes began to express glutamine synthetase only 4 days later, suggesting a requirement for prior periportal hepatocyte maturation. Additionally, c-met protein appeared in the connective tissue surrounding the large veins. The c-met protein/mRNA ratio was substantially higher in non-epithelial cells (hepatic connective tissue, heart) than in endoderm-derived epithelia, including hepatocytes, indicating important post-transcriptional regulation. CONCLUSIONS: The decline in c-met expression reflects the end of the embryonic phase and heralds the onset of the fetal, maturational phase of liver development.


Subject(s)
Hepatocyte Growth Factor/metabolism , Liver/embryology , Liver/metabolism , Proto-Oncogene Proteins c-met/metabolism , Animals , Carbamoyl-Phosphate Synthase (Ammonia)/metabolism , Fetus/metabolism , Glutamate-Ammonia Ligase/metabolism , Hepatocytes/cytology , Rats , Rats, Wistar
8.
Eur Cytokine Netw ; 11(2): 233-39, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10903802

ABSTRACT

Keratinocytes are increasingly recognized as key regulators of skin inflammation and remodeling, as they are capable of producing growth factors and cytokines that are important mediators in the wound healing process. We investigated the expression and distribution of TGF-beta 1 mRNA by mRNA in situ hybridization and of TGF-beta 1, TGF-beta 2, TGF-beta 3, bFGF and VEGF protein expression using immunohistochemistry in spontaneously healed, partial-thickness burns and compared this with the expression of these markers in matched unburned skin. This was done to assess their role in the remodeling phase of burn wound healing. Punch biopsies were taken from both partial-thickness burns after re-epithelialization and from matched, unburned skin. At 4 and 7 months post-burn, biopsies were taken of normotrophic and hypertrophic scars that had developed in these wounds. We observed a higher expression of all mentioned growth factors in keratinocytes in scars at 1 month post-burn compared with matched unburned skin. At 4 months, keratinocytes still displayed a higher expression of TGF-beta 3 and bFGF, but the expression of TGF-beta 1, TGF-beta 2 and VEGF was normalized. The expression of TGF-beta 3 in the epidermis of hypertrophic scars was slightly higher than in normotrophic scars. At 7 months post-burn, all growth factors studied showed a normal expression on keratinocytes. Our results suggest that keratinocytes are not only involved in re-epithelialization, but also in the scar maturation. The data support the idea that keratinocytes not only respond to cytokines and growth factors in an autocrine fashion, but also exert regulatory paracrine effects on contiguous cells.


Subject(s)
Burns/genetics , Burns/metabolism , Cicatrix/genetics , Cicatrix/metabolism , Endothelial Growth Factors/genetics , Endothelial Growth Factors/metabolism , Fibroblast Growth Factor 2/genetics , Fibroblast Growth Factor 2/metabolism , Lymphokines/genetics , Lymphokines/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Adult , Aged , Gene Expression , Humans , Immunohistochemistry , In Situ Hybridization , Keratinocytes/metabolism , Middle Aged , RNA, Messenger/genetics , RNA, Messenger/metabolism , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , Wound Healing/genetics , Wound Healing/physiology
10.
J Histochem Cytochem ; 47(12): 1507-12, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10567434

ABSTRACT

The expression of glutamine synthetase (GS) is confined to a rim of hepatocytes surrounding the efferent hepatic veins in all mammalian species investigated. In rat liver, a two- to three-cell thick layer of GS-positive (GS(+)) hepatocytes uniformly surrounds the two to four terminal branching generations of the hepatic vein that collect blood from sinusoids (central veins). With increasing diameter of the efferent vessel, this multilayered rim of GS(+) hepatocytes becomes confined to patches surrounding the decreasing number of central vein outlets. The remaining part of the wall of these sublobar hepatic veins is bordered by a one-cell thick layer of GS(+) hepatocytes. Around still larger veins, this single-cell layer of GS(+) hepatocytes gradually disappears. The expression pattern of GS is therefore a convenient biological parameter to delimit sinusoidal draining ("collecting") from nondraining ("conducting") surfaces in the wall of the efferent hepatic vessels. The hepatocytes surrounding a single tree of central veins together form a compound liver lobule. (J Histochem Cytochem 47:1507-1511, 1999)


Subject(s)
Glutamate-Ammonia Ligase/biosynthesis , Hepatic Veins/metabolism , Liver/metabolism , Animals , Hepatic Veins/anatomy & histology , Immunohistochemistry , Liver/blood supply , Liver/cytology , Male , Rats , Rats, Wistar
11.
Virchows Arch ; 434(3): 221-6, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10190301

