Human immunodeficiency virus type 1 (HIV-1) vaccine immunogens capable of inducing broadly neutralizing antibodies (bNAbs) remain obscure. HIV-1 evades immune responses through enormous diversity and hides its conserved vulnerable epitopes on the envelope glycoprotein (Env) by displaying an extensive immunodominant glycan shield. In elite HIV-1 viremic controllers, glycan-dependent bNAbs targeting conserved Env epitopes have been isolated and are utilized as vaccine design templates. However, immunological tolerance mechanisms limit the development of these antibodies in the general population. The well characterized bNAbs monoclonal variants frequently exhibit extensive levels of somatic hypermutation, a long third heavy chain complementary determining region, or a short third light chain complementarity determining region, and some exhibit poly-reactivity to autoantigens. This review elaborates on the obstacles to engaging and manipulating the Env glycoprotein as an effective immunogen and describes an alternative reverse vaccinology approach to develop a novel category of bNAb-epitope-derived non-cognate immunogens for HIV-1 vaccine design.
- MeSH
- epitopy imunologie MeSH
- genové produkty env - virus lidské imunodeficience imunologie MeSH
- HIV infekce imunologie MeSH
- HIV protilátky * imunologie MeSH
- HIV-1 * imunologie MeSH
- lidé MeSH
- ligandy MeSH
- molekulární mimikry imunologie MeSH
- neutralizující protilátky * imunologie MeSH
- polysacharidy imunologie MeSH
- vakcíny proti AIDS * imunologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
BACKGROUND: Among the non-traditional antibacterial agents in development, only a few targets critical Gram-negative bacteria such as carbapenem-resistant Pseudomonas aeruginosa, Acinetobacter baumannii or cephalosporin-resistant Enterobacteriaceae. Endolysins and their genetically modified versions meet the World Health Organization criteria for innovation, have a novel mode of antibacterial action, no known bacterial cross-resistance, and are being intensively studied for application against Gram-negative pathogens. METHODS: The study presents a multidisciplinary approach, including genetic engineering of LysECD7-SMAP and production of recombinant endolysin, its analysis by crystal structure solution following molecular dynamics simulations and evaluation of antibacterial properties. Two types of antimicrobial dosage forms were formulated, resulting in lyophilized powder for injection and hydroxyethylcellulose gel for topical administration. Their efficacy was estimated in the treatment of sepsis, and pneumonia models in BALB/c mice, diabetes-associated wound infection in the leptin receptor-deficient db/db mice and infected burn wounds in rats. RESULTS: In this work, we investigate the application strategies of the engineered endolysin LysECD7-SMAP and its dosage forms evaluated in preclinical studies. The catalytic domain of the enzyme shares the conserved structure of endopeptidases containing a putative antimicrobial peptide at the C-terminus of polypeptide chain. The activity of endolysins has been demonstrated against a range of pathogens, such as Klebsiella pneumoniae, A. baumannii, P. aeruginosa, Staphylococcus haemolyticus, Achromobacter spp, Burkholderia cepacia complex and Haemophylus influenzae, including those with multidrug resistance. The efficacy of candidate dosage forms has been confirmed in in vivo studies. Some aspects of the interaction of LysECD7-SMAP with cell wall molecular targets are also discussed. CONCLUSIONS: Our studies demonstrate the potential of LysECD7-SMAP therapeutics for the systemic or topical treatment of infectious diseases caused by susceptible Gram-negative bacterial species and are critical to proceed LysECD7-SMAP-based antimicrobials trials to advanced stages.
