Avian eggs contend with omnipresent microorganisms entering the egg interior, where they affect embryo viability and hatchling phenotype. The incubation behaviour and deposition of egg white antimicrobial proteins (AMPs) vary highly across the avian altricial-precocial spectrum. Experimental evidence of how these alterations in avian reproductive strategies affect the antimicrobial properties of the precocial and altricial egg interior is lacking, however. Here, we tested the egg white antimicrobial activity in eggs of two representative model species, from each end of the avian altricial-precocial spectrum, against potentially pathogenic and beneficial probiotic microorganisms. Eggs were experimentally treated to mimic un-incubated eggs in the nest, partial incubation during the egg-laying period, the onset of full incubation and the increased deposition of two main egg white AMPs, lysozyme and ovotransferrin. We moreover assessed to what extent egg antimicrobial components, egg white pH and AMP concentrations varied as a result of different incubation patterns. Fully incubated precocial and altricial eggs decreased their antimicrobial activity against a potentially pathogenic microorganism, whereas partial incubation significantly enhanced the persistence of a beneficial probiotic microorganism in precocial eggs. These effects were most probably conditioned by temperature-dependent alterations in egg white pH and AMP concentrations. While lysozyme concentration and pH decreased in fully incubated precocial but not altricial eggs, egg white ovotransferrin increased along with the intensity of incubation in both precocial and altricial eggs. This study is the first to experimentally demonstrate that different incubation patterns may have selective antimicrobial potential mediated by species-specific effects on antimicrobial components in the egg white.
- MeSH
- Anti-Infective Agents pharmacology MeSH
- Bacillus subtilis drug effects MeSH
- Columbidae physiology MeSH
- Coturnix physiology MeSH
- Micrococcus luteus drug effects MeSH
- Muramidase pharmacology MeSH
- Conalbumin pharmacology MeSH
- Ovum enzymology physiology MeSH
- Avian Proteins pharmacology MeSH
- Reproduction * MeSH
- Egg White chemistry MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Capillary electrophoresis integrated immobilized enzyme reactors are becoming an increasingly popular alternative for enzyme kinetic and inhibition assays thanks to their unique set of features including cost effectiveness, repeated use of the enzyme, minuscule sample consumption, rapid analysis time and easy automation. In this work we present the development and application of a capillary electrophoresis integrated immobilized enzyme reactor based on magnetic particles for kinetic and inhibition studies of β-secretase, a key enzyme in the development of Alzheimer's disease and a promising drug target. We document the optimization of the immobilization procedure, characterization of immobilized β-secretase, optimization of a mutually compatible incubation protocol and separation method as well as the production of the capillary electrophoresis integrated immobilized enzyme reactor. The applicability of the capillary electrophoresis integrated immobilized enzyme reactor was demonstrated by kinetic assay with an unlabelled substrate and by inhibition assays using three structurally different reference inhibitors. The resulting kinetic and inhibition parameters clearly support the applicability of the herein presented method as well as document the fundamental phenomena which need to be taken in account when comparing the results to other methods.
- MeSH
- Alzheimer Disease drug therapy metabolism MeSH
- Bioreactors * MeSH
- Electrophoresis, Capillary MeSH
- Enzymes, Immobilized antagonists & inhibitors chemistry metabolism MeSH
- HEK293 Cells MeSH
- Enzyme Inhibitors chemistry pharmacology MeSH
- Kinetics MeSH
- Humans MeSH
- Peptides chemistry pharmacology MeSH
- Amyloid Precursor Protein Secretases antagonists & inhibitors chemistry metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Transmissible spongiform encephalopathies are a group of fatal neurodegenerative diseases with long incubation time. This group includes Creutzfeld-Jakob disease, kuru, scrapie, chronic wasting disease, and bovine spongiform encephalopathy. Sensitive and specific detection of abnormal prion protein as "a source agent" of the above-mentioned diseases in blood could provide a diagnostic test or a screening assay for animal and human prion protein diseases diagnostics. Therefore, diagnostic tests for prion protein diseases represent unique challenge requiring development of novel assays exploiting properties of prion protein complex. Presently, diagnostic methods such as protein misfolding cyclic amplification, conformation-dependent immunoassay, dissociation-enhanced lanthanide fluorescent immunoassay, fluorescence correlation spectroscopy, and/or flow microbead immunoassay are used for abnormal prion protein (PrP(Sc) ) detection. On the other hand, using of CE for PrP(Sc) detection in body fluids is an attractive alternative; it has been already applied for the blood samples of infected sheep, elk, chimpanzee, as well as humans. In this review, assays for prion protein detection are summarized with special attention to capillary electromigration based techniques, such as CE, CIEF, and/or CGE. The potential of the miniaturized and integrated lab-on-chip devices is highlighted, emphasizing recent advances of this field in the proteomic analysis.