ABSTRACT

The reconstruction of epidermal architecture over time in normotrophic and hypertrophic scars in untransplanted, spontaneously healed partial-thickness burns has scarcely been studied, unlike the regeneration of epidermal grafts used to cover burn wounds and the regeneration of the dermis during hypertrophic scarring. The expression of markers of epidermal proliferation, differentiation and activation in normotrophic and hypertrophic scars in spontaneously healed partial-thickness burns was assessed and compared with the expression of these markers in normal control skin of healthy persons to determine whether hypertrophic scarring is associated with abnormalities in the phenotype of keratinocytes. Punch biopsies were taken both of partial-thickness burns after re-epithelialisation and of matched unburned skin. At 4 and 7 months post-burn, biopsies were taken of normotrophic and hypertrophic scars that had developed in these wounds. The biopsies were analysed using immunostaining for markers of keratinocyte proliferation, differentiation and activation (keratins 5, 10, 16 and 17, filaggrin, transglutaminase and CD36). We observed a higher expression of markers for proliferation, differentiation and activation in the epidermis of scars at 1 month post-burn than in normal control skin of healthy persons. There was a striking difference between normotrophic and hypertrophic scars at 4 months post-burn. Keratinocytes in hypertrophic scars displayed a higher level of proliferation, differentiation and activation than did normotrophic scars. At 7 months post-burn all keratinocyte proliferation and differentiation markers showed normal expression, but the activation marker CD36 remained upregulated in both normotrophic and hypertrophic scars. Surprisingly, in matched unburned skin of burn patients, a state of hyperactivation was observed at 1 month. Our results suggest that keratinocytes may be involved in the pathogenesis of hypertrophic scarring.


Subject(s)
Burns/physiopathology , Cicatrix, Hypertrophic/physiopathology , Epidermis/physiopathology , Adult , Aged , CD36 Antigens/metabolism , Cicatrix/metabolism , Cicatrix/pathology , Cicatrix, Hypertrophic/metabolism , Filaggrin Proteins , Follow-Up Studies , Humans , Immunohistochemistry , Keratins/metabolism , Middle Aged , Reference Values , Skin/metabolism , Skin/pathology , Time Factors
12.
Article in English | MEDLINE | ID: mdl-9752723

ABSTRACT

Glutamine synthetase, the enzyme that catalyzes the ATP-dependent conversion of glutamate and ammonia into glutamine, is expressed in a tissue-specific and developmentally controlled manner. The first part of this review focuses on its spatiotemporal pattern of expression, the factors that regulate its levels under (patho)physiological conditions, and its role in glutamine, glutamate, and ammonia metabolism in mammals. Glutamine synthetase protein stability is more than 10-fold reduced by its product glutamine and by covalent modifications. During late fetal development, translational efficiency increases more than 10-fold. Glutamine synthetase mRNA stability is negatively affected by cAMP, whereas glucocorticoids, growth hormone, insulin (all positive), and cAMP (negative) regulate its rate of transcription. The signal transduction pathways by which these factors may regulate the expression of glutamine synthetase are briefly discussed. The second part of the review focuses on the evolution, structure, and transcriptional regulation of the glutamine synthetase gene in rat and chicken. Two enhancers (at -6.5 and -2.5 kb) were identified in the upstream region and two enhancers (between +156 and +857 bp) in the first intron of the rat glutamine synthetase gene. In addition, sequence analysis suggests a regulatory role for regions in the 3' untranslated region of the gene. The immediate-upstream region of the chicken glutamine synthetase gene is responsible for its cell-specific expression, whereas the glucocorticoid-induced developmental appearance in the neural retina is governed by its far-upstream region.


Subject(s)
Gene Expression Regulation, Enzymologic , Glutamate-Ammonia Ligase/genetics , Aging , Animals , Base Sequence , Evolution, Molecular , Exons , Gene Expression Regulation, Developmental , Glutamate-Ammonia Ligase/biosynthesis , Humans , Molecular Sequence Data , Organ Specificity , Protein Biosynthesis , RNA Processing, Post-Transcriptional , Rats , Regulatory Sequences, Nucleic Acid , Sequence Alignment , Transcription, Genetic , Vertebrates
13.
Nucleic Acids Res ; 24(17): 3478-80, 1996 Sep 01.
Article in English | MEDLINE | ID: mdl-8811109

ABSTRACT

We have developed a fast and general method to obtain an enriched, full-length cDNA expression library with subtractively enriched cDNA fragments. The procedure relies on RecA-mediated triple-helix formation of single-stranded cDNA fragments with a double-stranded cDNA plasmid library. The complexes were then captured from the solutions using the digoxigenin-antidigoxigenin paramagnetic beads followed by recovery of the enriched double-stranded cDNA expression library. We have observed a linear relation between the capture of full-length cDNAs in the library and the fold enrichment in the subtracted cDNA population.