- MeSH
- antibakteriální látky farmakologie aplikace a dávkování MeSH
- endopeptidasy * farmakologie aplikace a dávkování MeSH
- gramnegativní bakteriální infekce * farmakoterapie MeSH
- gramnegativní bakterie * účinky léků MeSH
- krysa rodu rattus MeSH
- myši inbrední BALB C * MeSH
- myši MeSH
- proteinové inženýrství metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The expression of aquaporin 4 (AQP4) and intermediate filament (IF) proteins is altered in malignant glioblastoma (GBM), yet the expression of the major IF-based cytolinker, plectin (PLEC), and its contribution to GBM migration and invasiveness, are unknown. Here, we assessed the contribution of plectin in affecting the distribution of plasmalemmal AQP4 aggregates, migratory properties, and regulation of cell volume in astrocytes. METHODS: In human GBM, the expression of glial fibrillary acidic protein (GFAP), AQP4 and PLEC transcripts was analyzed using publicly available datasets, and the colocalization of PLEC with AQP4 and with GFAP was determined by immunohistochemistry. We performed experiments on wild-type and plectin-deficient primary and immortalized mouse astrocytes, human astrocytes and permanent cell lines (U-251 MG and T98G) derived from a human malignant GBM. The expression of plectin isoforms in mouse astrocytes was assessed by quantitative real-time PCR. Transfection, immunolabeling and confocal microscopy were used to assess plectin-induced alterations in the distribution of the cytoskeleton, the influence of plectin and its isoforms on the abundance and size of plasmalemmal AQP4 aggregates, and the presence of plectin at the plasma membrane. The release of plectin from cells was measured by ELISA. The migration and dynamics of cell volume regulation of immortalized astrocytes were assessed by the wound-healing assay and calcein labeling, respectively. RESULTS: A positive correlation was found between plectin and AQP4 at the level of gene expression and protein localization in tumorous brain samples. Deficiency of plectin led to a decrease in the abundance and size of plasmalemmal AQP4 aggregates and altered distribution and bundling of the cytoskeleton. Astrocytes predominantly expressed P1c, P1e, and P1g plectin isoforms. The predominant plectin isoform associated with plasmalemmal AQP4 aggregates was P1c, which also affected the mobility of astrocytes most prominently. In the absence of plectin, the collective migration of astrocytes was impaired and the dynamics of cytoplasmic volume changes in peripheral cell regions decreased. Plectin's abundance on the plasma membrane surface and its release from cells were increased in the GBM cell lines. CONCLUSIONS: Plectin affects cellular properties that contribute to the pathology of GBM. The observed increase in both cell surface and released plectin levels represents a potential biomarker and therapeutic target in the diagnostics and treatment of GBMs.
BACKGROUND: Cholinergic hypothesis of Alzheimer's disease (AD) is based on the findings that a reduced and/or perturbed cholinergic activity in the central nervous system correlates with cognitive decline in patients with Alzheimer's disease. The hypothesis resulted in the development of centrally-acting agents potentiating cholinergic neurotransmission; these drugs, however, only slowed down the cognitive decline and could not prevent it. Consequently, the perturbation of the central cholinergic signalling has been accepted as a part of the Alzheimer's aetiology but not necessarily the primary cause of the disease. In the present study we have focused on the rs3810950 polymorphism of ChAT (choline acetyltransferase) gene that has not been studied in Czech population before. METHODS: We carried out an association study to test for a relationship between the rs3810950 polymorphism and Alzheimer's disease in a group of 1186 persons; 759 patients with Alzheimer's disease and 427 control subjects. Furthermore, we performed molecular modelling of the terminal domain (1st-126th amino acid residue) of one of the ChAT isoforms (M) to visualise in silico whether the rs3810950 polymorphism (A120T) can change any features of the tertiary structure of the protein which would have a potential to alter its function. RESULTS: The AA genotype of CHAT was associated with a 1.25 times higher risk of AD (p < 0.002) thus demonstrating that the rs3810950 polymorphism can have a modest but statistically significant effect on the risk of AD in the Czech population. Furthermore, the molecular modelling indicated that the polymorphism is likely to be associated with significant variations in the tertiary structure of the protein molecule which may impact its enzyme activity. CONCLUSIONS: Our findings are consistent with the results of the meta-analytical studies of the relationship between rs3810950 polymorphism and AD and provide further material evidence for a direct (primary) involvement of cholinergic mechanisms in the etiopathogenesis of AD, particularly as a factor in cognitive decline and perturbed conscious awareness commonly observed in patients with AD.
