MAIN CONCLUSION: In tobacco, three sequence variants of the TERT gene have been described. We revealed unbalanced levels of TERT variant transcripts in vegetative tobacco tissues and enhanced TERT transcription and telomerase activity in reproductive tissues. Telomerase is a ribonucleoprotein complex responsible for the maintenance of telomeres, structures delimiting ends of linear eukaryotic chromosomes. In the Nicotiana tabacum (tobacco) allotetraploid plant, three sequence variants (paralogs) of the gene coding for the telomerase reverse transcriptase subunit (TERT) have been described, two of them derived from the maternal N. sylvestris genome (TERT_Cs, TERT_D) and one originated from the N. tomentosiformis paternal genome (TERT_Ct). In this work, we analyzed the transcription of TERT variants in correlation with telomerase activity in tobacco tissues. High and approximately comparable levels of TERT_Ct and TERT_Cs transcripts were detected in seedlings, roots, flower buds and leaves, while the transcript of the TERT_D variant was markedly underrepresented. Similarly, in N. sylvestris tissues, TERT_Cs transcript significantly predominated. A specific pattern of TERT transcripts was found in samples of tobacco pollen with the TERT_Cs variant clearly dominating particularly at the early stage of pollen development. Detailed analysis of TERT_C variants representation in functionally distinct fractions of pollen transcriptome revealed their prevalence in large ribonucleoprotein particles encompassing translationally silent mRNA; only a minority of TERT_Ct and TERT_Cs transcripts were localized in actively translated polysomes. Histones of the TERT_C chromatin were decorated predominantly with the euchromatin-specific epigenetic modification in both telomerase-positive and telomerase-negative tobacco tissues. We conclude that the existence and transcription pattern of tobacco TERT paralogs represents an interesting phenomenon and our results indicate its functional significance. Nicotiana species have again proved to be appropriate and useful model plants in telomere biology studies.
- MeSH
- Cell Nucleus genetics MeSH
- Chromatin Immunoprecipitation MeSH
- Euchromatin metabolism MeSH
- Transcription, Genetic MeSH
- Genetic Variation * MeSH
- Histones metabolism MeSH
- RNA, Messenger genetics metabolism MeSH
- Organ Specificity genetics MeSH
- Polyribosomes metabolism MeSH
- Protein Processing, Post-Translational MeSH
- Pollen Tube growth & development MeSH
- Gene Expression Regulation, Plant * MeSH
- Nicotiana genetics MeSH
- Telomerase genetics metabolism MeSH
- Publication type
- Journal Article MeSH
Piscine cytochrome P450 (CYP) enzymes play an important role in the metabolism of xenobiotics. Xenobiotics often act as inducers of CYP1A1 and CYP3A expression and activity in fish. We compared constitutive mRNA expression of CYP1A1, CYP3A27, and CYP3A45 and catalytic activity of CYP1A (7-ethoxyresorufin-O-deethylation, EROD) and CYP3A-like (benzyloxy-4-trifluoromethylcoumarin-O-debenzyloxylation, BFCOD) enzymes in the following six rainbow trout tissues: liver, gill, heart, brain, intestine, and gonad. mRNA expression and activity were present in all investigated tissues. The CYP1A1 mRNA expression was higher in the liver, gill, heart, and brain compared to gonad and intestine. The intestine was the main site of CYP3A27 and CYP3A45 expression. The highest EROD and BFCOD activity was observed in liver tissue followed in descending order by heart, brain, gill, intestine, and gonad. Such differences might be related to the role of CYP physiological functions in the specific tissue. Rainbow trout exposure to 50 mg/kg of β-naphthoflavone for 48 h resulted in a 7.5- and 5.9-fold increase in liver EROD and BFCOD activity, respectively. In vitro EROD activity inhibition with ellipticine showed tissue-specific inhibition, while ketoconazole decreased BFCOD activity by 50-98 % in all tissues. Further studies are needed to identify all CYP isoforms that are responsible for these activities and modes of regulation.
