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Biomedical subjects

J Kopecky

Publications and source records attributed to J Kopecky.

At least 19 recordsLinked to original sources
PubMed2006

Polyunsaturated fatty acids of marine origin induce adiponectin in mice fed a high-fat diet.

AIMS/HYPOTHESIS: Diets rich in n-3 polyunsaturated fatty acids, namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), protect against insulin resistance and obesity in rodents and increase insulin sensitivity in healthy humans. We tested whether the anti-diabetic effects of EPA and DHA involve enhanced production of the endogenous insulin sensitiser, adiponectin. METHODS: We studied the effects, in an obesity-promoting high-fat diet, of partial replacement of vegetable oils by EPA/DHA concentrate (6% EPA, 51% DHA) over a 5-week period in adult male C57BL/6J mice that either had free access to food or had their food intake restricted by 30%. At the end of the treatment, systemic markers of lipid and glucose metabolism and full-length adiponectin and leptin were measured. Adiponectin (Adipoq) and leptin (Lep) gene expression in dorsolumbar and epididymal white adipose tissue (WAT) and isolated adipocytes was quantified and adipokine production from WAT explants evaluated. RESULTS: In mice with free access to food, plasma triacylglycerols, NEFA, and insulin levels were lower in the presence of EPA/DHA, while glucose and leptin levels were not significantly altered. Food restriction decreased plasma triacylglycerols, glucose, insulin and leptin, but not adiponectin. EPA/DHA increased plasma adiponectin levels, independent of food intake, reflecting the stimulation of Adipoq expression in adipocytes and the release of adiponectin from WAT, particularly from epididymal fat. Expression of Lep and the release of leptin from WAT, while being extremely sensitive to caloric restriction, was unaltered by EPA/DHA. CONCLUSIONS/INTERPRETATION: Intake of diets rich in EPA and DHA leads to elevated systemic concentrations of adiponectin, largely independent of food intake or adiposity and explain, to some extent, their anti-diabetic effects.

AMP-Activated Protein Kinase Kinases↗
PubMed2005

Polyunsaturated fatty acids of marine origin upregulate mitochondrial biogenesis and induce beta-oxidation in white fat.

AIMS/HYPOTHESIS: Intake of n-3 polyunsaturated fatty acids reduces adipose tissue mass, preferentially in the abdomen. The more pronounced effect of marine-derived eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids on adiposity, compared with their precursor alpha-linolenic acid, may be mediated by changes in gene expression and metabolism in white fat. METHODS: The effects of EPA/DHA concentrate (6% EPA, 51% DHA) admixed to form two types of high-fat diet were studied in C57BL/6J mice. Oligonucleotide microarrays, cDNA PCR subtraction and quantitative real-time RT-PCR were used to characterise gene expression. Mitochondrial proteins were quantified using immunoblots. Fatty acid oxidation and synthesis were measured in adipose tissue fragments. RESULTS: Expression screens revealed upregulation of genes for mitochondrial proteins, predominantly in epididymal fat when EPA/DHA concentrate was admixed to a semisynthetic high-fat diet rich in alpha-linolenic acid. This was associated with a three-fold stimulation of the expression of genes encoding regulatory factors for mitochondrial biogenesis and oxidative metabolism (peroxisome proliferator-activated receptor gamma coactivator 1 alpha [Ppargc1a, also known as Pgc1alpha] and nuclear respiratory factor-1 [Nrf1] respectively). Expression of genes for carnitine palmitoyltransferase 1A and fatty acid oxidation was increased in epididymal but not subcutaneous fat. In the former depot, lipogenesis was depressed. Similar changes in adipose gene expression were detected after replacement of as little as 15% of lipids in the composite high-fat diet with EPA/DHA concentrate, while the development of obesity was reduced. The expression of Ppargc1a and Nrf1 was also stimulated by n-3 polyunsaturated fatty acids in 3T3-L1 cells. CONCLUSIONS/INTERPRETATION: The anti-adipogenic effect of EPA/DHA may involve a metabolic switch in adipocytes that includes enhancement of beta-oxidation and upregulation of mitochondrial biogenesis.

Adipocytes↗
PubMed2005

Challenges and opportunities in Pan-European collaboration for researchers from Central and Eastern Europe.

Ten Central and Eastern [NLG4] European countries have recently joined the European Union. This historical enlargement provided a good opportunity to discuss the challenges and opportunities in Pan-European Research Collaboration for researchers from Central/Eastern Europe. This paper summarises examples of productive research collaboration between East and West, current challenges [NLG5], and ideas on how to facilitate better collaboration. A short overview of training, mobility and career development opportunities, covered by the Marie Curie actions, is also presented.

Europe↗
PubMed2005

Thermal dissipation of light energy is regulated differently and by different mechanisms in lichens and higher plants.

