The patatin-like phospholipase domain containing 3 (PNPLA3) gene (viz. its I148M variant) is one of the key players in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). We have identified a novel insertion/deletion variant of 1114 bp, localized in the second intron of the PNPLA3 gene, which corresponds to the 3' terminal sequence of the long-interspersed element (LINE-1). DNA analysis of 122 NAFLD patients and 167 control subjects as well as RNA analysis of 19 liver biopsies revealed that the novel variant is very common (frequency = 0.41), fully linked to the clinically important I148M variant, and clinically silent. Although the LINE-1 insertion does not seem to have any biological effect, it can impede genotyping of the I148M variant. If insertion prevents the attachment of the diagnostic primer, then the non-insertion allele will be selectively amplified; and thus the frequency of the 148M "risk" allele will be significantly overestimated due to the complete linkage of the LINE-1 insertion and the 148I allele of the PNPLA3 gene. Therefore, our findings underline the importance of careful design and consistent documentation of the methodology, including primer sequences. Critical revisions of the results of some studies that have already been reported may therefore be needed.
- MeSH
- acyltransferasy genetika MeSH
- alely MeSH
- dlouhé rozptýlené jaderné elementy genetika MeSH
- fosfolipasy A2 nezávislé na vápníku genetika MeSH
- genetická predispozice k nemoci genetika MeSH
- genotyp MeSH
- játra patologie MeSH
- jednonukleotidový polymorfismus genetika MeSH
- lidé MeSH
- nealkoholová steatóza jater genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Various endocrine factors that regulate energy homeostasis are also implicated in the reproductive physiology of mammals. However, the hormonal link between metabolism and reproduction in fish is poorly understood. Ghrelin is a multifunctional hormone with both metabolic and reproductive roles in vertebrates. Post-translational acylation by ghrelin-O-acyltransferase (GOAT) is critical for its biological actions. The expression of ghrelin, ghrelin or growth hormone secretagogue receptor (GHSR), and GOAT (which forms the ghrelinergic system) in fish under metabolic stress remains unclear. In this research, we used RT-qPCR and Western blot analysis to determine the expression of the ghrelinergic system in goldfish (during the reproductively active phase) hypothalamus and gonads under 7 and 28 days of fasting. We found a significant increase in preproghrelin mRNA expresson in the ovary, and GOAT mRNA expression in the testis of goldfish deprived of food for 7 days. In fish deprived of food for 28 days, preproghrelin, GHSR and GOAT mRNA expression was significantly increased in the hypothalamus of male goldfish. Such differences were not observed in the hypothalamus of female fish, and in the testis of 28 days fasted fish. Meanwhile, preproghrelin, GHSR, and GOAT expression (both mRNA and protein) was significantly increased in the ovary of female fish fasted for 28 days. Ghrelin has been shown to suppress oocyte maturation in fish. The upregulation of a system that has ovarian inbititory roles suggests a role for ghrelin in maintaining reduced reproductive capability during metabolically challenging periods.
- MeSH
- acyltransferasy genetika MeSH
- fyziologický stres genetika MeSH
- ghrelin genetika MeSH
- gonády růst a vývoj metabolismus MeSH
- hypothalamus metabolismus MeSH
- karas zlatý genetika MeSH
- messenger RNA genetika MeSH
- omezení příjmu potravy MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lecithin:retinol acyltransferase (LRAT) is the major enzyme responsible for retinol esterification in the mammalian body. LRAT exhibits specific activity in the cells with active retinol metabolism where it converts retinols into retinyl esters, which represents the major storage form of retinol. Besides hepatic stellate cells in the liver, LRAT appears to have a key physiologic role in several other tissues. In this study, we generated a transgenic reporter mouse expressing green fluorescence protein (EGFP) under the control of region containing -1166 bps from promoter upstream from the putative transcriptional start site and 262 bps downstream of this start. Transgenic reporter mice exhibited specific expression in eyes and testes. In eyes, expression of EGFP-reporter is found in lens and lens epithelium and fibers from embryo to adulthood. In testes, LRAT-EGFP reporter is expressed both in Sertoli and in spermatocytes marking initiation of spermatogenesis in prepubertal mice. Our data show that the examined LRAT regulatory region is sufficient to achieve strong and selective expression in the eye and testes but not in liver and other organs.
