ABSTRACT
AIMS/HYPOTHESIS: Diabetic retinopathy is a progressive neurodegenerative disease, but the underlying mechanism is still obscure. Here, we focused on oxidative stress in the retina, and analysed its influence on retinal neurodegeneration, using an antioxidant, lutein. METHODS: C57BL/6 mice with streptozotocin-induced diabetes were constantly fed either a lutein-supplemented diet or a control diet from the onset of diabetes, and their metabolic data were recorded. In 1-month-diabetic mice, reactive oxygen species (ROS) in the retina were measured using dihydroethidium and visual function was evaluated by electroretinograms. Levels of activated extracellular signal-regulated kinase (ERK), synaptophysin and brain-derived neurotrophic factor (BDNF) were also measured by immunoblotting in the retina of 1-month-diabetic mice. In the retinal sections of 4-month-diabetic mice, histological changes, cleaved caspase-3 and TUNEL staining were analysed. RESULTS: Lutein did not affect the metabolic status of the diabetic mice, but it prevented ROS generation in the retina and the visual impairment induced by diabetes. ERK activation, the subsequent synaptophysin reduction, and the BDNF depletion in the diabetic retina were all prevented by lutein. Later, in 4-month-diabetic mice, a decrease in the thickness of the inner plexiform and nuclear layers, and ganglion cell number, together with increase in cleaved caspase-3- and TUNEL-positive cells, were avoided in the retina of lutein-fed mice. CONCLUSIONS/INTERPRETATION: The results indicated that local oxidative stress that has a neurodegenerative influence in the diabetic retina is prevented by constant intake of a lutein-supplemented diet. The antioxidant, lutein may be a potential therapeutic approach to protect visual function in diabetes.
Subject(s)
Diabetes Mellitus, Experimental/metabolism , Diabetic Retinopathy/metabolism , Dietary Supplements , Lutein/administration & dosage , Nerve Degeneration/metabolism , Retina/metabolism , Analysis of Variance , Animals , Blotting, Western , Brain-Derived Neurotrophic Factor/metabolism , Diabetes Mellitus, Experimental/pathology , Diabetic Retinopathy/pathology , Diabetic Retinopathy/prevention & control , Extracellular Signal-Regulated MAP Kinases/metabolism , Lutein/metabolism , Mice , Nerve Degeneration/pathology , Oxidative Stress/drug effects , Reactive Oxygen Species/metabolism , Retina/drug effects , Retina/pathology , Synaptophysin/metabolismABSTRACT
BACKGROUND: It is known that auditory input, such as comforting music or sound, blunts the human response to surgical stress in conscious patients under regional anaesthesia. As auditory perception has been demonstrated to remain active under general anaesthesia, playing comforting sounds to patients under general anaesthesia might also modulate the response of these patients to surgical stress. METHODS: Fifty-nine patients scheduled for laparoscopic cholecystectomy were anaesthetized with propofol general anaesthesia in combination with epidural anaesthesia. Natural sounds, chosen preoperatively by each patient as being comforting, were played to 29 patients using headphones during surgery (S group) and the remainder of the patients (n = 30) were fitted with dummy open-type headphones (N group). We compared the haemodynamic change during anaesthesia and the acceptability of anaesthetic practice between the two groups in a randomized double-blind design. RESULTS: There were no differences in haemodynamics between the S and N groups during surgery. During the emergence from anaesthesia, the mean blood pressure and heart rate gradually increased; both parameters were significantly higher in the N group than in the S group. Postoperatively, patients in the S group perceived the experience of anaesthesia as significantly more acceptable than did those in the N group. CONCLUSION: These findings indicate that allowing patients comforting background sounds during general anaesthesia may blunt haemodynamic changes upon emergence from general anaesthesia and increase the acceptability of the experience of anaesthesia.