- MeSH
- Cytochrome P-450 CYP1A1 genetics metabolism MeSH
- Cytochrome P-450 CYP3A genetics metabolism MeSH
- Liver enzymology MeSH
- RNA, Messenger genetics metabolism MeSH
- Brain enzymology MeSH
- Myocardium enzymology MeSH
- Oncorhynchus mykiss metabolism MeSH
- Sex Characteristics MeSH
- Gene Expression Regulation, Enzymologic physiology MeSH
- Intestines enzymology MeSH
- Gills enzymology MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Prostate specific membrane antigen (PSMA), also called glutamate carboxypeptidase II (GCPII), is a target enzyme for diagnosis and treatment of prostate cancer. Moreover, it is upregulated in the vasculature of most solid tumors and is therefore a potential target for the generation of novel antineoplastics. In this context, we analyze the possibility of using rat and pig as animal models for enzymologic and in vivo studies. METHODS: We prepared the recombinant extracellular part of human, rat, and pig GCPII in S2 cell media and characterized the activity and inhibition profiles of the three orthologs by radioenzymatic assay. We performed Western blot analysis of GCPII expression in human, rat, and pig tissues using the monoclonal antibody GCP-04 and confirmed these findings by activity measurements and immunohistochemistry. RESULTS: The three recombinant proteins show similar specific enzymatic activities and inhibition profiles. Tissue expression analysis revealed that most of the pig and human tissues show at least some GCPII-positivity, while the expression pattern in rat is more restricted. Moreover, tissues such as prostate and testes exhibit different GCPII expression levels among the species studied. CONCLUSIONS: The rat and pig orthologs of GCPII seem to be suitable to approximate human GCPII in enzymologic studies. However, the diffuse expression pattern of GCPII in animal and human tissues could be a caveat for the potential utilization of GCPII-targeted anticancer drugs. Furthermore, variations in GCPII tissue distribution among the species studied should be considered when using rat or pig as models for antineoplastic drug discovery.
- MeSH
- Species Specificity MeSH
- Financing, Organized MeSH
- Rats MeSH
- Kidney enzymology pathology MeSH
- Humans MeSH
- Spinal Cord enzymology pathology MeSH
- Swine, Miniature MeSH
- Models, Animal MeSH
- Molecular Sequence Data MeSH
- Rats, Inbred Lew MeSH
- Swine MeSH
- Prostate enzymology pathology MeSH
- Prostate-Specific Antigen analysis genetics metabolism MeSH
- Gene Expression Regulation, Enzymologic MeSH
- Amino Acid Sequence MeSH
- Testis enzymology pathology MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Comparative Study MeSH
BACKGROUND: The angiotensin receptor blocker telmisartan has unique chemical properties that enable it to partially activate the peroxisome proliferator activated receptor gamma (PPARG) as well as block angiotensin II type 1 receptors. METHODS: To directly test whether some of the metabolic effects of telmisartan require the presence of PPARG, we studied mice in which the gene (Pparg) for PPARG had been deleted in fat or in muscle. RESULTS: We found that knockout of Pparg in fat tissue greatly impaired the ability of telmisartan to increase adiponectin levels and to enhance sensitivity to insulin-stimulated glucose incorporation into adipose tissue lipids. In contrast, muscle-specific Pparg knockout had relatively little or no impact on the ability of telmisartan to increase adiponectin levels or affect glucose metabolism either in fat or muscle. These findings provide compelling evidence that the ability of telmisartan to increase adiponectin levels and stimulate glucose use in adipose tissue may depend on the presence of PPARG in fat. CONCLUSIONS: We conclude that PPARG in adipose tissue is required for at least several of the metabolic actions of telmisartan.