Modulated chlorophyll fluorescence was used to compare dissipation of light energy as heat in photosystem II of homoiohydric and poikilohydric photosynthetic organisms which were either hydrated or dehydrated. In hydrated chlorolichens with an alga as the photobiont, fluorescence quenching revealed a dominant mechanism of energy dissipation which was based on a protonation reaction when zeaxanthin was present. CO2 was effective as a weak protonating agent and actinic light was not necessary. In a hydrated cyanobacterial lichen, protonation by CO2 was ineffective to initiate energy dissipation. This was also true for leaves of higher plants. Thus, regulation of zeaxanthin-dependent energy dissipation by protonation was different in leaves and in chlorolichens. A mechanism of energy dissipation different from that based on zeaxanthin became apparent on dehydration of both lichens and leaves. Quenching of maximum or Fm fluorescence increased strongly during dehydration. In lichens, this was also true for so-called basal or Fo fluorescence. In contrast to zeaxanthin-dependent quenching, dehydration-induced quenching could not be inhibited by dithiothreitol. Both zeaxanthin-dependent and dehydration-induced quenching cooperated in chlorolichens to increase thermal dissipation of light energy if desiccation occurred in the light. In cyanolichens, which do not possess a zeaxanthin cycle, only desiccation-induced thermal energy dissipation was active in the dry state. Fluorescence emission spectra of chlorolichens revealed stronger desiccation-induced suppression of 685-nm fluorescence than of 720-nm fluorescence. In agreement with earlier reports of , fluorescence excitation data showed that desiccation reduced flow of excitation energy from chlorophyll b of the light harvesting complex II to emitting centres more than flow from chlorophyll a of core pigments. The data are discussed in relation to regulation and localization of thermal energy dissipation mechanisms. It is concluded that desiccation-induced fluorescence quenching of lichens results from the reversible conversion of energy-conserving to energy-dissipating photosystem II core complexes.

Ascomycota↗
PubMed2004

Role of energy charge and AMP-activated protein kinase in adipocytes in the control of body fat stores.

As indicated by in vitro studies, both lipogenesis and lipolysis in adipocytes depend on the cellular ATP levels. Ectopic expression of mitochondrial uncoupling protein 1 (UCP1) in the white adipose tissue of the aP2-Ucp1 transgenic mice reduced obesity induced by genetic or dietary manipulations. Furthermore, respiratory uncoupling lowered the cellular energy charge in adipocytes, while the synthesis of fatty acids (FA) was inhibited and their oxidation increased. Importantly, the complex metabolic changes triggered by ectopic UCP1 were associated with the activation of AMP-activated protein kinase (AMPK), a metabolic master switch, in adipocytes. Effects of several typical treatments that reduce adiposity, such as administration of leptin, beta-adrenoceptor agonists, bezafibrate, dietary n-3 polyunsaturated FA or fasting, can be compared with a phenotype of the aP2-Ucp1 mice. These situations generally lead to the upregulation of mitochondrial UCPs and suppression of the cellular energy charge and FA synthesis in adipocytes. On the other hand, FA oxidation is increased. Moreover, it has been shown that AMPK in adipocytes can be activated by adipocyte-derived hormones leptin and adiponectin, and also by insulin-sensitizes thiazolidinediones. Thus, it is evident that metabolism of adipose tissue itself is important for the control of body fat content and that the cellular energy charge and AMPK are involved in the control of lipid metabolism in adipocytes. The reciprocal link between synthesis and oxidation of FA in adipocytes represents a prospective target for the new treatment strategies aimed at reducing obesity.

AMP-Activated Protein Kinases↗
PubMed2003

LmbJ and LmbIH protein levels correlate with lincomycin production in Streptomyces lincolnensis.

AIMS: To demonstrate the expression of two overlapping genes lmbJ and lmbIH in Streptomyces lincolnensis and to document LmbJ and LmbIH protein levels during the lincomycin production phase. To analyse presumable function of the LmbIH protein. METHODS AND RESULTS: Lincomycin production was monitored by thin-layer chromatography, proteins LmbJ and LmbIH were assayed in the cell-free extracts of S. lincolnensis by immunodetection. LmbJ occurred at stable level (2-4 mg x g(-1) of total proteins) for a long time period (36-96 h of cultivation) covering the whole production phase. This fairly corresponds to the catalytic function of the protein in the antibiotic biosynthesis (N-demethyllincomycin methyltransferase). On the contrary, LmbIH reached the detectable level (0.1 and 0.7 mg x g(-1)) just for a short period at 60-72 h. CONCLUSIONS: The absence of LmbIH protein at a detectable level during the major part of the antibiotic production phase casts doubt on its possible catalytic function. Rather a different connection with the final biosynthetic steps, e.g. regulatory, can be envisaged. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of a newly found putative regulatory gene was demonstrated during production of industrial antibiotic, lincomycin.