Subject(s)
Anesthesia, General , Blood Pressure , Heart Rate , Propofol/pharmacology , Relaxation , Aged , Double-Blind Method , Humans , Middle Aged , Music , Patient Acceptance of Health Care , Stress, Psychological/prevention & controlSubject(s)
Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Drugs, Chinese Herbal/therapeutic use , Muscular Diseases/drug therapy , Pain/drug therapy , Carboplatin/administration & dosage , Carboplatin/adverse effects , Drug Combinations , Female , Glycyrrhiza , Humans , Muscular Diseases/chemically induced , Muscular Diseases/complications , Ovarian Neoplasms/drug therapy , Paclitaxel/administration & dosage , Paclitaxel/adverse effects , Paeonia , Pain/chemically induced , Pain/etiologyABSTRACT
Pitch glides of a continuous tone elicit auditory N1-like responses. However, their characteristics have not well been investigated, and it remained unclear whether the response is an auditory true N1 or the mismatch negativity (MMN). We found here that a rapid pitch glide activates almost the same response as a true N1. On the contrary, as the rate of the pitch glide decreases, the response continuously varies the characteristics from true N1 to MMN. This suggests that there would exist intermediate responses between auditory N1 and MMN.
Subject(s)
Auditory Cortex/physiology , Evoked Potentials, Auditory/physiology , Magnetoencephalography , Pitch Perception/physiology , Acoustic Stimulation , Adult , Female , Humans , Male , Reaction Time/physiologyABSTRACT
Offset auditory responses were investigated by electroencephalography mainly in the 1970s, but since then no particular attention has been paid to them. Among the studies using magnetoencephalography (MEG) devices there are, to our knowledge, only three studies of the auditory off-response, and no significant variance has ever been observed between the source locations of on- and off-responses elicited from pure tones. We measured auditory evoked magnetic fields (AEFs) to various frequency pure tone stimulation in 5 healthy subjects with a 122-channel helmet-shaped magnetometer, and compared the distributions of the source locations of auditory N100m-Off (magnetic off-response around 100 ms) with those of N100m-On. Their spatial distributions were quite close to each other, and yet they were significantly different.
Subject(s)
Evoked Potentials, Auditory/physiology , Acoustic Stimulation , Adult , Auditory Cortex/anatomy & histology , Auditory Cortex/physiology , Electroencephalography , Humans , Magnetic Resonance Imaging , Male , Middle AgedABSTRACT
We have investigated the inhibitory action of nimesulide (4-nitro-2-phenoxymethanesulfonanilide) on release of prostaglandin E2 (PGE2) from rat peritoneal exudated macrophages (macrophages) and its mechanism of action. PGE2 release from macrophages stimulated with opsonized zymosan (OPZ) were increased in the 20 h after stimulation, whereas no significant increase was noted in PGE2 release from unstimulated macrophages. Nimesulide caused a weak inhibition of PGE2 release from macrophages at 15 min after OPZ stimulation as compared with indomethacin, but nimesulide caused approximately the same strong inhibition as indomethacin at 10 h after OPZ stimulation. Cellular cyclooxygenase (COX) activity in macrophage at 10 h after OPZ stimulation was increased approximately seven times the COX activity in macrophages before OPZ stimulation. Nimesulide caused approximately the same strong inhibition of cellular COX activity as indomethacin at 10 h after OPZ stimulation. COX-1 mRNA was expressed in macrophages irrespective of OPZ stimulation, but COX-2 mRNA was expressed only after OPZ stimulation, and COX-2 protein was simultaneously induced. Nimesulide affected neither the levels of COX-1 mRNA and COX-2 mRNA at 4 h after OPZ stimulation nor the levels of COX-2 protein at 10 h after OPZ stimulation. In contrast, actinomycin D caused strong inhibition of COX-2 mRNA expression and protein induction. These results suggest that inhibition by nimesulide of PGE2 release from macrophages, namely inflammatory cells, would be neither due to inhibition of COX-2 mRNA expression nor COX-2 induction, but to the selective inhibition of COX-2 activity itself.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cyclooxygenase Inhibitors/pharmacology , Dinoprostone/metabolism , Macrophages, Peritoneal/drug effects , Sulfonamides/pharmacology , Zymosan/pharmacology , Animals , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Dinoprostone/biosynthesis , Drug Evaluation, Preclinical , Enzyme Induction , Isoenzymes/biosynthesis , Macrophages, Peritoneal/metabolism , Male , Prostaglandin-Endoperoxide Synthases/biosynthesis , Rats , Rats, Wistar , Stimulation, ChemicalABSTRACT
Oral administration of dry powder of Chlorella vulgaris (CVP) showed clear prophylactic effects in water-immersion restraint stress-induced and in cysteamine-induced peptic ulcer models, but not in Shay's rat model. Drugs that enhance the protective factors of ulcer formation are effective in the first two models. CVP may prevent ulcer formation mainly through the "immune-brain-gut" axis and protection of gastric mucosa by its own characteristics.