- MeSH
- Benzimidazoles pharmacokinetics MeSH
- Benzoates pharmacokinetics MeSH
- Angiotensin II Type 1 Receptor Blockers pharmacokinetics MeSH
- Hypertension drug therapy metabolism MeSH
- Insulin Resistance MeSH
- Blood Glucose metabolism MeSH
- Disease Models, Animal MeSH
- Mice, Knockout MeSH
- Mice MeSH
- PPAR gamma biosynthesis MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
The biogenesis of eukaryotic COX (cytochrome c oxidase) requires several accessory proteins in addition to structural subunits and prosthetic groups. We have analysed the assembly state of COX and SCO2 protein levels in various tissues of six patients with mutations in SCO2 and SURF1. SCO2 is a copper-binding protein presumably involved in formation of the Cu(A) centre of the COX2 subunit. The function of SURF1 is unknown. Immunoblot analysis of native gels demonstrated that COX holoenzyme is reduced to 10-20% in skeletal muscle and brain of SCO2 and SURF1 patients and to 10-30% in heart of SCO2 patients, whereas liver of SCO2 patients' contained normal holoenzyme levels. The steady-state levels of mutant SCO2 protein ranged from 0 to 20% in different SCO2 patient tissues. In addition, eight distinct COX subcomplexes and unassembled subunits were found, some of them identical with known assembly intermediates of the human enzyme. Heart, brain and skeletal muscle of SCO2 patients contained accumulated levels of the COX1.COX4.COX5A subcomplex, three COX1-containing subcomplexes, a COX4.COX5A subcomplex and two subcomplexes composed of only COX4 or COX5A. The accumulation of COX1.COX4.COX5A subcomplex, along with the virtual absence of free COX2, suggests that the lack of the Cu(A) centre may result in decreased stability of COX2. The appearance of COX4.COX5A subcomplex indicates that association of these nucleus-encoded subunits probably precedes their addition to COX1 during the assembly process. Finally, the consequences of SCO2 and SURF1 mutations suggest the existence of tissue-specific functional differences of these proteins that may serve different tissue-specific requirements for the regulation of COX biogenesis.
- MeSH
- Fibroblasts enzymology MeSH
- Financing, Organized MeSH
- Liver enzymology MeSH
- Infant MeSH
- Muscle, Skeletal enzymology MeSH
- Humans MeSH
- Membrane Proteins MeSH
- Mitochondrial Proteins MeSH
- Brain enzymology MeSH
- Mutation genetics MeSH
- Myocardium enzymology MeSH
- Organ Specificity MeSH
- Protein Subunits chemistry metabolism MeSH
- Child, Preschool MeSH
- Proteins genetics metabolism MeSH
- Gene Expression Regulation, Enzymologic MeSH
- Electron Transport Complex IV biosynthesis chemistry metabolism MeSH
- Carrier Proteins MeSH
- Check Tag
- Infant MeSH
- Humans MeSH
- Child, Preschool MeSH
Fatty acid esters of hydroxy fatty acids (FAHFAs) are endogenous bioactive lipids known for their anti-inflammatory and anti-diabetic properties. Despite their therapeutic potential, little is known about the sex-specific variations in FAHFA metabolism. This study investigated the role of sex and Androgen Dependent TFPI Regulating Protein (ADTRP), a FAHFA hydrolase. Additionally, tissue-specific differences in FAHFA levels, focusing on the perigonadal white adipose tissue (pgWAT), subcutaneous white adipose tissue (scWAT), brown adipose tissue (BAT), plasma, and liver, were evaluated using metabolomics and lipidomics. We found that female mice exhibited higher FAHFA levels in pgWAT, scWAT, and BAT compared to males. FAHFA levels were inversely related to testosterone and Adtrp mRNA, which showed significantly lower expression in females compared with males in pgWAT and scWAT. However, no significant differences between the sexes were observed in plasma and liver FAHFA levels. Adtrp deletion had minimal impact on both sexes' metabolome and lipidome of pgWAT. However, we discovered higher endogenous levels of triacylglycerol estolides containing FAHFAs, a FAHFA metabolic reservoir, in the pgWAT of female mice. These findings suggest that sex-dependent differences in FAHFA levels occur primarily in specific WAT depots and may modulate local insulin sensitivity in adipocytes, and the role of ADTRP is limited to adipose depots. However, further investigations are warranted to fully comprehend the underlying mechanisms and implications of sex-dependent regulation of human FAHFA metabolism.
