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1.
Biochemistry (Mosc) ; 84(3): 310-320, 2019 Mar.
Article in English | MEDLINE | ID: mdl-31221069

ABSTRACT

Ionotropic glutamate and GABA receptors regulate the differentiation and determine the functional properties of mature neurons. Both insufficient and excessive activity of these neurotransmission systems are associated with various nervous system diseases. Our knowledge regarding the expression profiles of these receptors and the mechanisms of their regulation during the differentiation of specialized human neuron subtypes is limited. Here the expression profiles of the NMDA and GABAA receptor subunits were explored during in vitro differentiation of human induced pluripotent stem cells (iPSCs) into ventral mesencephalic neurons. The correlation between the neuronal maturation and the expression dynamics of these genes was investigated, and the functional activity of these receptors was assessed by calcium imaging. The role of NMDA and GABAA receptors in neurite outgrowth and the development of spontaneous activity was analyzed using the viral transduction of neural progenitors with the reporter genes TagGFP and TagRFP. The data indicate that agonists of the investigated receptors can be employed for optimization of existing protocols for neural differentiation of iPSCs, in particular for acceleration of neuronal maturation.


Subject(s)
Induced Pluripotent Stem Cells/metabolism , Mesencephalon/metabolism , Neurons/metabolism , Receptors, GABA-A/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Cell Differentiation , Cells, Cultured , Humans , Induced Pluripotent Stem Cells/cytology , Mesencephalon/cytology , Neurons/cytology , Receptors, GABA-A/genetics , Receptors, N-Methyl-D-Aspartate/genetics
2.
Bull Exp Biol Med ; 162(5): 679-683, 2017 Mar.
Article in English | MEDLINE | ID: mdl-28364186

ABSTRACT

The cholinergic, GABAergic, and catecholaminergic neurons derived from mouse embryonic stem cells in a culture medium supplemented with recombinant NGF were phenotyped and scored. NGF stimulated generation of neurons, but not neuronal progenitors from embryonic stem cells and affected the proportion of specific types of neurons in cultures of differentiating embryonic stem cells. These findings open vista to employ NGF for generation of specific neuron types from embryonic stem cells for fundamental and toxicological studies.


Subject(s)
Mouse Embryonic Stem Cells/physiology , Nerve Growth Factor/physiology , Animals , Cell Proliferation , Cells, Cultured , Fibroblast Growth Factor 2/physiology , Humans , Mice , Nerve Growth Factor/pharmacology , Neurogenesis
3.
Dokl Biol Sci ; 470(1): 244-246, 2016 Sep.
Article in English | MEDLINE | ID: mdl-27822752

ABSTRACT

The influence of GABA receptor agonists on the terminal differentiation in vitro of dopaminergic (DA) neurons derived from IPS cells was investigated. GABA-A agonist muscimol induced transient elevation of intracellular Ca2+ level ([Ca2+] i ) in the investigated cells at days 5 to 21 of differentiation. Differentiation of cells in the presence of muscimol reduced tyrosine hydroxylase expression. Thus, the presence of active GABA-A receptors, associated with phenotype determination via Ca2+-signalling was demonstrated in differentiating human DA neurons.


Subject(s)
Dopaminergic Neurons/cytology , Dopaminergic Neurons/metabolism , GABA Agonists/administration & dosage , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Receptors, GABA-A/metabolism , Baclofen/administration & dosage , Calcium/metabolism , Calcium Signaling/drug effects , Calcium Signaling/physiology , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cells, Cultured , Dopaminergic Neurons/drug effects , Humans , Induced Pluripotent Stem Cells/drug effects , Muscimol/administration & dosage
4.
Bull Exp Biol Med ; 151(5): 615-8, 2011 Sep.
Article in English | MEDLINE | ID: mdl-22462059

ABSTRACT

Immunogenic characteristics of filaggrin protein molecule as an antigen for antibodies to filaggrin, markers of early rheumatoid arthritis, were studied. Two new peptide motives, possible epitopes for antibodies to filaggrin, were shown in the filaggrin molecule by predictive analysis using programmed algorithms. Only IMG-3 and its cyclic form IMG-4 exhibited antigenic reactivity with sera from rheumatoid arthritis patients, differing significantly from the reactivity with donor sera. The immunogenic characteristics of IMG-3 differed from the characteristics of a previously described epitope.