Subject(s)
Chlorella , Gastric Mucosa/physiopathology , Plants, Medicinal , Stomach Ulcer/prevention & control , Stress, Psychological , Administration, Oral , Animals , Cysteamine , Gastric Mucosa/pathology , Histamine , Rats , Restraint, Physical , Stomach Ulcer/chemically induced , Stomach Ulcer/physiopathologyABSTRACT
We evaluated the effect of acarbose, an alpha-glucosidase inhibitor, on glucose intolerance in patients with non-insulin-dependent diabetes mellitus (NIDDM). Acarbose was given orally (300 mg/day) for 24 weeks to 20 NIDDM patients. Data in an oral glucose tolerance test (OGTT) were evaluated before and after 24 weeks of treatment using principal component analysis. Acarbose administration significantly reduced the postprandial plasma glucose level over 24 weeks of treatment. Principal component analysis suggested that the patients were separated into responders and non-responders. There was a significant improvement of fasting and postprandial glucose levels after 12 and 24 weeks in the responders, but not in the non-responders. Plasma glucose level following the OGTT improved significantly after 24 weeks of treatment in the responders (Hotelling T2 value = 47.098, P = 0.022500), but not in the non-responders. The immunoreactive insulin level did not change in either group. Results thus suggest that acarbose improved insulin resistance in some patients with NIDDM (responders as classified by principal component analysis).
Subject(s)
Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/drug therapy , Glucose Intolerance/drug therapy , Hypoglycemic Agents/therapeutic use , Trisaccharides/therapeutic use , Acarbose , Adult , Aged , Analysis of Variance , Blood Glucose/drug effects , Blood Glucose/metabolism , Eating , Fasting , Female , Glucose Tolerance Test , Glycated Hemoglobin/analysis , Humans , Insulin Resistance , Male , Middle Aged , Time Factors , Trisaccharides/adverse effectsABSTRACT
OBJECTIVE: To determine the nature and distribution of afferent fibers to the interspinous tissues and facet joints of the lumbar spine in the rat. DESIGN: Dissection and photography of medial branch of the primary dorsal ramus; histological and electron microscopic examination of the medial branch; measurement of conduction velocities of fibers within the medial branch; recording of compound nerve activity in medial branch in response to mechanical and chemical stimulation of interspinous tissues and facet joints. RESULTS: In the rat, the medial branch of the primary dorsal ramus of lumbar spinal nerves is normally distributed to the facet joints and interspinous tissues one and two segmental levels caudad to its origin. This nerve contains unmyelinated and myelinated afferents with conduction velocities within the ranges of C fibers, and A-delta and A-beta fibers. The tissues served by this nerve are sensitive to mechanical and noxious chemical stimulation. CONCLUSIONS: There are many structural and functional similarities in the innervation of the lumbar spine in rats and humans. However, there are anatomical variants and, in rats, the medial branch of the primary dorsal ramus, which serves the interspinous tissues and facet joints, is distributed more caudally than in humans. This information should be taken into account in extrapolating experimental results from rats to the human situation.