Subject(s)
Cloning, Molecular/methods , DNA, Complementary/genetics , Gene Expression , Gene Library , Affinity Labels , Animals , DNA , Digoxigenin , Liver Regeneration , Nucleic Acid Conformation , Nucleic Acid Hybridization , Rats , Rec A Recombinases
14.
Biochim Biophys Acta ; 1315(3): 169-75, 1996 Apr 12.
Article in English | MEDLINE | ID: mdl-8611655

ABSTRACT

Identification of gene products exclusively or abundantly expressed in hepatocellular carcinoma (HCC) and in normal liver may yield novel tumor markers. We have isolated 36 up- and down-regulated cDNAs from diethylnitrosamine-induced rat hepatocellular carcinoma and normal liver tissue by using the subtraction-enhanced display technique. Nucleotide sequence analysis revealed that the majority of 20 subtraction-enriched cDNA fragments were well-characterized oncogenes and tumor-associated genes, like c-myc, alpha-prothymosin, p21, glutathione-S transferase (G-ST) and alpha 1-acid glycoprotein (AGP). As demonstrated by Northern blot detection, all of them were preferentially expressed either in HCC or in normal liver (2- to 7-fold). As paradigm, G-ST and AGP were shown to be exclusively overexpressed in tumor nodules by in situ hybridization. In addition, 14 of the remaining 16 novel genes were analysed on Northern blot, 10 of which were differentially expressed in HCC.


Subject(s)
DNA, Complementary/isolation & purification , DNA, Neoplasm/isolation & purification , Gene Expression Regulation, Neoplastic , Liver Neoplasms, Experimental/genetics , Neoplasm Proteins/genetics , RNA, Messenger/genetics , RNA, Neoplasm/genetics , Subtraction Technique , Animals , Base Sequence , Biomarkers, Tumor/genetics , Blotting, Northern , DNA, Neoplasm/genetics , Diethylnitrosamine , In Situ Hybridization , Liver/metabolism , Molecular Sequence Data , Neoplasm Proteins/biosynthesis , Oncogenes , RNA, Messenger/biosynthesis , RNA, Neoplasm/biosynthesis , Rats , Rats, Wistar
16.
Proc Natl Acad Sci U S A ; 87(15): 5729-33, 1990 Aug.
Article in English | MEDLINE | ID: mdl-2165601

ABSTRACT

To examine the amino acid sequence requirements for the biphasic association of Drosophila melanogaster apocytochrome c with mouse liver mitochondria in vitro, recombinant constructs of the protein were prepared. Removal of the C-terminal sequence to residue 58 had little influence, but truncation to residue 50 decreased the association to low levels and removal to residue 36 was even more effective. However, a mutant missing the segment between residues 35 and 66 was fully functional, but, when the C-terminal segment from residue 36 was replaced with a noncytochrome c sequence, the high-affinity phase of the association was lost. A mutant in which residues 90, 91, 92, 96, and 100 were replaced by lysine, leucine, proline, proline, and proline, respectively, to prevent the possible formation of the C-terminal alpha-helix and another mutant in which the C-terminal segment from residue 90 to residue 120 was a noncytochrome c sequence had normal association. In contrast, replacing lysine-5, -7, and -8 by glutamine, glutamic acid, and asparagine, respectively, resulted in loss of the high-affinity phase. The same mutations in the apoprotein lacking the segment between residues 35 and 66 caused, in addition, a decrease of the low-affinity phase association. Thus, the N-terminal region is most critical for apocytochrome c association, but alternative segments of the central and/or C-terminal region can be utilized, where noncytochrome c sequences are ineffective. These results emphasize the wide disparity between the structural requirements for association with mitochondria and for the production of a functional holoprotein.


Subject(s)
Apoproteins/genetics , Cytochrome c Group/genetics , Mitochondria, Liver/metabolism , Amino Acid Sequence , Animals , Apoproteins/metabolism , Chromosome Deletion , Cloning, Molecular , Cytochrome c Group/metabolism , Cytochromes c , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Kinetics , Mice , Molecular Sequence Data , Mutation , Protein Conformation , Rats
17.
Proc Natl Acad Sci U S A ; 87(13): 4996-5000, 1990 Jul.
Article in English | MEDLINE | ID: mdl-2164215

ABSTRACT

35S-labeled Drosophila melanogaster apocytochrome c was made by in vitro transcription/translation of the gene and purified to the monomeric, fully reduced form. It was found that in the presence of a wheat germ extract factor there was a high-affinity phase of the uptake into mouse liver mitochondria at 10-300 pM apocytochrome c, and a lower-affinity phase through 4000 pM. Without the factor, the high-affinity phase was absent. The stimulatory effect of the factor could not be elicited with various reductants, such as NADH, FMN, and ferrous protoheme IX. Conversely, when mitochondria loaded with apocytochrome c were resuspended in fresh medium, the protein readily reequilibrated. Successive washings depleted greater than 95% of the associated apoprotein but removed no holoprotein. Proteases (proteinase K or trypsin) added to a suspension of mitochondria loaded with apoprotein digested an amount of apoprotein similar to that which would have been dissociated during the same time, as measured by successive washings in the absence of protease. Mitochondria loaded with apoprotein and similarly treated with protease continued exporting the apoprotein, even after the protease was inhibited and removed, suggesting that most of the apoprotein associated with the organelle was in a protease-resistant compartment. Apocytochrome c mutants in which serines or alanines replaced cysteines 14 and 17, which bind the prosthetic group, behaved like the cysteine-containing protein, indicating that the covalent attachment of the heme is unrelated to the translocation of the apoprotein.