- MeSH
- acyltransferasy genetika MeSH
- epitel metabolismus MeSH
- genetická transkripce genetika MeSH
- genotyp MeSH
- meióza genetika MeSH
- myši transgenní MeSH
- myši MeSH
- oční čočka metabolismus MeSH
- promotorové oblasti (genetika) genetika MeSH
- regulace genové exprese enzymů genetika MeSH
- Sertoliho buňky metabolismus MeSH
- spermatocyty ultrastruktura MeSH
- spermatogeneze MeSH
- testis metabolismus MeSH
- vitamin A metabolismus MeSH
- vývojová regulace genové exprese genetika MeSH
- zelené fluorescenční proteiny MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cytokinins (CKs) regulate plant development and growth via a two-component signaling pathway. By forward genetic screening, we isolated an Arabidopsis mutant named grow fast on cytokinins 1 (gfc1), whose seedlings grew larger aerial parts on MS medium with CK. gfc1 is allelic to a previously reported cutin mutant defective in cuticular ridges (dcr). GFC1/DCR encodes a soluble BAHD acyltransferase (a name based on the first four enzymes characterized in this family: Benzylalcohol O-acetyltransferase, Anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase and Deacetylvindoline 4-O-acetyltransferase) with diacylglycerol acyltransferase (DGAT) activity in vitro and is necessary for normal cuticle formation on epidermis in vivo. Here we show that gfc1 was a CK-insensitive mutant, as revealed by its low regeneration frequency in vitro and resistance to CK in adventitious root formation and dark-grown hypocotyl inhibition assays. In addition, gfc1 had de-etiolated phenotypes in darkness and was therefore defective in skotomorphogenesis. The background expression levels of most type-A Arabidopsis Response Regulator (ARR) genes were higher in the gfc1 mutant. The gfc1-associated phenotypes were also observed in the cutin-deficient glycerol-3-phosphate acyltransferase 4/8 (gpat4/8) double mutant [defective in glycerol-3-phosphate (G3P) acyltransferase enzymes GPAT4 and GPAT8, which redundantly catalyze the acylation of G3P by hydroxyl fatty acid (OH-FA)], but not in the cutin-deficient mutant cytochrome p450, family 86, subfamily A, polypeptide 2/aberrant induction of type three 1 (cyp86A2/att1), which affects the biosynthesis of some OH-FAs. Our results indicate that some acyltransferases associated with cutin formation are involved in CK responses and skotomorphogenesis in Arabidopsis.
- MeSH
- acyltransferasy genetika metabolismus MeSH
- Arabidopsis genetika růst a vývoj metabolismus účinky záření MeSH
- cytokininy metabolismus farmakologie MeSH
- fenotyp MeSH
- membránové lipidy biosyntéza MeSH
- meristém účinky léků genetika růst a vývoj účinky záření MeSH
- morfogeneze * účinky léků účinky záření MeSH
- mutace MeSH
- proteiny huseníčku genetika metabolismus MeSH
- regulace genové exprese u rostlin účinky léků účinky záření MeSH
- semenáček účinky léků genetika růst a vývoj účinky záření MeSH
- tma MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Nuclear magnetic resonance (NMR) is a powerful technique for solving protein structures or studying their interactions. However, it requires molecules labeled with NMR sensitive isotopes like carbon (13)C and nitrogen (15)N. The recombinant expression of labeled proteins is simple to perform but requires quite expensive chemicals. When there is a need for special labeled chemicals, like uniformly (13)C-labeled myristic acid, the price significantly rises. Here we describe a cost-effective method for the recombinant expression of uniformly labeled myristoylated proteins in Escherichia coli demonstrated on the production of Mason-Pfizer monkey virus matrix protein. We used the ability of E. coli to naturally synthetize myristic acid. When grown in isotopically labeled medium the myristic acid will be labelled as well. Bacteria were co-transfected with plasmid carrying gene for yeast N-myristoyltransferase which ensures myristoylation of expressed protein. This process provided 1.8mg of the myristoylated, doubly labeled ((13)C/(15)N)M-PMV matrix protein from 1L of (15)N/(13)C labeled M9 medium. The price represents approximately 50% cost reduction of conventional method using commercially available [U-(13)C]myristic acid.