Subject(s)
Lumbosacral Region/innervation , Spinal Nerves/anatomy & histology , Spinal Nerves/physiology , Animals , Back Pain , Capsaicin/pharmacology , Evoked Potentials/physiology , Histocytochemistry , Male , Microscopy, Electron , Nerve Fibers/ultrastructure , Neural Conduction/drug effects , Neurons, Afferent/physiology , Neurons, Afferent/ultrastructure , Physical Stimulation , Potassium Chloride/pharmacology , Rats , Rats, Wistar , Stimulation, ChemicalABSTRACT
The antipyretic action and the mechanism of action of 4-nitro-2-phenoxymethanesulfonanilide (nimesulide), a new nonsteroidal antiinflammatory drug, were investigated in yeast-induced febrile rats. Yeast-injected rats developed marked fever and exhibited an approximately 7-fold increase in brain levels of prostaglandin E2 and an approximately 2-fold increase in the expression of cyclooxygenase-2 mRNA despite an almost unchanged expression of cyclooxygenase-1 mRNA. Nimesulide produced a dose dependent antipyretic action, which was stronger than that of indomethacin and ibuprofen, and decreased dose dependently the increased brain prostaglandin E2 levels, whereas it did not influence the expression of cyclooxygenase-2 mRNA. It inhibited markedly the enhanced brain cyclooxygenase activity, primarily cyclooxygenase-2, in vivo and dose dependently increased brain cyclooxygenase activity in vitro. These results suggest that the marked antipyretic action of nimesulide is primarily mediated through the selective inhibition of the activity of brain cyclooxygenase-2 induced under febrile conditions.
Subject(s)
Analgesics, Non-Narcotic/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Brain/enzymology , Cyclooxygenase Inhibitors/pharmacology , Isoenzymes/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Sulfonamides/pharmacology , Animals , Body Temperature , Cyclooxygenase 1 , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Dinoprostone/metabolism , Female , Fever/drug therapy , Fever/enzymology , Fever/metabolism , Hypothalamus/metabolism , Isoenzymes/biosynthesis , Male , Membrane Proteins , Prostaglandin-Endoperoxide Synthases/biosynthesis , RNA, Messenger/metabolism , Rats , Rats, WistarABSTRACT
In 1994, the Japanese Pharmaceutical Manufacturers Association (JPMA) conducted a questionnaire survey on case studies of toxicokinetics which were recently conducted its member companies. The present paper summarizes study designs and results of data analysis of 102 cases of repeated oral dose toxicity studies with dogs, monkeys, rats or mice. The statistical analysis of toxicokinetic data introduced in this paper, e.g. log-transformation of concentration data, analysis of variance and regression analysis, may be useful in clarifying the comprehensive effects of test doses, duration of repeated dosing, and sex of test animal on the systemic exposure. Suggestions are made on how experimental design can aid the efficient conduct of toxicokinetic studies in the face of limits on the number of animals.
Subject(s)
Drug Evaluation, Preclinical/methods , Drug Evaluation, Preclinical/statistics & numerical data , Drug Industry , Pharmacokinetics , Toxicology/methods , Toxicology/statistics & numerical data , Animals , Biotransformation , Japan , Research DesignABSTRACT
An active substance with antitumor activity (ARS2) was purified from the culture media of Chlorella vulgaris and found to be a glycoprotein with a molecular weight of 63,100 amu, as determined by matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry. ARS2 contains 66.9% carbohydrate, mainly D-galactose, and 35.2% protein. The carbohydrate moiety has a beta-1,6-D-galactopyranose backbone, as determined by methylation analysis and 13C-NMR. Apparently, the protein moiety, whose 15 amino acid sequence at the NH2-terminus, we determined as DVGEAFPTVVDALVA, is necessary for the antitumor activity, as assessed by hydrazinolysis, periodate oxidation, and proteolysis.