Subject(s)
Apoproteins/metabolism , Cytochrome c Group/metabolism , Drosophila melanogaster/metabolism , Mitochondria, Liver/metabolism , Alanine , Animals , Apoproteins/genetics , Apoproteins/isolation & purification , Biological Transport , Cysteine , Cytochrome c Group/genetics , Cytochrome c Group/isolation & purification , Cytochromes c , Drosophila melanogaster/genetics , Kinetics , Mice , Mice, Inbred ICR , Mutation , Protein Binding , Protein Biosynthesis , Serine , Transcription, Genetic
19.
Biochim Biophys Acta ; 894(3): 347-54, 1987 Dec 17.
Article in English | MEDLINE | ID: mdl-2825776

ABSTRACT

The stability of monomeric and dimeric bovine heart cytochrome c oxidase in laurylmaltoside-containing buffers of high ionic strength allowed separation of the two forms by gel-filtration high-performance liquid chromatography (HPLC). A solution of the dimeric oxidase could be diluted without monomerisation. Both monomeric and dimeric cytochrome c oxidase showed biphasic steady-state kinetics when assayed spectrophotometrically at low ionic strength. Thus, the biphasic kinetics did not result from negative cooperativity between the two adjacent cytochrome c binding sites of the monomers constituting the dimeric oxidase. On polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS) a fraction of subunit III of the dimeric enzyme migrated as a dimer, a phenomenon not seen with the monomeric enzyme. This might suggest that in the dimeric oxidase subunit III lies on the contact surface between the protomers. If so, the presumably hydrophobic interaction between the two subunits III resisted dissociation by SDS to some extent. Addition of sufficient ascorbate and cytochrome c to the monomeric oxidase to allow a few turnovers induced slow dimerisation (on a time-scale of hours). This probably indicates that one of the transient forms arising upon reoxidation of the reduced enzyme is more easily converted to the dimeric state than the resting enzyme. Gel-filtration HPLC proved to be a useful step in small-scale purification of cytochrome c oxidase. In the presence of laurylmaltoside the monomeric oxidase eluted after the usual trace contaminants, the dimeric Complex III and the much larger Complex I. The procedure is fast and non-denaturing, although limited by the capacity of available columns.


Subject(s)
Electron Transport Complex IV/isolation & purification , Mitochondria, Heart/enzymology , Animals , Ascorbic Acid/metabolism , Cattle , Chromatography, High Pressure Liquid , Electron Transport Complex IV/metabolism
20.
Biochim Biophys Acta ; 893(2): 251-8, 1987 Sep 10.
Article in English | MEDLINE | ID: mdl-3040092

ABSTRACT

(1) Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate of purified cytochrome c oxidase preparations revealed that bovine kidney, skeletal muscle and heart contain different cytochrome c oxidase isoenzymes, which show differences in mobility of the subunits encoded by the nuclear genome. No differences in subunit pattern were observed between the oxidase preparations isolated from kidney and liver. (2) The kinetics of the steady-state reactions between bovine ferrocytochrome c and the four types of bovine cytochrome c oxidase preparation were compared under conditions of both high- and low-ionic strength. Also the pre-steady-state kinetics were studied. Only minor differences were observed in the electron-transfer activity of the isoenzymes. Thus, our experiments do not support the notion that the subunits encoded by the nuclear genome act as modulators conferring different activities to the isoenzymes of cytochrome c oxidase. (3) The cytochrome c oxidase preparation from bovine skeletal muscle was found to consist mainly of dimers, whereas the enzymes isolated from bovine kidney, liver and heart were monomeric.


Subject(s)
Cytochrome c Group/metabolism , Electron Transport Complex IV/isolation & purification , Isoenzymes/isolation & purification , Kidney/enzymology , Mitochondria, Heart/enzymology , Mitochondria, Liver/enzymology , Muscles/enzymology , Animals , Cattle , Electron Transport Complex IV/genetics , Electron Transport Complex IV/metabolism , Electrophoresis, Polyacrylamide Gel , Isoenzymes/genetics , Isoenzymes/metabolism , Kinetics
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