- MeSH
- Alzheimer Disease diagnosis metabolism MeSH
- Electrophoresis, Capillary methods MeSH
- Humans MeSH
- Prion Diseases diagnosis MeSH
- Prions analysis chemistry metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
The objective of this study was to elucidate the pharmacokinetics and metabolite formation of newly developed non-selective AChE/BChE 7-MEOTA-donepezil-like inhibitors for potential therapeutic use in Alzheimer's disease (AD) patients. The chemical structures of metabolites were defined during incubation with human liver microsomes, and subsequently, the metabolization was verified in in vivo study. In vitro metabolic profiling revealed the formation of nine major metabolites in the case of PC-37 and eight metabolites of PC-48. Hydroxylation and the enzymatic hydrolysis of bonds close to the piperazine ring appeared to be the principal metabolic pathways in vitro. Of these metabolites, M1-M7 of PC-37 and M1-M6 of PC-48 were confirmed under in vivo conditions. Pilot pharmacokinetic experiments in rats were focused on the absorption, distribution and elimination of these compounds. Absorption after i.m. application was relatively fast; the bioavailability expressed as AUCtotal was 28179 ± 4691 min.ng/mL for PC-37 and 23374 ± 4045 min.ng/mL for PC-48. Both compounds showed ability to target the central nervous system, with brain concentrations exceeding those in plasma. The maximal brain concentrations are approximately two times higher than the plasma concentrations. The relatively high brain concentrations persisted throughout the experiment until 24 hr after application. Elimination via the kidneys (urine) significantly exceeded elimination via the liver (bile). All these characteristics are crucial for new candidates intended for AD treatment. The principle metabolic pathways that were verified in the in vivo study do not show any evidence for formation of extremely toxic metabolites, but this needs to be confirmed by further studies.
- MeSH
- Alzheimer Disease drug therapy MeSH
- Biological Availability MeSH
- Cholinesterase Inhibitors chemical synthesis metabolism pharmacokinetics therapeutic use MeSH
- Hydrolysis MeSH
- Hydroxylation MeSH
- Microsomes, Liver metabolism MeSH
- Rats MeSH
- Humans MeSH
- Metabolic Networks and Pathways MeSH
- Brain drug effects MeSH
- Pilot Projects MeSH
- Piperazines chemical synthesis metabolism pharmacokinetics therapeutic use MeSH
- Rats, Wistar MeSH
- Tacrine analogs & derivatives chemical synthesis metabolism pharmacokinetics therapeutic use MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Huntington disease (HD) is a fatal neurodegenerative disorder involving reduced muscle coordination, mental and behavioral changes, and testicular degeneration. In order to further clarify the decreased fertility and penetration ability of the spermatozoa of transgenic HD minipig boars (TgHD), we applied a set of mitochondrial metabolism (MM) parameter measurements to this promising biological material, which can be collected noninvasively in longitudinal studies. OBJECTIVE: We aimed to optimize methods for MM measurements in spermatozoa and to establish possible biomarkers of HD in TgHD spermatozoa expressing the N-terminal part of mutated human huntingtin. METHODS: Semen samples from 12 TgHD and wild-type animals, aged 12-65 months, were obtained repeatedly during the study. Respiration was measured by polarography, MM was assessed by the detection of oxidation of radiolabeled substrates (mitochondrial energy-generating system; MEGS), and the content of the oxidative phosphorylation system subunits was detected by Western blot. Three possibly interfering factors were statistically analyzed: the effect of HD, generation and aging. RESULTS: We found 5 MM parameters which were significantly diminished in TgHD spermatozoa and propose 3 specific MEGS incubations and complex I-dependent respiration as potential biomarkers of HD in TgHD spermatozoa. CONCLUSIONS: Our results suggest a link between the gain of toxic function of mutated huntingtin in TgHD spermatozoa and the observed MM and/or glycolytic impairment. We determined 4 biomarkers useful for HD phenotyping and experimental therapy monitoring studies in TgHD minipigs.
