Optimalization of preparation of apo-cytochrome b5 utilizing apo-myoglobin

Cytochrome b5 (cyt b5), a component of endoplasmic reticulum membrane, plays a role in modulation of enzymatic activity of some cytochrome P450 (CYP) enzymes. The effect of apo-cytochrome b5 on this enzymatic system has not been investigated in details, because preparation of cyt b5 as a pure protein failed in many laboratories. In order to prepare the native apo-cytochrome b5 in a large scale we utilized a protein with higher affinity toward the heme; the apo-myoglobin from the equine skeletal muscle. In the first step, we extracted heme moiety from the native myoglobin by butanone extraction. Than the effect of pH on spontaneous heme release from both proteins was investigated: purified rabbit cyt b5 as well as equine skeletal muscle myoglobin. The prepared apo-myoglobin was incubated with the cyt b5 and heme transfer was monitored as a shift of absorption maximum from 413 to 409 nm in pH varying between 3–6 (10 mM KH2PO4, pH 3–6). Here, we obtained 43 mg of the equine skeletal muscle apo-myoglobin (43% yield). The optimal pH range for heme transfer from cyt b5 into apo-myoglobin was between 4.2 and 5. Native apo-cytochrome b5 was successfully prepared using procedure described here

Genotoxic mechanisms for the carcinogenicity of the environmental pollutants and carcinogens o-anisidine and 2-nitroanisole follow from adducts generated by their metabolite N-(2-methoxyphenyl)-hydroxylamine with deoxyguanosine in DNA

An aromatic amine, o-anisidine (2-methoxyaniline) and its oxidative counterpart, 2-nitroanisole (2-methoxynitrobenzene), are the industrial and environmental pollutants causing tumors of the urinary bladder in rats and mice. Both carcinogens are activated to the same proximate carcinogenic metabolite, N-(2-methoxyphenyl)hydroxylamine, which spontaneously decomposes to nitrenium and/or carbenium ions responsible for formation of deoxyguanosine adducts in DNA in vitro and in vivo. In other words, generation of N-(2-methoxyphenyl)hydroxylamine is responsible for the genotoxic mechanisms of the o-anisidine and 2-nitroanisole carcinogenicity. Analogous enzymes of human and rat livers are capable of activating these carcinogens. Namely, human and rat cytochorme P4502E1 is the major enzyme oxidizing o-anisidine to N-(2-methoxyphenyl)hydroxylamine, while xanthine oxidase of both species reduces 2-nitroanisole to this metabolite. Likewise, O-demethylation of 2-nitroanisole, which is the detoxication pathway of its metabolism, is also catalyzed by the same human and rat enzyme, cytochorme P450 2E1. The results demonstrate that the rat is a suitable animal model mimicking the fate of both carcinogens in humans and suggest that both compounds are potential carcinogens also for humans

Cytochrome P450-mediated metabolism of N-(2-methoxyphenyl)-hydroxylamine, a human metabolite of the environmental pollutants and carcinogens o-anisidine and o-nitroanisole

N-(2-methoxyphenyl)hydroxylamine is a human metabolite of the industrial and environmental pollutants and bladder carcinogens 2-methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-nitroanisole). Here, we investigated the ability of hepatic microsomes from rat and rabbit to metabolize this reactive compound. We found that N-(2-methoxyphenyl)hydroxylamine is metabolized by microsomes of both species mainly to o-aminophenol and a parent carcinogen, o-anisidine, whereas 2-methoxynitrosobenzene (o-nitrosoanisole) is formed as a minor metabolite. Another N-(2-methoxyphenyl)hydroxylamine metabolite, the exact structure of which has not been identified as yet, was generated by hepatic microsomes of rabbits, but its formation by those of rats was negligible. To evaluate the role of rat hepatic microsomal cytochromes P450 (CYP) in N-(2-methoxyphenyl)hydroxylamine metabolism, we investigated the modulation of its metabolism by specific inducers of these enzymes. The results of this study show that rat hepatic CYPs of a 1A subfamily and, to a lesser extent those of a 2B subfamily, catalyze N-(2-methoxyphenyl)hydroxylamine conversion to form both its reductive metabolite, o-anisidine, and o-aminophenol. CYP2E1 is the most efficient enzyme catalyzing conversion of N-(2-methoxyphenyl)hydroxylamine to o-aminophenol

Creation of the plan of care for locality Velky Kamen in Novohradske foothills

The Novohradske Mountains and their foothills belong in a group of well-preserved areas of the Czech Republic. The value of this area lies in its unimpaired landscape and in presence of cryogenic mesoforms of the landscape. The nature park Sobenovska Highlands ensures a widespread protection for the Novohradske Mountains. We can find some smaller protected areas too. They mostly exist in order to protect forest districts. The aim of this bachelor thesis is to project a plan of care for a nature sanctuary called Velky Kamen, including other parts of the landscape like cryogenic mesoforms as the object of protection. The work is divided into two basic sections - physical-geographic characteristicof Novohradske Mountains' foothills and the plan of care for the nature sanctuary Velky Kamen. Maps and photographs are included as supplements acquired during a field research in the key locality

Examples of good practice in teaching English grammar through the inductive metod

The master thesis describes in its introduction the deductive and inductive approach to teaching English grammar, compares them, and also lists the pros and cons of each approach. The main content of the theoretical part is the presentation of key concepts and principles of inductive teaching of English grammar. The practical part converts theoretical knowledge to real grammar teaching. Pupils learned how to express past events within four lessons. These sample lessons are examples of good practice of teaching grammar inductively. The reflection of these lessons is provided at the end of this part

Is ATP the Only Nucleoside Triphosphate among ATP, CTP, GTP, and UTP to Have a Role in Kinase Catalysis of Heme-Regulated Inhibitor toward eIF2α during Lung Cancer Development?