- MeSH
- Alzheimerova nemoc genetika MeSH
- cholin-O-acetyltransferasa genetika metabolismus MeSH
- genotyp MeSH
- jednonukleotidový polymorfismus * MeSH
- lidé MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
BACKGROUND: The brain stem contains important nuclei that control cardiovascular function via the sympathetic nervous system (SNS), which is strongly influenced by nitric oxide. Its biological activity is also largely determined by oxygen free radicals. Despite many experimental studies, the role of AT1R-NAD(P)H oxidase-superoxide pathway in NO-deficiency is not yet sufficiently clarified. We determined changes in free radical signaling and antioxidant and detoxification response in the brain stem of young and adult Wistar rats during chronic administration of exogenous NO inhibitors. METHODS: Young (4 weeks) and adult (10 weeks) Wistar rats were treated with 7-nitroindazole (7-NI group, 10 mg/kg/day), a specific nNOS inhibitor, with NG-nitro-L-arginine-methyl ester (L-NAME group, 50 mg/kg/day), a nonspecific NOS inhibitor, and with drinking water (Control group) during 6 weeks. Systolic blood pressure was measured by non-invasive plethysmography. Expression of genes (AT1R, AT2R, p22phox, SOD and NOS isoforms, HO-1, MDR1a, housekeeper GAPDH) was identified by real-time PCR. NOS activity was detected by conversion of [3H]-L-arginine to [3H]-L-citrulline and SOD activity was measured using UV VIS spectroscopy. RESULTS: We observed a blood pressure elevation and decrease in NOS activity only after L-NAME application in both age groups. Gene expression of nNOS (youngs) and eNOS (adults) in the brain stem decreased after both inhibitors. The radical signaling pathway triggered by AT1R and p22phox was elevated in L-NAME adults, but not in young rats. Moreover, L-NAME-induced NOS inhibition increased antioxidant response, as indicated by the observed elevation of mRNA SOD3, HO-1, AT2R and MDR1a in adult rats. 7-NI did not have a significant effect on AT1R-NADPH oxidase-superoxide pathway, yet it affected antioxidant response of mRNA expression of SOD1 and stimulated total activity of SOD in young rats and mRNA expression of AT2R in adult rats. CONCLUSION: Our results show that chronic NOS inhibition by two different NOS inhibitors has age-dependent effect on radical signaling and antioxidant/detoxificant response in Wistar rats. While 7-NI had neuroprotective effect in the brain stem of young Wistar rats, L-NAME- induced NOS inhibition evoked activation of AT1R-NAD(P)H oxidase pathway in adult Wistar rats. Triggering of the radical pathway was followed by activation of protective compensation mechanism at the gene expression level.
- MeSH
- antioxidancia metabolismus MeSH
- indazoly farmakologie MeSH
- inhibitory enzymů farmakologie MeSH
- krysa rodu rattus MeSH
- metabolická inaktivace * MeSH
- mozkový kmen účinky léků metabolismus MeSH
- NG-nitroargininmethylester farmakologie MeSH
- oxid dusnatý nedostatek MeSH
- potkani Wistar MeSH
- synthasa oxidu dusnatého antagonisté a inhibitory MeSH
- věkové faktory MeSH
- volné radikály metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Recent studies reported the critical role of the intrauterine environment of a fetus in growth or the development of disease in adulthood. In this article we discussed the implications of salt restriction in growth of a fetus and the development of growth-related disease in adulthood. Salt restriction causes retardation of fatal growth or intrauterine death thereby leading to low birth weight or decreased birth rate. Such retardation of growth along with the upregulation of the renin angiotensin system due to salt restriction results in the underdevelopment of cardiovascular organs or decreases the number of the nephron in the kidney and is responsible for onset of hypertension in adulthood. In addition, gestational salt restriction is associated with salt craving after weaning. Moreover, salt restriction is associated with a decrease in insulin sensitivity. A series of alterations in metabolism due to salt restriction are probably mediated by the upregulation of the renin angiotensin system and an epigenetic mechanism including proinflammatory substances or histone methylation. Part of the metabolic disease in adulthood may be programmed through such epigenetic changes. The modification of gene in a fetus may be switched on through environment factors or life style after birth. The benefits of salt restriction have been assumed thus far; however, more precise investigation is required of its influence on the health of fetuses and the onset of various diseases in adulthood.