Subject(s)
Arthritis, Rheumatoid/immunology , Epitopes/immunology , Intermediate Filament Proteins/immunology , Peptide Fragments/immunology , Amino Acid Motifs/immunology , Amino Acid Sequence , Arthritis, Rheumatoid/blood , Case-Control Studies , Epitope Mapping , Epitopes/chemistry , Fibrin/chemistry , Filaggrin Proteins , Humans , Intermediate Filament Proteins/chemistry , Molecular Sequence Data , Peptide Fragments/chemistry , Protein Binding , Sequence Analysis, Protein , Sequence Homology, Amino Acid
5.
Int J Artif Organs ; 14(8): 508-14, 1991 Aug.
Article in English | MEDLINE | ID: mdl-1937940

ABSTRACT

We have developed an immunoadsorbent (IA) for ex vivo removal of IgE after in vitro screening of matrix (Sepharose and tresyl-activated Toyopearl) and ligand (monospecific rabbit polyclonal anti-IgE antiserum and monoclonal antibodies (Abs) or their Fab fragments). Specific adsorptive capacity (SAC) for IgE was maximal in Sepharose-based IA with both types of Abs. Fab-containing IA on Sepharose retained 70-90% of the SAC of native Ab-containing IA. Toyopearl-based IA showed comparable SAC under static conditions but worked unsatisfactorily under continuous flow conditions. To assess the complement-activating capacity (CAC) of IA in vitro anaphylatoxin (C3a, C4a, C5a) generation was applied. CAC was directly related with the amount of immobilized Ab ligand, without depending on Ab specific activity. Fab-containing IA showed more CAC than native Ab-containing IA, and polyclonal IA more than monoclonal IA. Therefore, IA for IgE apheresis were prepared from native monoclonal Abs and CNBr-activated Sepharose CL 4B under aseptic conditions and packed into a glass column. This IA was used in 17 clinical IgE apheresis treatments of five atopic asthma patients. No substantial side effects were observed; in vivo IA effectively removed IgE from plasma (83 to 98%).


Subject(s)
Asthma/therapy , Immunoglobulin E , Immunosorbent Techniques , Immunosorbents , Plasma , Polymers , Adult , Asthma/immunology , Blood Component Removal/methods , Humans , Male , Perfusion
6.
Int J Neurosci ; 47(3-4): 217-30, 1989 Aug.
Article in English | MEDLINE | ID: mdl-2553634

ABSTRACT

The extract of left (LVC) and right (RVC) turtle visual cortex locally applied to the surface of turtle visual cortex inhibited the orthodromic evoked potentials (EP; fast negative component N1). The inhibition was partially (70-90%) prevented by opiate antagonist naloxone. LVC-extract proved to be a more potent inhibitor of the left cortex EP, whereas RVC-extract was slightly more effective when applied to the right cortex. The extracts of the left and right hemispheres of rat brain, but not the extract of the whole brain, applied subarachnoidally into the caudal portion of a transected spinal cord at the T7-T9 level, induce postural asymmetry of hind limbs in rats. The extract of the left hemisphere predominantly induce flexion of the left leg, and the extract of the right hemisphere induce flexion of the right leg. The factors of the right brain hemisphere able to induce postural asymmetry (PAFs) have been studied in detail. PAFs are thermostable compounds inactivated by trypsin and papain. Naloxone significantly reduces the number of asymmetries. HPLC analysis of the rat and pig right hemisphere extract has revealed two PAFs which differ from enkephalins, endorphins and dynorphin. The activity of PAFs in rat pituitary is significantly lower than in the brain. These findings suggest the existence of side-specific mechanism for selective neurohormonal regulation of neuronal activity and other processes in the left and right halves of the brain which involve lateralized factors of turtle visual cortex and PAFs.


Subject(s)
Functional Laterality/physiology , Muscles/innervation , Receptors, Cell Surface/physiology , Spinal Cord/physiology , Tissue Extracts/pharmacology , Visual Cortex/metabolism , Animals , Evoked Potentials, Visual/drug effects , Male , Muscles/drug effects , Muscles/physiology , Naloxone/pharmacology , Posture , Rats , Rats, Inbred Strains , Receptors, Cell Surface/metabolism , Receptors, Cell Surface/pharmacology , Spinal Cord/drug effects , Turtles , Visual Cortex/drug effects , Visual Cortex/physiology
7.
Brain Res ; 480(1-2): 277-89, 1989 Feb 20.
Article in English | MEDLINE | ID: mdl-2540878

ABSTRACT

The kappa-agonists bremazocine and dynorphin(1-13), the sigma-agonist SKF 10.047 as well as the delta-agonists [D-Ala2,D-Leu5]-enkephalin (DADL) and Met-enkephalin, but not the mu-agonist morphine, applied subarachnoidally to the caudal portion of the transected spinal cord (at the T3-T4 level) induced postural asymmetry of the hind limbs in rats. Asymmetry was registered visually. The leg was regarded as flexed if its projection on the longitudinal axis of the animal was smaller than that of its counterpart. The side of the flexed leg depended upon the type of drug: bremazocine, dynorphin(1-13) and Met-enkephalin predominantly induced flexion of the right leg, SKF 10.047 induced flexion of the left leg (at some doses there is no side preference), while in the case of DADL the side of the flexed leg depended upon the dose of the drug. Comparison of electromyographic activity of the symmetric biceps and quadriceps femoris revealed that bremazocine considerably facilitates the flexion reflex of the right hind limb without affecting the left limb reflex. As a rule, a flexed leg determined visually exhibited higher EMG activity of the biceps femoris as compared with a symmetric one. The opiate antagonist naloxone significantly reduced the percentage of animals with postural asymmetry. The magnitude of asymmetry and the side of flexion were not constant in some animals, but changed with time. However, the mean magnitude of asymmetry, the percentage of animals with asymmetry and the left/right flexion ratio in each group of animals remained constant. The side of flexion also depended upon the level of spinal cord transection: bremazocine and Met-enkephalin injected subarachnoidally following transection at the T1-T4 and T5-T6 levels predominantly induced flexion of the right and the left leg, respectively. Asymmetry did not develop in physiologically intact animals given bremazocine, even if the spinal cord was cut later, i.e. transection of the spinal cord was necessary for the development of asymmetry. These data indicate that the neurons which maintain muscular tone of the hind limbs and which are located symmetrically to the sagittal plane, have different sensitivities to kappa-, sigma-, and delta-agonists. In most animals, neurons with a higher specificity to some agonist are localized on one side of the sagittal plane.