The heme-regulated eukaryotic initiation factor 2α (eIF2α) kinase, also known as heme-regulated inhibitor (HRI), detects misfolded proteins and induces cytoprotective response to stress, mainly caused by heme-shortage. The nucleoside triphosphate ATP serves as the main donor of phosphate for the phosphorylation of eIF2α by HRI in human cells. However, the other main nucleoside triphosphates (CTP, GTP, UTP) are also present at relatively high concentrations, especially in human tumor cells. Therefore, in this short communication we evaluate the role of four substrates (namely ATP, CTP, GTP, and UTP) on human HRI kinase activity. Additionally, for the first time, we perform a detailed kinetics study of the HRI G202S mutant, whose presence in the human lung is associated with cancer development. Here, the role of all four tested nucleoside triphosphates during cancer development is discussed from the point of view of the HRI activity. The results showed that the kcat value of GTP was lower than that of ATP but was significantly higher than those of CTP and UTP. Additionally, the kcat value of GTP for G202S was approximately 20% higher than that for wild-type, while the kcat values of ATP, CTP, and UTP for G202S were lower than those for wild-type

Is ATP the Only Nucleoside Triphosphate among ATP, CTP, GTP, and UTP to Have a Role in Kinase Catalysis of Heme-Regulated Inhibitor toward eIF2α during Lung Cancer Development?

The heme-regulated eukaryotic initiation factor 2α (eIF2α) kinase, also known as heme-regulated inhibitor (HRI), detects misfolded proteins and induces cytoprotective response to stress, mainly caused by heme-shortage. The nucleoside triphosphate ATP serves as the main donor of phosphate for the phosphorylation of eIF2α by HRI in human cells. However, the other main nucleoside triphosphates (CTP, GTP, UTP) are also present at relatively high concentrations, especially in human tumor cells. Therefore, in this short communication we evaluate the role of four substrates (namely ATP, CTP, GTP, and UTP) on human HRI kinase activity. Additionally, for the first time, we perform a detailed kinetics study of the HRI G202S mutant, whose presence in the human lung is associated with cancer development. Here, the role of all four tested nucleoside triphosphates during cancer development is discussed from the point of view of the HRI activity. The results showed that the kcat value of GTP was lower than that of ATP but was significantly higher than those of CTP and UTP. Additionally, the kcat value of GTP for G202S was approximately 20% higher than that for wild-type, while the kcat values of ATP, CTP, and UTP for G202S were lower than those for wild-type

Transcriptomic responses of bat cells to European bat lyssavirus 1 infection under conditions simulating euthermia and hibernation

Abstract Background Coevolution between pathogens and their hosts decreases host morbidity and mortality. Bats host and can tolerate viruses which can be lethal to other vertebrate orders, including humans. Bat adaptations to infection include localized immune response, early pathogen sensing, high interferon expression without pathogen stimulation, and regulated inflammatory response. The immune reaction is costly, and bats suppress high-cost metabolism during torpor. In the temperate zone, bats hibernate in winter, utilizing a specific behavioural adaptation to survive detrimental environmental conditions and lack of energy resources. Hibernation torpor involves major physiological changes that pose an additional challenge to bat-pathogen coexistence. Here, we compared bat cellular reaction to viral challenge under conditions simulating hibernation, evaluating the changes between torpor and euthermia. Results We infected the olfactory nerve-derived cell culture of Myotis myotis with an endemic bat pathogen, European bat lyssavirus 1 (EBLV-1). After infection, the bat cells were cultivated at two different temperatures, 37 °C and 5 °C, to examine the cell response during conditions simulating euthermia and torpor, respectively. The mRNA isolated from the cells was sequenced and analysed for differential gene expression attributable to the temperature and/or infection treatment. In conditions simulating euthermia, infected bat cells produce an excess signalling by multitude of pathways involved in apoptosis and immune regulation influencing proliferation of regulatory cell types which can, in synergy with other produced cytokines, contribute to viral tolerance. We found no up- or down-regulated genes expressed in infected cells cultivated at conditions simulating torpor compared to non-infected cells cultivated under the same conditions. When studying the reaction of uninfected cells to the temperature treatment, bat cells show an increased production of heat shock proteins (HSPs) with chaperone activity, improving the bat’s ability to repair molecular structures damaged due to the stress related to the temperature change. Conclusions The lack of bat cell reaction to infection in conditions simulating hibernation may contribute to the virus tolerance or persistence in bats. Together with the cell damage repair mechanisms induced in response to hibernation, the immune regulation may promote bats’ ability to act as reservoirs of zoonotic viruses such as lyssaviruses