- MeSH
- Respiration MeSH
- Animals, Genetically Modified MeSH
- Huntington Disease complications genetics pathology MeSH
- Tricarboxylic Acids metabolism MeSH
- Humans MeSH
- Swine, Miniature MeSH
- Mitochondrial Proteins metabolism MeSH
- Mitochondria metabolism MeSH
- Mutation genetics MeSH
- Oxidative Phosphorylation MeSH
- Swine MeSH
- Huntingtin Protein genetics MeSH
- Pyruvate Dehydrogenase Complex metabolism MeSH
- Semen metabolism MeSH
- Spermatozoa metabolism pathology MeSH
- Trinucleotide Repeats genetics MeSH
- Age Factors MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
We evaluated the effects of exercise on the vascular constrictor responses to ?-adrenergic stimulation in the db/db mice. Twenty male db/db and their age-matched wild-type (WT) mice were exercised (1 hour/day, five days a week). Mice were anesthetized 7 weeks later, thoracic aortae were mounted in wire myograph and constrictor responses to phenylephrine (PE, 1 nM-10 µM) were obtained. Citrate synthase activity measured in the thigh adductor muscle was significantly increased in db/db mice that were exercise trained. Maximal force generated by PE was markedly greater in db/db aortae and exercise did not attenuate this augmented contractile response. Vessels were incubated with inhibitors of nitric oxide synthase (L-NAME, 200 µM), endothelin receptors (bosentan, 10 µM), protein kinase C (PKC) (calphostin C, 5 µM), cyclooxygenase (indomethacin, 10 µM) or Rho-kinase (Y-27632, 0.1 µM). Only calphostin-C normalized the augmented PE-induced constriction in db/db and db/dbexercised mice to that observed in WT (p<0.05). Cumulative additions of indolactam, a PKC activator, induced significantly greater constrictor responses in aortic rings of db/db mice compared to WT and exercise did not affect this response. Our data suggest that the augmented vasoconstriction observed in the aorta of db/db mice is likely due to increased PKC activity and that exercise do not ameliorate this increased PKC-mediated vasoconstriction.
- Keywords
- Exercise, Vasoconstriction, Diabetes,
- MeSH
- Enzyme Activation MeSH
- Enzyme Activators pharmacology MeSH
- Endothelin A Receptor Antagonists MeSH
- Aorta, Thoracic enzymology physiopathology drug effects MeSH
- Citrate (si)-Synthase metabolism MeSH
- Diabetes Mellitus, Type 2 enzymology physiopathology MeSH
- Financing, Organized MeSH
- Cyclooxygenase Inhibitors pharmacology MeSH
- Protein Kinase Inhibitors pharmacology MeSH
- Insulin blood MeSH
- rho-Associated Kinases antagonists & inhibitors metabolism MeSH
- Muscle, Skeletal enzymology MeSH
- Blood Glucose metabolism MeSH
- Lipids blood MeSH
- Disease Models, Animal MeSH
- Mice MeSH
- Protein Kinase C antagonists & inhibitors metabolism MeSH
- Receptor, Endothelin A metabolism MeSH
- Nitric Oxide Synthase antagonists & inhibitors metabolism MeSH
- Body Weight MeSH
- Physical Exertion MeSH
- Vasodilation MeSH
- Vasodilator Agents pharmacology MeSH
- Vasoconstriction drug effects MeSH
- Vasoconstrictor Agents pharmacology MeSH
- Dose-Response Relationship, Drug MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Animals MeSH
Parkinson's disease (PD) is associated with the formation and deposition of amyloid fibrils of the protein alpha-synuclein (AS). It has been proposed that oligomeric intermediates on the pathway to fibrilization rather than the fibrils themselves are the pathogenic agents of PD, but efficient methods for their detection are lacking. We have studied the interfacial properties of wild-type AS and the course of its aggregation in vitro using electrochemical analysis and dynamic light scattering. The oxidation signals of tyrosine residues of AS at carbon electrodes and the ability of fibrils to adsorb and catalyze hydrogen evolution at hanging mercury drop electrodes (HMDEs) decreased during incubation. HMDEs were particularly sensitive to pre-aggregation changes in AS. Already after 1 h of a standard aggregation assay in vitro (stirring at 37 degrees C), the electrocatalytic peak H increased greatly and shifted to less negative potentials. Between 3 and 9 h of incubation, an interval during which dynamic light scattering indicated AS oligomerization, peak H diminished and shifted to more negative potentials, and AS adsorbability decreased. We tentatively attribute the very early changes in the interfacial behavior of the protein after the first few hours of incubation to protein destabilization with disruption of long-range interactions. The subsequent changes can be related to the onset of oligomerization. Our results demonstrate the utility of electrochemical methods as new and simple tools for the investigation of amyloid formation.
