Purinergic receptors play an important role in regulating gastrointestinal (GI) motility. Interstitial cells of Cajal (ICCs) are pacemaker cells that regulate GI smooth muscle activity. We studied the functional roles of external adenosine 5′-triphosphate (ATP) on pacemaker activity in cultured ICCs from mouse small intestines by using the whole-cell patch clamp technique and intracellular Ca²⁺ ([Ca²⁺]ᵢ) imaging. External ATP dose-dependently depolarized the resting membrane and produced tonic inward pacemaker currents, and these effects were antagonized by suramin, a purinergic P2 receptor antagonist. ATP-induced effects on pacemaker currents were suppressed by an external Na⁺-free solution and inhibited by the nonselective cation channel blockers, flufenamic acid and niflumic acid. The removal of external Ca²⁺ or treatment with thapsigargin (inhibitor of Ca²⁺ uptake into endoplasmic reticulum) inhibited the ATP-induced effects on pacemaker currents. Spontaneous [Ca²⁺]ᵢ oscillations were enhanced by external ATP. These results suggest that external ATP modulates pacemaker activity by activating nonselective cation channels via external Ca²⁺ influx and [Ca²⁺]ᵢ release from the endoplasmic reticulum. Thus, it seems that activating the purinergic P2 receptor may modulate GI motility by acting on ICCs in the small intestine.
Adenosine ; Adenosine Triphosphate ; Animals ; Endoplasmic Reticulum ; Flufenamic Acid ; Interstitial Cells of Cajal ; Intestine, Small ; Membranes ; Mice ; Muscle, Smooth ; Niflumic Acid ; Pacemaker, Artificial ; Receptors, Purinergic ; Receptors, Purinergic P2 ; Suramin ; Thapsigargin

To understand the expression of hoth TGF-beta l and II ligands and the receptors, artificial fracture was made on rat femur. Fracture callus and epiphyseul plate were stained immunohistochemically on 3rd. 7th, 14th, 21st, 42nd and 56th day after trauma. Polyclonal antibody was used to stain TGF-beta I and II ligands and receptors. At epiphyseal plate, both ligand and receptor were expressed from each cell in proliferating and maturing zone. But there was no difference between type I and II except expression time. TGF-beta II ligand and receptor were expressed earlier: they were expressed mostly by the cells at the zone of proliferating cartilage but TGF-beta1 ligand and receptor were expressed mostly hy the cells at zone of maturing cartilage. At fracture site, TGF-beta expression was observed from 3rd day after trauma and it reached its maximum intensity at 2 weeks. It decreased thereafter and disappeared at 6 weeks after trauma. In enchondral ossification area, TGF-beta expressing cells were scattered throughout the enchondral mass. In intramembranous ossification area, the ligands and receptors were expressed from the osteohlasts just heneath the periosteum. ln summary, TGF-beta ligands and receptors were expressed at epiphyseal plate and fracture callus. There was no difference between TGF-beta 1 and 2 expres.ion except the appearance time at epiphyseal plate. We could not draw any conclusion about ligand and rcceptor mechanism with this immunohistochemical staining.
Animals ; Bony Callus* ; Cartilage ; Femur ; Growth Plate* ; Ligands* ; Periosteum ; Rats ; Receptors, Artificial ; Transforming Growth Factor beta ; Transforming Growth Factor beta1

OBJECTIVETo study the effect of different CO2 pneumoperitoneum pressures on the expression of adhesion molecules of human gastric cancer cell line MNK-45.

METHODSMKN-45 cells in the experimental groups were exposed to simulated CO2 environment maintained at different pressures (1.2, 1.6, 2.0 kPa) for 4 hours. Control groups were exposed to room air. At the 0, 24, 48, 72, 96 hours after treatment, CD44v6, ICAM-1 and E-cadherin were detected by flow cytometry method.

RESULTSCD44v6 and ICAM-1 expressions showed pattern of firstly elevating, then descending to normal under the pressures of 1.2 kPa and 1.6 kPa. The expressions were different from control group significantly at 24 and 48 hours (P<0.01), while the 72 hours expression showed no difference compared with the controls (P>0.05). E-cadherin expression decreased significantly right after treatment compared to the control (P<0.01), but recovered to the level of control at 48 hours (P>0.05). In the 2.0 kPa group the expression changes of CD44v6, ICAM-1 and E-cadherin were more remarkable. CD44v6 and ICAM-1 expressions were increased significantly compared to control right after treatment (P<0.05). E-cadherin expression was significantly decreased even at 48 hours compared to the controls (P<0.01).

CONCLUSIONIn vitro CO2 pneumoperitoneum pressures have transient influence on the adhesion molecules expression of gastric cancer cell MKN-45, then those expressions can recover in a short-time.