Subject(s)
Decerebrate State/physiopathology , Functional Laterality/physiology , Narcotics/pharmacology , Posture , Receptors, Opioid/physiology , Spinal Cord/physiopathology , Animals , Benzomorphans/pharmacology , Decerebrate State/metabolism , Enkephalin, Leucine/analogs & derivatives , Enkephalin, Leucine/pharmacology , Enkephalin, Leucine-2-Alanine , Male , Muscles/drug effects , Muscles/innervation , Naloxone/pharmacology , Phenazocine/analogs & derivatives , Phenazocine/pharmacology , Rats , Rats, Inbred Strains , Receptors, Opioid/drug effects , Spinal Cord/drug effects , Spinal Cord/metabolism , Time Factors
8.
Brain Res ; 480(1-2): 268-76, 1989 Feb 20.
Article in English | MEDLINE | ID: mdl-2540877

ABSTRACT

The opioid mu-agonist morphine, the delta-agonist [D-Ala2,D-Leu5]enkephalin (DADL) and the kappa-agonist bremazocine locally applied to the surface of turtle visual cortex inhibited the orthodromic evoked potential (EP; fast negative component N1). After application of the sigma-agonist SKF 10.047 the inhibition was followed by facilitation of EP. The lack of cross-desensitization to the inhibitory action of opioids upon EP indicates that the drugs exert their effects via different opioid receptors. Bremazocine and morphine predominantly inhibited the left cortical EP, whereas DADL was a potent inhibitor of the right cortical EP. Thus, the opioid receptors which modulate evoked electrical activity of the left visual cortex (LVC) apparently belong mostly to the kappa- and mu-type, while delta-receptors were predominantly responsible for the modulation of electrical activity in the right visual cortex (RVC). Besides, we have studied in vivo binding of the kappa-agonist [3H]ethylketocyclazocine ([3H]EKC) and the delta-agonist [3H]DADL to LVC and RVC. The binding was specific and could be accounted for by the interaction with membrane opioid receptors and/or specific uptake of these drugs by cortex cells. [3H]EKC and [3H]DADL exhibited a more effective binding to LVC and RVC membranes, respectively. The results obtained in vitro apparently indicate that LVC and RVC differ in the number of kappa- and delta-binding sites. Thus, turtle brain may have a side-specific mechanism for selective neurochemical regulation of neuron activity in LVC and RVC realized predominantly via kappa- and delta-receptors, respectively.


Subject(s)
Functional Laterality/physiology , Receptors, Opioid/metabolism , Turtles/metabolism , Visual Cortex/metabolism , Animals , Benzomorphans/pharmacology , Enkephalin, Leucine/analogs & derivatives , Enkephalin, Leucine/pharmacology , Enkephalin, Leucine-2-Alanine , Enkephalins/metabolism , Evoked Potentials, Visual/drug effects , Morphine/pharmacology , Naloxone/pharmacology , Receptors, Opioid/physiology , Turtles/physiology , Visual Cortex/physiology
9.
Peptides ; 7(4): 551-6, 1986.
Article in English | MEDLINE | ID: mdl-3763433

ABSTRACT

Met- and Leu-enkephalin applied subarachnoidally into the rostral portion of a transected spinal cord (at the T6-T7 level) induce postural asymmetry of the hind limbs in rats, Met-enkephalin being predominantly responsible for the flexion of the right, and Leu-enkephalin of the left, hind leg. The blood serum of rats injected with Met-enkephalin contains a factor which, when administered subarachnoidally into the caudal portion of the transected spinal cord, is capable of inducing the hind limb postural asymmetry--predominantly, with the right leg flexion. This factor is inactivated by papain and differs from Met- and Leu-enkephalin in chromatographic properties. Apparently, Met-enkephalin induces the release of a peptide factor into the blood, from the brain or organs innervated by the neurons lying above the cut. It is then carried with the blood to the hind limbs and effects the hind limb postural asymmetry.


Subject(s)
Blood Proteins/isolation & purification , Enkephalin, Methionine/pharmacology , Posture , Animals , Blood Proteins/physiology , Enkephalin, Leucine/pharmacology , Male , Naloxone/pharmacology , Rats , Rats, Inbred Strains , Spinal Cord/physiology
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