OBJECTIVE: Currently, it is thought that uterine cervix mucosal samples present a low risk of SARS-CoV-2 exposure. So far, there is no evidence of SARS-CoV-2 detection in Papanicolaou (Pap) smears. Nevertheless, clinicians could be exposed unaware to the coronavirus while performing and handling a Pap smear. We aimed to retrospectively evaluate the presence of SARS-CoV-2 RNA in cervical liquid-based cytology (LBC) samples in women who tested positive for a nasopharyngeal COVID-19 PCR test. METHODS: From our laboratory database, we identified patients with data on a cervical cancer screening LBC sample paired with a positive nasopharyngeal COVID-19 PCR test. Relevant LBC samples taken within an incubation period of 14 days and post-onset RNA shedding interval of 25 days were subsequently tested for SARS-CoV-2 RNA using RT-PCR tests. RESULTS: The study group consisted of 102 women. Of those, 23 LBC samples were tested. SARS-CoV-2 RNA was detected in one LBC sample from a 26-year-old asymptomatic woman taken six days before reporting headaches and knee arthralgia with a positive nasopharyngeal SARS-CoV-2 RT-PCR test. CONCLUSIONS: It is possible to detect SARS-CoV-2 RNA in cervical LBC samples at an early asymptomatic stage of COVID-19. In general, this finding is infrequent in asymptomatic women who tested SARS-CoV-2 positive within an incubation of 14 days and a post-onset RNA shedding period of 25 days. We fully support the current thinking that cervical LBC samples from asymptomatic women pose a low risk of SARS-CoV-2 exposure and can be handled in the frame of good microbiological practice and procedures.
- MeSH
- COVID-19 * diagnosis genetics metabolism MeSH
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Uterine Cervical Neoplasms diagnosis genetics metabolism virology MeSH
- Papanicolaou Test * MeSH
- Retrospective Studies MeSH
- SARS-CoV-2 * genetics metabolism MeSH
- COVID-19 Nucleic Acid Testing * MeSH
- Vaginal Smears * MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Je uvedeno obecné schéma patogeneze a klinický průběh onemocnění vzteklinou u zvířat. Inkubační doba je u různých druhů v průměru 3 až 8 týdnů. Klinicky se rozlišuje lichá a zuřivá forma a vlastní průběh má stádium prodromální, excitační a paralytické. Vylučování viru ve slině nemocných zvířat představuje hlavní riziko a může k němu docházet již několik dní před nástupem klinických příznaků. Onemocnění zvířat vzteklinou je neléčitelné.
The general schema of pathogenesis together with the clinical course of rabies in animals is given. The incubation period differs from 3 to 8 weeks according to the animal genus. There are two formo f dinase (furious and paralytic) and the course usually has a prodromal, excitation and paralytic stages. The secretion of the virus into the saliva of ill animals represents the main risk and it can occur already several days before the onset of clinical symptom manifestation. Rabies is incurable in animals.
- MeSH
- Animal Diseases MeSH
- Pathological Conditions, Signs and Symptoms MeSH
- Vaccination veterinary MeSH
- Rabies physiopathology therapy veterinary MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Review MeSH
Familial occurrence of three definitive and two possible cases of Creutzfeldt-Jakob disease (CJD) with temporal and spatial separation in the area of focal CJD accumulation in Slovakia is reported. Incubation period is 51 and 53 years respectively, if spatial and temporal separation of affected siblings is considered, and 51 years when the time interval between the death of the affected mother and the clinical onset in the first affected child is determined. Affected children tend to die at the same time (mean difference 3.3 years) and not at the same age (mean difference 6 years). Due to separation of the affected children, a possible common exposure to CJD infection was limited to approximately seven years during their childhood. Potential endo- and exogenous risk factors and a possible mode of CJD transmission in the described family, as well as in the CJD focus, is discussed.
- MeSH
- Creutzfeldt-Jakob Syndrome epidemiology etiology genetics transmission MeSH
- Middle Aged MeSH
- Humans MeSH
- Cerebral Cortex pathology MeSH
- Family MeSH
- Aged MeSH
- Animals MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Case Reports MeSH
- Geographicals
- Czechoslovakia MeSH