Cadherins ; metabolism ; Carbon Dioxide ; Cell Adhesion Molecules ; metabolism ; Cell Line, Tumor ; Humans ; Hyaluronan Receptors ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Pneumoperitoneum, Artificial ; Pressure ; Stomach Neoplasms ; metabolism

G-protein-coupled receptors (GPCRs), the largest family of cell surface receptors, play an important role in the production of therapeutic drugs. The functions of GPCRs are closely related to their classification and subclassification. It is difficult to obtain the spatial structure of GPCRs sequence by experimental approaches. It is highly desired to develop powerful tools and effective algorithms for classifying the family of GPCRs. In this study, based on the concept of pseudo amino acid composition (PseAA) originally introduced by Chou, approximate entropy (ApEn) of protein sequence as an additional characteristic is used to construct PseAA. A 21-D (dimensional) PseAA is formulated to represent the sample of a protein. Fuzzy K nearest neighbors (FKNN) classifier is adopted as prediction engine. The datasets in low homology are used to validate the performance of the proposed method. Compared with prior works, the successful rates achieved of our research are the highest. The test results indicate that the novel approach can play the role of a compliment to many of the existing methods, which promises to be a useful tool for GPCRs function prediction.
Algorithms ; Amino Acids ; chemistry ; Artificial Intelligence ; Chemical Phenomena ; Entropy ; Fuzzy Logic ; Humans ; Hydrophobic and Hydrophilic Interactions ; Receptors, G-Protein-Coupled ; chemistry ; classification ; Sequence Analysis, Protein ; methods

The effects of presynaptic nicotinic acetylcholine receptors (nAChRs) on excitatory synaptic transmission in CA1 pyramidal neurons of the rat hippocampus were examined by blind whole-cell patch clamp recording from hippocampal slice preparations. Local application of the nAChRs agonist dimethylphenyl-piperazinium iodide (DMPP) did not induce a postsynaptic current response in CA1 pyramidal cells. However, DMPP enhanced the frequency and amplitude of spontaneous excitatory postsynaptic current (sEPSC) in these cells in a dose-dependent manner. This enhancement was blocked by the selective nicotinic alpha-7 receptor antagonist alpha-bungarotoxin, but not by the antagonist mecamylamine, hexamethonium or dihydro-beta-erythroidine. The frequency of miniature excitatory postsynaptic current (mEPSC) in CA1 pyramidal neurons was also increased by application of DMPP, indicating a presynaptic site of action of the agonist. Taken together, these results suggest that activation of presynaptic nAChRs in CA1 pyramidal neurons, which contain alpha-7 subunits, potentiates presynaptic glutamate release and consequently modulate excitatory synaptic transmission in the hippocampus.
Animals ; Bungarotoxins ; physiology ; Dimethylphenylpiperazinium Iodide ; pharmacology ; Glutamic Acid ; pharmacology ; Hippocampus ; physiology ; Male ; Neurons ; physiology ; Nicotinic Agonists ; pharmacology ; Pacemaker, Artificial ; Patch-Clamp Techniques ; Rats ; Rats, Wistar ; Receptors, Nicotinic ; physiology ; Receptors, Presynaptic ; physiology ; Synapses ; physiology ; Synaptic Transmission ; alpha7 Nicotinic Acetylcholine Receptor

Acute Disease ; Antigens, CD ; blood ; Antigens, Differentiation, Myelomonocytic ; blood ; Hepatitis, Viral, Human ; classification ; pathology ; therapy ; Humans ; Liver Failure, Acute ; etiology ; pathology ; therapy ; Liver Transplantation ; Liver, Artificial ; Prognosis ; Receptors, Cell Surface ; blood ; Troponin I ; blood

We isolated mouse embryonic cardiomyocytes derived from timed-pregnant females at different periods and used patch-clamp technique to investigate the muscarinic cholinergic modulation of pacemaker current I(f) in different developmental stages. In early development stage (EDS), muscarinic agonist carbachol (CCh) significantly decreased the magnitude of the pacemaker current I(f) but had no effect in late development stage (LDS). Forskolin (a direct adenylate cyclase activator) and IBMX (a non-selective phosphodiesterase inhibitor) increased I(f) in both EDS and LDS cells. Interestingly, although both forskolin and IBMX increased basal I(f), their effects on CCh-inhibited I(f) were different. Forskolin did not reverse the inhibitory action of CCh until intermediate development stage (IDS). In contrast, IBMX reversed the inhibitory action of CCh on I(f) in EDS but not in IDS. It is suggested that a decrease in intracellular cAMP is a possible mechanism for CCh to modulate I(f). During the EDS and IDS CCh controls the cytoplasmic cAMP level by different pathways: In EDS, CCh modulates I(f) possibly by activating PDE which accelerates the breakdown of cAMP, but in IDS possibly by inhibiting adenylate cyclase (AC) which then reduces the synthesis of cAMP.
Animals ; Carbachol ; pharmacology ; Colforsin ; metabolism ; pharmacology ; Female ; Heart ; embryology ; physiology ; Mice ; Muscarinic Agonists ; pharmacology ; Myocytes, Cardiac ; drug effects ; physiology ; Pacemaker, Artificial ; Phosphodiesterase Inhibitors ; metabolism ; pharmacology ; Pregnancy ; Receptors, Muscarinic ; metabolism

OBJECTIVETo delete IL-11 receptor alpha chain gene from the Bacterial Artificial Chromosome (BAC) chimeric DNA by RecA protein mediated homologous recombination method and establish the transgenic mice model containing whole beta-globin gene cluster.