- MeSH
- epigeneze genetická * MeSH
- kardiovaskulární systém embryologie patologie MeSH
- kuchyňská sůl * MeSH
- lidé MeSH
- renin-angiotensin systém * MeSH
- růstová retardace plodu metabolismus patologie MeSH
- těhotenství MeSH
- vývojová regulace genové exprese * MeSH
- zpožděný efekt prenatální expozice metabolismus patologie MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
BACKGROUND: Morphine is used in clinical practice as a highly effective painkiller as well as the drug of choice for treatment of certain heart diseases. However, there is lack of information about its effect on protein expression in the heart. Therefore, here we aimed to identify the presumed alterations in rat myocardial protein levels after prolonged morphine treatment. METHODS: Morphine was administered to adult male Wistar rats in high doses (10 mg/kg per day) for 10 days. Proteins from the plasma membrane- and mitochondria-enriched fractions or cytosolic proteins isolated from left ventricles were run on 2D gel electrophoresis, scanned and quantified with specific software to reveal differentially expressed proteins. RESULTS: Nine proteins were found to show markedly altered expression levels in samples from morphine-treaded rats and these proteins were identified by mass spectrometric analysis. They belong to different cell pathways including signaling, cytoprotective, and structural elements. CONCLUSIONS: The present identification of several important myocardial proteins altered by prolonged morphine treatment points to global effects of this drug on heart tissue. These findings represent an initial step toward a more complex view on the action of morphine on the heart.
- MeSH
- krysa rodu rattus MeSH
- morfin aplikace a dávkování farmakologie MeSH
- myokard metabolismus MeSH
- opioidní analgetika aplikace a dávkování farmakologie MeSH
- potkani Wistar MeSH
- proteiny metabolismus MeSH
- proteomika MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
During apoptosis several mitochondrial proteins are released. Some of them participate in caspase-independent nuclear DNA degradation, especially apoptosis-inducing factor (AIF) and endonuclease G (endoG). Another interesting protein, which was expected to act similarly as AIF due to the high sequence homology with AIF is AIF-homologous mitochondrion-associated inducer of death (AMID). We studied the structure, cellular localization, and interactions of several proteins in silico and also in cells using fluorescent microscopy. We found the AMID protein to be cytoplasmic, most probably incorporated into the cytoplasmic side of the lipid membranes. Bioinformatic predictions were conducted to analyze the interactions of the studied proteins with each other and with other possible partners. We conducted molecular modeling of proteins with unknown 3D structures. These models were then refined by MolProbity server and employed in molecular docking simulations of interactions. Our results show data acquired using a combination of modern in silico methods and image analysis to understand the localization, interactions and functions of proteins AMID, AIF, endonuclease G, and other apoptosis-related proteins.