Bacterial Proteins↗
PubMed2001

A few molecules of zeaxanthin per reaction centre of photosystem II permit effective thermal dissipation of light energy in photosystem II of a poikilohydric moss.

The relationship between thermal dissipation of light energy (as indicated by the quenching of chlorophyll fluorescence), zeaxanthin availability and protonation reactions was investigated in the moss Rhytidiadelphus squarrosus (Hedw.) Warnst. In the absence of zeaxanthin and actinic illumination, acidification by 20% CO2 in air was incapable of quenching basal, so-called F0 fluorescence either in the moss or in spinach (Spinacia oleracea L.) leaves. However, 1-s light pulses given either every 40, 60 or 200 s increased thermal dissipation as indicated by F0 and Fm quenching in the presence of 20% CO2 in air in the moss, but not in spinach while reaction centres of photosystem II (PSII) were photochemically open. In the moss, a few short light pulses, which were separated by prolonged dark times, were sufficient to raise zeaxanthin levels in the presence of 20% CO2 in air. Simultaneously, quantum efficiency of charge separation in PSII was decreased. Increasing the CO2 concentration beyond 20% further decreased quantum efficiency even in the absence of short light pulses. Under conditions optimal for fluorescence quenching, one molecule of zeaxanthin per reaction centre of PSII was sufficient to decrease quantum efficiency of charge separation in PSII by 50%. Thus, in combination with a protonation reaction, one molecule of zeaxanthin was as efficient at capturing excitation energy as a photochemically open reaction centre. The data are discussed in relation to the interaction between zeaxanthin and thylakoid protonation, which enables effective thermal dissipation of light energy in the antennae of PSII in the moss but not in higher plants when actinic illumination is absent.

Bryopsida↗
PubMed1995

Expression of the mitochondrial uncoupling protein gene from the aP2 gene promoter prevents genetic obesity.

The brown fat-specific mitochondrial uncoupling protein (UCP) provides a mechanism for generating heat by uncoupling respiration and oxidative phosphorylation. It has been suggested that this system of thermogenesis can provide a defense against obesity. To test this idea, we created a transgenic mouse in which the fat-specific aP2 gene promoter directed Ucp expression in white fat and provided for the constitutive expression of Ucp in brown fat. Transgenic mice showed both Ucp mRNA and immunoreactive UCP in white fat at 2-10% the level normally measured in brown fat. A reduction in subcutaneous fat of aP2-Ucp C57BL/6J mice was observed at 3 mo of age. When the transgene was expressed in Avy genetically obese mice reductions in total body weight and subcutaneous fat stores were observed. Female transgenic Avy mice at 13 mo of age weighed 35 grams, a weight indistinguishable from nontransgenic C57BL/6J mice. Gonadal fat showed an increase in a novel adipocyte derivative that did not accumulate lipids and that constituted approximately 80% of the mass of the tissue in Avy transgenic. A major effect of aP2-Ucp in brown fat was to reduce endogenous gene expression by as much as 95%. The results suggest that UCP synthesized from the aP2 gene promoter is thermogenically active and capable of reducing fat stores.

Adipose Tissue↗
PubMed1994

An upstream enhancer regulating brown-fat-specific expression of the mitochondrial uncoupling protein gene.

Previous studies on the regulation of a Ucp minigene in transgenic mice demonstrated that the sequences necessary for brown-fat-specific expression and inducibility by norepinephrine were located in the 5' flanking region between 1 and 2.8 kb from the transcriptional start site. We have investigated this region in more detail in cultured mouse brown adipocyte tumor cells. Deletion analysis of two types of chloramphenicol acetyltransferase reporter gene constructs under control of either the Ucp promoter or a heterologous herpes simplex virus-tk promoter defined an enhancer in a 220-bp HindIII-XbaI fragment which was essential for both brown fat specificity and norepinephrine inducibility. Site-directed mutagenesis of the reporter gene constructs established that independent mutations to a cyclic AMP-responsive element (CRE-2) or one of two TTCC motifs (BRE [brown fat regulatory element]), all within 17 bp, eliminated transient expression. Competitive DNA mobility shift assays with probes of the CRE and BRE motifs indicate that nuclear proteins interact with these motifs in a cooperative, synergistic manner. While these CRE-BRE probes do not show changes in binding which is dependent on norepinephrine treatment, a probe containing a third TTCC motif located 130 bp downstream of BRE-1 does show this dependency. The results indicate that a complex interaction of the CRE and BRE motifs, which cannot be functionally separated, control Ucp expression.

Adipose Tissue, Brown↗
PubMed1992

Cloning and mutational analysis of the gene encoding subunit C of yeast vacuolar H(+)-ATPase.