Subject(s)
Antineoplastic Agents, Phytogenic/isolation & purification , Chlorella , Fibrosarcoma/drug therapy , Glycoproteins/isolation & purification , Plants, Medicinal , Amino Acid Sequence , Animals , Antibodies , Antineoplastic Agents, Phytogenic/therapeutic use , Galactose/analysis , Glycoproteins/chemistry , Glycoproteins/therapeutic use , Mice , Mice, Inbred BALB C , Mice, Inbred DBA , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/immunology , Phytotherapy , Plant Extracts , Solubility , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Effect of Ginkgo biloba extract was examined on dissociated rat cerebellar neurons suffering from oxidative stress induced by hydrogen peroxide using a flow cytometer and ethidium bromide. Hydrogen peroxide at a concentration of 3 mM increased the number of neurons stained with ethidium (presumably dead neurons) in a time-dependent manner. Pretreatment of neurons with G. biloba extract (10 micrograms/ml) greatly delayed a time-dependent increase in number of dead neurons during exposure to hydrogen peroxide. It was true, but less effective, in the case of treatment with G. biloba extract immediately or 60 min after start of oxidative stress. Results implicate G. biloba extract as a potential agent in protecting the neurons suffering from oxidative stress induced by hydrogen peroxide.
Subject(s)
Hydrogen Peroxide/toxicity , Neurons/metabolism , Oxidants/toxicity , Oxidative Stress/physiology , Plant Extracts/pharmacology , Animals , Cell Survival/drug effects , Cerebellum/cytology , Cerebellum/drug effects , Ethidium , Flow Cytometry , Neurons/drug effects , Oxidative Stress/drug effects , RatsABSTRACT
The amino acid sequence of chicken liver xanthine dehydrogenase (EC 1.1.1.204) was determined by cDNA cloning and partial amino acid sequencing of the purified enzyme. The enzyme consisted of 1358 amino acids with calculated molecular mass of 149,633 Da. In order to compare the structure of the chicken and rat enzymes, limited proteolysis was performed with the purified chicken liver xanthine dehydrogenase. When the enzyme was digested with subtilisin, it was not converted from the NAD-dependent dehydrogenase type to the O2-dependent oxidase type, in contrast with the mammalian enzyme. However, the enzyme was cleaved mainly into three fragments in a manner similar to that for the rat enzyme at pH 8.2 (20, 37, and 84 kDa) and retaining a full complement of redox centers. The cleavage sites were identified by determination of amino-terminal sequences of the produced fragments. It was concluded that the 20-kDa fragment was amino-terminal, the 84-kDa fragment carboxyl-terminal, and the 37-kDa fragment an intermediate portion in the enzyme protein. On the other hand, when the enzyme was digested with the same protease at pH 10.5, the sample contained only the 20- and 84-kDa portions and lacked the 37-kDa portion. The resultant sample possessed xanthine dichlorophenol indophenol reductase activity, indicating that the molybdenum center remained intact. The absorption spectrum showed the sample was very similar to deflavo-enzyme. From these results and sequence analyses, the domain structure of the enzyme is discussed.
Subject(s)
Liver/enzymology , Xanthine Dehydrogenase/chemistry , Amino Acid Sequence , Animals , Base Sequence , Chickens , Cloning, Molecular , DNA, Complementary , Hydrolysis , Iron-Sulfur Proteins/chemistry , Molecular Sequence Data , Molybdenum/chemistry , Peptide Fragments/chemistry , Sequence Homology, Amino Acid , Substrate Specificity , Subtilisins/chemistry , Xanthine Dehydrogenase/geneticsABSTRACT
Cervical adenocarcinoma and adenosquamous cell carcinoma are low in radiation sensitivity, and the prognosis is said to be inferior to that of squamous cell carcinoma. Eleven facilities nationwide participated in the historical control study on the recurrence prevention effect of carmofur (HCFU) administered at 300 mg/day for more than two years postoperatively as an adjuvant chemotherapy to patients with cervical adenocarcinoma or adenosquamous cell carcinoma for which radical operation is possible. Registered for the study was a total of 252 patients: 77 patients for whom administration of carmofur was begun during the period from January 1987 and March 1989, 71 patients (control 1) who were treated for the cancer after January 1982 but did not receive adjuvant chemotherapy, and 104 patients who received adjuvant chemotherapies with other than carmofur (control 2). In analyses with the Kaplan-Meier method, the carmofur administration group demonstrated a better cumulative survival rate and disease free rate than control 1 (no adjuvant chemotherapy), suggesting carmofur was effective in preventing the recurrence of cervical adenocarcinoma and adenosquamous cell carcinoma.