- MeSH
- acylace MeSH
- acyltransferasy genetika metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- izotopové značení ekonomika metody MeSH
- izotopy dusíku MeSH
- izotopy uhlíku MeSH
- kyselina myristová chemie metabolismus MeSH
- Masonův-Pfizerův opičí virus genetika MeSH
- nukleární magnetická rezonance biomolekulární metody MeSH
- proteiny virové matrix biosyntéza genetika izolace a purifikace MeSH
- rekombinantní proteiny biosyntéza izolace a purifikace MeSH
- transfekce MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Viroid-derived small RNAs generated during hop stunt viroid (HSVd) pathogenesis may induce the symptoms found in the hop cultivar "Admiral", including observed shifts in phenylpropanoid metabolites and changes in petiole coloration. Using quantitative RT-PCR, we examined hop lupulin gland-specific genes that have been shown to be involved in phenylpropanoid metabolism, for altered expression in response to infection with two HSVd isolates, HSVd-g and CPFVd. Most notably, the expression of a gene encoding a key enzyme for phenylpropanoid synthesis, naringenin-chalcone synthase H1 (chs_H1), decreased up to 40-fold in infected samples. In addition, a marked decrease in the expression of HlbHLH2 and an increase in the expression of HlMyb3 were observed. These two genes encode transcription factors that form a ternary complex with HlWDR1 for chs_H1 promoter activation. In a transient expression assay, a decrease in the ternary complex potential to activate the chs_H1 promoter was observed upon the decrease of HlbHLH2 expression. In addition, targeting of the chs_H1 transcript by vd-sRNAs may contribute to these expression changes. Our data show that HSVd infection causes a significant imbalance in the expression of phenylpropanoid metabolite-affecting genes via a complex mechanism, possibly involving regulatory disorders and direct targeting by vd-sRNA.
- MeSH
- acyltransferasy genetika metabolismus MeSH
- down regulace MeSH
- exprese genu MeSH
- Humulus enzymologie genetika virologie MeSH
- listy rostlin enzymologie genetika virologie MeSH
- messenger RNA chemie genetika MeSH
- nemoci rostlin virologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- propanoly metabolismus MeSH
- regulace genové exprese enzymů * MeSH
- regulace genové exprese u rostlin MeSH
- RNA interference MeSH
- RNA rostlin chemie genetika MeSH
- RNA virová chemie genetika MeSH
- rostlinné proteiny genetika metabolismus MeSH
- stonky rostlin enzymologie genetika virologie MeSH
- transkripční faktory genetika metabolismus MeSH
- upregulace MeSH
- viroidy patogenita fyziologie MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Matrix proteins play multiple roles both in early and late stages of the viral replication cycle. Their N-terminal myristoylation is important for interaction with the host cell membrane during virus budding. We used Escherichia coli, carrying N-myristoyltransferase gene, for the expression of the myristoylated His-tagged matrix protein of Mason-Pfizer monkey virus. An efficient, single-step purification procedure eliminating all contaminating proteins including, importantly, the non-myristoylated matrix protein was designed. The comparison of NMR spectra of matrix protein with its myristoylated form revealed substantial structural changes induced by this fatty acid modification.
- MeSH
- acyltransferasy chemie genetika izolace a purifikace MeSH
- Escherichia coli enzymologie genetika MeSH
- exprese genu MeSH
- kyselina myristová chemie MeSH
- Masonův-Pfizerův opičí virus chemie genetika MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- proteiny virové matrix chemie genetika izolace a purifikace MeSH
- rekombinantní fúzní proteiny chemie genetika izolace a purifikace MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- MeSH
- acyltransferasy genetika chemie MeSH
- chalkonoidy analýza MeSH
- financování organizované MeSH
- fylogeneze MeSH
- Humulus enzymologie chemie MeSH
- klonování DNA MeSH
- messenger RNA analýza MeSH
- prenylace proteinů MeSH
- promotorové oblasti (genetika) MeSH
- protoonkogenní proteiny c-myb genetika chemie MeSH
- rostlinné proteiny genetika chemie MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