- MeSH
- apoptóza MeSH
- biologické modely MeSH
- endonukleasy MeSH
- fluorescenční mikroskopie metody MeSH
- kaspasy metabolismus MeSH
- konformace proteinů MeSH
- lidé MeSH
- molekulární modely MeSH
- nádorové buněčné linie MeSH
- počítačová simulace MeSH
- proteomika metody MeSH
- software MeSH
- terciární struktura proteinů MeSH
- vazba proteinů MeSH
- výpočetní biologie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Acyclic nucleoside phosphonates are potent antiviral agents effective against replication of DNA viruses and retroviruses including human immunodeficiency virus (HIV). In addition to their antimetabolic mode of antiviral action, acyclic nucleoside phosphonates also possess immunomodulatory properties. We have shown recently that a number of them stimulate secretion of cytokines including chemokines RANTES/CCL5 ("regulated upon activation, normal T cell expressed and secreted") and MIP-1 alpha/CCL3 (macrophage inflammatory protein-1 alpha) that may inhibit entry of HIV in cells. In present experiments we analyzed effects of acyclic nucleoside phosphonates on gene expression of other members of the beta family of chemokines, monocyte chemotactic proteins (MCPs), which have also been implicated in the control of HIV infection. The following compounds differing at the type of heterocyclic base, i.e. adenine (A), or 2,6-diaminopurine (DAP), at the 6-amino group of the base, and at the N ( 9 )-side chain represented by 9-[2-(phosphonomethoxy)ethyl] (PME) and 9-[2-(phosphonomethoxy)propyl] (PMP) moieties were included in the study: (1) (R)-PMPA, ie. tenofovir, (2) N ( 6 )-cyclopropyl-(R)-PMPDAP, (3) N ( 6 )-cyclopentyl-(R)-PMPDAP, (4) N ( 6 )-dimethylaminoethyl-(R)-PMPDAP, (5) N ( 6 )-cyclopentyl-PMEDAP, (6) N ( 6 )-isobutyl-PMEDAP, (7) N ( 6 ) -cyclohexylmetyl-PMEDAP, and (8) N ( 6 ) -cyclooctyl-PMEDAP. These compounds are able to activate production of MCP-1 and MCP-3, and none of them influences gene expression of MCP-2, and MCP-5. Enhancement of monocyte chemotactic protein expression was found to be mediated by transcriptional factor nuclear factor-kappaB (NF-kappaB).
- MeSH
- chemokin CCL2 biosyntéza imunologie MeSH
- HIV infekce farmakoterapie imunologie metabolismus MeSH
- HIV imunologie metabolismus MeSH
- karboxypeptidasy A biosyntéza imunologie MeSH
- kultivované buňky MeSH
- látky proti HIV farmakologie MeSH
- lidé MeSH
- myši MeSH
- nukleosidy farmakologie MeSH
- peritoneální makrofágy imunologie metabolismus MeSH
- replikace viru účinky léků účinky záření MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
Interference of antiviral agent adefovir, i.e. 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) with microsomal drug metabolizing system was investigated in rats. The content of total liver cytochrome P450 (CYP) was lowered while that of its denaturated form, P420, was elevated in animals intraperitoneally treated with PMEA (25 mg/kg). Similar effect was observed after treatment with a prodrug of adevofir, adefovir dipivoxil (bisPOM-PMEA). The CYP2E1-dependent formation of 4-nitrocatechol from p-nitrophenol was diminished, though the specific activity of p-nitrophenol hydroxylase remained unchanged. PMEA had no influence on expression of CYP2E1 protein and mRNA and mRNAs of other P450 isoenzymes (1A1, 1A2, 2C11, 3A1, 3A2, and 4A1). It may be concluded that repeated systemic administration of higher doses of PMEA results in a partial degradation of rat CYP protein to inactive P420.
- MeSH
- adenin analogy a deriváty farmakologie chemie MeSH
- antivirové látky farmakologie MeSH
- financování organizované MeSH
- jaterní mikrozomy účinky léků MeSH
- katecholy farmakologie MeSH
- krysa rodu rattus MeSH
- messenger RNA metabolismus MeSH
- nitrofenoly chemie MeSH
- organofosfonáty farmakologie chemie MeSH
- potkani inbrední LEW MeSH
- systém (enzymů) cytochromů P-450 chemie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- ženské pohlaví MeSH
- zvířata MeSH