Subject(s)
Adenocarcinoma/drug therapy , Antineoplastic Agents/administration & dosage , Carcinoma, Squamous Cell/drug therapy , Fluorouracil/analogs & derivatives , Uterine Cervical Neoplasms/drug therapy , Adenocarcinoma/mortality , Administration, Oral , Adult , Aged , Carcinoma, Squamous Cell/mortality , Chemotherapy, Adjuvant , Female , Fluorouracil/administration & dosage , Humans , Middle Aged , Neoplasm Recurrence, Local/prevention & control , Postoperative Period , Registries , Survival Rate , Uterine Cervical Neoplasms/mortalityABSTRACT
The upper first molars of adult male rats were moved for 7 days and unfixed, undecalcified frozen sections of the molar periodontal ligament were prepared and observed. The upper jaws of the rats were immersed rapidly in liquid nitrogen and sectioned with a cryostat using a super hard knife. Five micrometer serial sections were cut, collected, freeze-dried and observed with both light and scanning electron microscopy. Electron probe microanalysis (EPMA) was also performed on the sections. On the tension side of the periodontal ligament, periodontal fibers were stretched and the osteoblasts were aligned on the osteoid, which showed metamasia with the toluidine blue stain. On the pressure side where the periodontal ligament was extremely compressed, tissue degeneration was caused by tooth movement and the osteoclasts were observed on the bone surface adjacent to the degenerating tissues. Scanning electron microscopy revealed a network arrangement of the collagen fiber bundles on the tension side, but not on the pressure side of the periodontal ligament. The spectrum obtained from EPMA of the osteoid demonstrated X-ray (Ka) peaks of Na, P, S, K and Ca.
Subject(s)
Periodontal Ligament/anatomy & histology , Tooth Mobility/pathology , Animals , Bone Demineralization Technique , Bone and Bones/chemistry , Bone and Bones/metabolism , Calcium/analysis , Collagen/analysis , Electron Probe Microanalysis , Frozen Sections , Male , Microscopy, Electron, Scanning , Periodontal Ligament/pathology , Phosphorus/analysis , Rats , Rats, Wistar , Tissue Fixation , Tolonium Chloride , Tooth Mobility/metabolismABSTRACT
Flower colour is determined primarily by the production of pigments, usually anthocyanins or carotenoids, but the shade and intensity of the colour are often changed by other factors such as vacuolar compounds, pH and metal ions. Pigmentation can also be affected by the shape of epidermal cells, especially those facing prospective pollinators. A conical shape is believed to increase the proportion of incident light that enters the epidermal cells, enhancing light absorption by the floral pigments, and thus the intensity of their colour. We have identified a gene (mixta) that affects the intensity of pigmentation of epidermal cells in Antirrhinum majus petals. The cells of the corolla lobes fail to differentiate into their normal conical form in mixta mutants. We have cloned the mixta gene by transposon tagging; its sequence reveals that it encodes a Myb-related protein that probably participates in the transcriptional control of epidermal cell shape.