- MeSH
- Adipose Tissue, White * metabolism MeSH
- Esters metabolism MeSH
- Adipose Tissue, Brown metabolism MeSH
- Liver metabolism MeSH
- Fatty Acids * metabolism MeSH
- Lipid Metabolism MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Organ Specificity MeSH
- Sex Characteristics MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Lithium (Li) represents a first choice mood stabilizer for bipolar disorder (BD). Despite extensive clinical use, questions regarding its mechanism of action and pathological mechanism of renal function impairment by Li remain open. The present study aimed to improve our knowledge in this area paying special attention to the relationship between the length of Li action, lipid peroxidation (LP), and Na+/K+-ATPase properties. The effects of therapeutic Li doses, administered daily to male Wistar rats for 1 (acute), 7 (short term) and 28 days (chronic), were studied. For this purpose, Na+/K+-ATPase activity measurements, [3H]ouabain binding and immunoblot analysis of α-Na+/K+-ATPase were performed. Li-induced LP was evaluated by determining the malondialdehyde concentration by HPLC. Sleep deprivation (SD) was used as an experimental approach to model the manic phase of BD. Results obtained from the kidney were compared to those obtained from erythrocytes and different brain regions in the same tested animals. Whereas treatment with therapeutic Li concentration did not bring any LP damage nor significant changes of Na+/K+-ATPase expression and [3H]ouabain binding in the kidney, it conferred strong protection against this type of damage in the forebrain cortex. Importantly, the observed changes in erythrocytes indicated changes in forebrain cortices. Thus, different resistance to SD-induced changes of LP and Na+/K+-ATPase was detected in the kidney, erythrocytes and the brain of Li-treated rats. Our study revealed the tissue-specific protective properties of Li against LP and Na+/K+-ATPase regulation.
- MeSH
- Antimanic Agents administration & dosage pharmacology MeSH
- Bipolar Disorder drug therapy MeSH
- Time Factors MeSH
- Erythrocytes drug effects metabolism MeSH
- Rats MeSH
- Kidney drug effects metabolism MeSH
- Lithium Carbonate administration & dosage pharmacology MeSH
- Disease Models, Animal MeSH
- Brain drug effects metabolism MeSH
- Lipid Peroxidation drug effects MeSH
- Rats, Wistar MeSH
- Sodium-Potassium-Exchanging ATPase metabolism MeSH
- Sleep Deprivation psychology MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Numerous chemicals in the environment have the ability to interact with the endocrine system. These compounds are called endocrine disruptors (EDs). Exposure to EDs represents one of the hypotheses for decreasing fertility, the increased risk of numerous cancers and obesity, metabolic syndrome and type 2 diabetes. There are various mechanisms of ED action, one of which is their interference in the action of 11β-hydroxysteroid dehydrogenase (11βHSD) that maintains a balance between active and inactive glucocorticoids on the intracellular level. This enzyme has two isoforms and is expressed in various tissues. Inhibition of 11βHSD in various tissues can have different consequences. In the case of EDs, the results of exposure are mainly adverse; on the other hand pharmaceutically developed inhibitors of 11βHSD type 1 are evaluated as an option for treating metabolic syndrome, as well as related diseases and depressive disorders. This review focuses on the effects of 11βHSD inhibitors in the testis, colon, adipose tissue, kidney, brain and placenta.
- MeSH
- 11-beta-Hydroxysteroid Dehydrogenase Type 1 antagonists & inhibitors metabolism MeSH
- 11-beta-Hydroxysteroid Dehydrogenase Type 2 antagonists & inhibitors metabolism MeSH
- Diabetes Mellitus chemically induced enzymology pathology MeSH
- Endocrine Disruptors pharmacology MeSH
- Glucocorticoids metabolism MeSH
- Enzyme Inhibitors pharmacology MeSH
- Colon drug effects enzymology MeSH
- Humans MeSH
- Metabolic Syndrome chemically induced enzymology pathology MeSH
- Brain drug effects enzymology MeSH
- Neoplasms chemically induced enzymology pathology MeSH
- Obesity chemically induced enzymology pathology MeSH
- Organ Specificity MeSH
- Placenta drug effects enzymology MeSH
- Pregnancy MeSH
- Testis drug effects enzymology MeSH
- Adipose Tissue drug effects enzymology MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Pregnancy MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
In the central nervous system (CNS), monocarboxylate transporter 1 (MCT1) is expressed in astrocytes and endothelial cells but also in oligodendroglia. Oligodendroglia support neurons and axons through lactate transportation by MCT1. Limited information is available on the MCT1 expression changes in candidate cells in the developing rat brain, especially in corpus callosum which is the most vulnerable area in demyelinating diseases. In the present study, we investigated the expression pattern of MCT1 during postnatal development in the rat corpus callosum using immunofluorescene staining, Western blotting analysis and RT-PCR. We reported that MCT1 gene and protein were consistently expressed in the rat corpus callosum from birth to adult. MCT1/CNPase and MCT1/GFAP immunofluorescence staining demonstrated that most of MCT1 positive cells were co-labeled with cyclic nucleotide 3´ phosphodiesterase (CNPase) in rat corpus callosum from P7 to adult, whereas MCT1(+)/GFAP(+) cells preserve the dominate position before P7. Moreover, there were significant associations between the expression of MCT1 protein and the expression of myelin basic protein (MBP) (correlation coefficient: r=0.962, P=0.009) from P7 to adult. Similarly, the MCT1 mRNA expression was also significantly associated with MBP mRNA expression (r=0.976, P=0.005). Our results are proposing that in the developing brain white matter, MCT1 is predominately expressed in oligodendrocyte though it mainly expressed in astrocyte in early postnatal, which indicate that MCT1 may involve in the oligodendrocyte development and myelination.