METHODSTwo 500 bp homologous sequences (A and B) located at the upstream and downstream of IL-11 receptor alpha chain gene respectively were cloned into the Hind III and Xba I sites of pBV vector, then the 1 kb A + B fragment was recovered from the building vector and inserted into the Sal I site of the shuttle vector pSV-RecA. After transforming the shuttle vector into the competent DH10B E. Coli containing BAC DNA, the IL-11 receptor alpha chain gene was finally deleted from the BAC DNA through chloramphenicol positive selection and fusaic acid negative selection. The new BAC clone was characterized by Pulse Field Gel Electrophoresis (PFGE). Then, we microinjected the linearized and purified BAC DNA into the mouse fertilized eggs and prepared the transgenic mice.

RESULTSBy RecA protein mediated homologous recombination method, we deleted the IL-11 receptor alpha chain gene from the BAC DNA containing the complete beta-globin Gene Cluster and established 3 respective transgenic mice lines.

CONCLUSIONHuman beta-globin gene cluster in the transgenic mice mediated by new BAC expresses in a correct mode and level as compared with previous transgenic mice.

Animals ; Chromosomes, Artificial, Bacterial ; genetics ; DNA ; Globins ; biosynthesis ; genetics ; Humans ; Interleukin-11 ; genetics ; Mice ; Mice, Transgenic ; Models, Animal ; Multigene Family ; genetics ; Receptors, Interleukin ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transcription, Genetic

Eisenmenger syndrome is a severe form of pulmonary arterial hypertension related to congenital cardiac defects. Many patients die at a young age from such complications. The treatment of primary pulmonary hypertension is being applied to Eisenmenger syndrome such as endothelin receptor antagonists, phosphodiesterase-5 blockers, and prostacyclin. We experienced a case of 29-year female with ventricular septal defect-related Eisenmenger syndrome complicated with Down syndrome and Moyamoya disease, who was admitted to intensive care unit due to enteritis-associated septic shock. After the combination treatment with iloprost and sildenafil within the intensive care unit, the patient was able to wean mechanical ventilation without further applications of invasive rescue therapy such as extracorporeal membrane oxygenator. She was later discharged with bosentan. She maintained bosentan therapy for 34 months continuously without aggravations of symptom but eventually died with intracranial hemorrhage, a complication of Moyamoya disease. To our knowledge, this is the first case report of Eisenmenger syndrome accompanied by mosaic Down syndrome and Moyamoya disease.
Cyclic Nucleotide Phosphodiesterases, Type 5 ; Down Syndrome ; Eisenmenger Complex ; Epoprostenol ; Female ; Humans ; Hypertension ; Hypertension, Pulmonary ; Iloprost ; Critical Care ; Intensive Care Units ; Intracranial Hemorrhages ; Moyamoya Disease ; Oxygenators, Membrane ; Piperazines ; Purines ; Receptors, Endothelin ; Respiration, Artificial ; Shock, Septic ; Sulfonamides ; Sulfones ; Sildenafil Citrate

Human immunodeficiency virus (HIV) infects the host cells by the fusion of viral and cell membranes. Blocking the combining between HIV and the receptors can prevent HIV from entering the host cells. We designed an invasion-inhibitor for HIV-1 targeting dendritic cell (DC), including 2 important HIV-1 receptors CD4 and CCR5, and 2 molecules Flt3-L and Mip-3alpha. With the synthetic gene of the inhibitor, 2 eukaryotic expression vectors pABK-CKR5-CD4/Flt3L-Mip3alpha (pABK-HIV-MF) and pABK-CKR5-CD4 (pABK-HIV-MT) were constructed and transfected into HEK 293 cells for expression. Results from RT-PCR, immunofluorescent assay, ELISA and Western blot approved that the invasion-inhibitor for HIV-1 was successfully and exactly expressed in the eukaryotic cells. Current study formed a solid base for the further research on the function of inhibitors for HIV-1 and elimination targeting DC.
Artificial Gene Fusion ; CD4 Antigens ; biosynthesis ; genetics ; Chemokine CCL20 ; biosynthesis ; genetics ; Dendritic Cells ; immunology ; metabolism ; Genetic Vectors ; genetics ; HEK293 Cells ; HIV Envelope Protein gp120 ; genetics ; HIV-1 ; physiology ; Humans ; Receptors, CCR5 ; biosynthesis ; genetics ; Receptors, HIV ; antagonists & inhibitors ; Transfection ; fms-Like Tyrosine Kinase 3 ; biosynthesis ; genetics