A DNA fragment containing the gene encoding subunit C of vaculor H(+)-ATPase (V-ATPase) was cloned from a yeast library. The predicted amino acid sequence indicated that the C subunit consists of 373 amino acids with a calculated molecular mass of 42,287 Da. The protein from yeast is 37% identical in its amino acid sequence to the C subunit of bovine V-ATPase. The DNA fragment that was cloned in this study contained two additional reading frames. At the 5' end an amino acid sequence that is homologous to Artemia elongation factor 1 was detected. At the 3' end the N-terminal part of a kinesin-like protein was observed. The gene encoding subunit C of the V-ATPase was interrupted, and the resulting mutant could not grow at high pH and was sensitive to low and high Ca2+ concentrations in the growth medium. Transformation of the mutant by a plasmid containing the gene encoding subunit C repaired the phenotype of the mutant. Substitution of more than half of the coding region by a corresponding DNA fragment encoding the bovine subunit C resulted in a phenotype indistinguishable from wild type. Immunological studies with the disruptant mutant revealed that subunit C is necessary for the assembly of the catalytic sector of the enzyme.

Amino Acid Sequence↗
PubMed1992

Ophthalmic drugs and anesthesia interactions.

Ophthalmic medications are often overlooked in the preoperative evaluation for patients experiencing surgery and anesthesia as "just eye drops." Both acute and chronic administration of these agents affects the anesthesia practitioner. Virtually every patient experiencing anesthesia for cataract extraction will have other nonanesthetic medications administered perioperatively. Medications are dispensed throughout the age continuum for the treatment of glaucoma on a chronic basis. All of these medications have the potential to cause morbidity and mortality by themselves or in combination with other drugs, to include anesthetic agents. This article focuses on the more common agents the anesthetist will encounter.

Anesthetics↗
PubMed1986

Thyroxine 5'-deiodinase in hamster and rat brown adipose tissue: effect of cold and diet.

Thyroxine 5'-deiodinase (type II) is present in a microsomal fraction of brown adipose tissue (BAT) of Syrian hamsters. Cold exposure increased specific activity in homogenates after a lag period of approximately 2 h to reach a maximum by 1-3 days. Total activity increased 80 times in cold-acclimated hamsters. During deacclimation, a rapid decrease occurred again after a lag period of approximately 2 h. The increase did not parallel increases in protein or in thermogenic state of mitochondria (mitochondrial GDP binding), which occurred more slowly. Changes in serum 3,5,3'-triiodothyronine concentration paralleled changes in BAT 5'-deiodinase during both acclimation to cold and deacclimation. The cold-induced increase in BAT 5'-deiodinase activity occurred more slowly but was much larger in hamsters than in rats. Eating a palatable diet increased BAT protein content and thermogenic activity in both hamsters and rats but did not alter total 5'-deiodinase activity in either species. We conclude that the trophic response of brown adipose tissue to cold in both hamsters and rats is accompanied by an enhanced endogenous production of triiodothyronine that may play a role in control of tissue growth. The trophic response to a palatable diet differs in that enhanced endogenous production of triiodothyronine is not an obligatory accompaniment. Specific stimulation of 5'-deiodinase by cold might modify thermogenesis in tissues other than BAT by providing triiodothyronine and be of greater significance in hamsters than in rats.

Acclimatization↗
PubMed1986

Thyroxine 5'-deiodinase in brown adipose tissue of myopathic hamsters.

Myopathic Syrian hamsters (BIO 14.6) have less brown adipose tissue (BAT) than normal. The trophic response of this tissue to cold is smaller than normal and trophic responses to diet and to photoperiod are absent. The objective was to find out whether activity of thyroxine 5'-deiodinase in their BAT was increased normally in response to cold and thus whether a defect in endogenous production of 3,5,3'-triiodothyronine might underlie the attenuated trophic response. The effect of feeding a high-fat diet on activity of 5'-deiodinase was also studied. Cold acclimation increased thyroxine 5'-deiodinase activity in BAT of the myopathic hamster, but the total remained smaller than normal because of the smaller size. The cold-induced increase in concentration of mitochondrial uncoupling protein was also smaller than normal. The level of serum 3,5,3'-triiodothyronine was low in myopathic hamsters and remained lower than normal when they were cold-exposed or cold acclimated. Feeding the high-fat diet to myopathic hamsters resulted in a greater than normal suppression of thyroxine 5'-deiodinase activity than in normal hamsters; the normal increases in protein content and in concentration of mitochondrial uncoupling protein were absent. We conclude that the defective trophic response of BAT of the myopathic hamster is not secondary to defective regulation of its thyroxine 5'-deiodinase activity because this activity does not appear to be obligatorily linked to hypertrophy of BAT. The low level of serum 3,5,3'-triiodothyronine in the myopathic hamster may be secondary to reduced capacity for peripheral thyroxine deiodination in its BAT.

Acclimatization↗