- MeSH
- White Matter metabolism MeSH
- Corpus Callosum metabolism MeSH
- Rats MeSH
- Animals, Newborn MeSH
- Organ Specificity MeSH
- Rats, Sprague-Dawley MeSH
- Monocarboxylic Acid Transporters metabolism MeSH
- Gene Expression Regulation physiology MeSH
- Aging metabolism MeSH
- Symporters metabolism MeSH
- Tissue Distribution MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Jedním z ukazatelů proliferační aktivity nádorů je apoptóza. Ke studiu apoptózy na tkáňových řezech je nyní používána řada metodik zahrnující světelnou a elektronovou mikroskopii, in situ nick end-labeling (ISEL), TdT-mediated dUTP nick-end labeling (TUNEL) a imunohistochemický průkaz antigénu spojených s apoptózou. K průkazu apoptózy lze použít také monoklonální protilátku M30 CytoDEATH (M30), která se váže na neo-epitopy vzniklé při štěpení cytokeratinu 18 kaspazami v průběhu časných stadií apoptózy. K faktorům regulujícím apoptózu u řady nádoro vých onemocnění patří také protein bcl-2. V naší práci bylo imunohistochemicky vyšetřeno 26 invazivních duktálních adenokarcinomů pankreatu pomocí protilátek M30 a bcl-2. Průměmý apoptotický index (AI, procento apoptotických buněk z celkového počtu nádorových buněk) u vyšetřených karcinomů pankreatu byl 2,75 %. Vysoký AI (>10 %) byl zaznamenán ve 4 případech (15 %). Exprese proteinu bcl-2 byla imunohistochemicky prokázána ve 3 pnpadech (11,5 %). Korelace mezi AI a expresí proteinu bcl-2 nebyla prokázána. Ověřili jsme, že monoklonální protilátku M30 proti neo-epitopu cytokeratinu 18 lze použít k průkazu apoptotických buněk na tkáňových řezech a v tomto směru představuje nový přístup, který dosud nebyl u pankreatických karcinomů aplikován. Nízká pozitivita exprese bcl-2 ukazuje, že bcl-2 protein pravděpodobně nehraje hlavní roli v kancerogenezi a progresi duktálních karcinomů pankreatu.
Apoptosis plays a central role in the development and/or progression of cancer. There are several methods for detection of apoptotic cells in tissue sections including light and electron microscopy, in situ nick end-labeling (ISEL), TdT-mediated dUTP nick-end labeling (TUNED and immunohistochemical detection of proteins associated with apoptosis. Apoptosis was assessed by the monoclonal antibody M30 CytoDEATH (M30), which is specific for neo-epitope in cytokeratin 18 that becomes available at an early caspase cleavage during apoptosis. Expression of bcl-2 protein was evaluated, because bcl-2 protein plays an important role in the regulation of apoptosis. Twenty-six invasive ductal adenocarcinomas of the pancreas were studied immunohistochemically with antibodies M30 and bcl-2. The mean apoptotic index (AI, the percentage of apoptotic cells of the total tumor cells number) was 2.75%. High AI (>10%) was observed in 4 cases of the 26 pancreatic carcinomas (15%). Protein bcl-2 was expressed in 3 cases (11,5 %). The AI did not correlate with the expression of protein bcl-2. In conclusion, the detection of neo-epitope in y okeratin 18 by monoclonal antibody M30 can be used for quantification of apoptotic cells with immunohistochemical techniques in tissue sections. It is a new approach to evaluate apoptosis in pancreatic carcinomas. The low positivity of bcl-2 expression in pancreatic adenocarcinomas suggests that bcl-2 protein does not play a central role in pancreatic tumorigenesis and cancer progression.