Subject(s)
Genes, Plant , Pigmentation/genetics , Plant Proteins/genetics , Plants/genetics , Transcription Factors/genetics , Amino Acid Sequence , Base Sequence , Crosses, Genetic , DNA, Complementary , Microscopy, Electron, Scanning , Molecular Sequence Data , Mutation , Pigmentation/physiology , Plants/ultrastructure , Proto-Oncogene Proteins/classification , Proto-Oncogene Proteins c-myb , Transcription Factors/classification , Transcription Factors/physiologyABSTRACT
A disulfide bond between two extracellular cysteines, conserved in all G-protein-coupled receptors, is believed to be critical for stabilization of the ligand-binding pocket. The beta 2-adrenergic receptor (beta 2-AR) contains two conserved cysteines (Cys106 and Cys184) as well as two other extracellular cysteines (Cys190 and Cys191). The specificity of the interactions between these four cysteines has not yet been clearly established. Mutants encoding alanines for specific extracellular cysteines in the beta 2-AR gene were constructed and expressed in COS-1 and Chinese hamster ovary cells. Ala106, Ala184,190,191, and Ala106,184,190,191 mutants displayed low affinity for the beta-antagonist, 125I-cyanopindolol and insensitivity to dithiothreitol (DTT). The Ala106,191 mutant displayed an intermediate affinity and DTT sensitivity. Mutants Ala184, Ala184,190, and Ala184,191 displayed high affinity and DTT sensitivity, indicating that a solvent-accessible disulfide bond(s) is present in these mutant receptors as in the wild-type beta 2-AR. Additionally, thermal stability studies provided evidence that the extracellular disulfide bonds are essential for stabilization of the high affinity state of the receptor. These studies indicate that the covalent linkage between loops 1 and 2 of the beta 2-AR extracellular domains involves the formation of disulfide bonds, uniquely between Cys106 and Cys191, and Cys184 and Cys190, and is, thus, distinct from that of other G-protein-coupled receptors.
Subject(s)
Cysteine , Protein Structure, Secondary , Receptors, Adrenergic, beta-2/chemistry , Receptors, Adrenergic, beta-2/metabolism , Amino Acid Sequence , Animals , Binding Sites , CHO Cells , Cloning, Molecular , Conserved Sequence , Cricetinae , DNA, Complementary/metabolism , Dithiothreitol/pharmacology , Electrophoresis, Polyacrylamide Gel , Immunoblotting , Iodocyanopindolol , Kinetics , Lung/metabolism , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Pindolol/analogs & derivatives , Pindolol/pharmacology , Point Mutation , Receptors, Adrenergic, beta-2/isolation & purification , TransfectionABSTRACT
The amino acid sequences of two subtilisin inhibitors (CLSI-II and -III) from Canavalia lineata seeds were determined by manual Edman degradation using the DABITC/PITC double coupling method after enzymatic digestions and CNBr degradation. CLSI-II and -III consist of 190 and 183 amino acids, respectively, and have identical amino acid sequences except for in the C-terminal regions: an elongated sequence, Gly-Thr-Ile-Arg- Ser-Asp-Gly, was found at the C-terminus of CLSI-II. A short-chain analog protein without an N-terminal Asn residue was also detected in each inhibitor preparation. The inhibitors showed significant homology to Kunitz type inhibitors, but differed from them with respect to the half-cystine content. Among five half-cystine residues present in CLSI-III, two disulfide bonds link Cys44 to Cys88 and Cys142 to Cys149, Cys106 being present as a free cysteine residue. Phenylglyoxal treatment abolished the inhibitory activity of CLSI-III, indicating the participation of an Arg residue in the interaction with the enzyme. The reactive-site peptide bond was deduced to be Arg68-Gly69.
Subject(s)
Fabaceae/chemistry , Plant Proteins/chemistry , Plants, Medicinal , Seeds/chemistry , Subtilisins/antagonists & inhibitors , Amino Acid Sequence , Arginine/chemistry , Binding Sites , Chemical Fractionation , Chromatography, High Pressure Liquid , Cysteine/chemistry , Glycine/chemistry , Molecular Sequence Data , Plant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
The antioxidant action of myricetin and quercetin, the flavonoid constituents of the extract of Ginkgo biloba (EGb), on oxidative metabolism of brain neurons dissociated from the rats was examined using 2',7'-dichlorofluorescin (DCFH) which is retained within the neuron and then is oxidized by cellular hydrogen peroxide to be highly fluorescent. Incubation with myricetin or quercetin reduced the oxidation of DCFH in resting brain neurons, more profoundly than EGb. Myricetin decreased the oxidative metabolism at concentrations of 3 nM or more. It was 10 nM or more for the case of quercetin. Incubation with each flavonoid constituent also reduced the Ca(2+)-induced increase in the oxidative metabolism without affecting the cellular content of DCFH or the intracellular concentrations of Ca2+. Such an antioxidant action of myricetin or quercetin may be responsible for a part of the beneficial effects of EGb on brain neurons subject to ischemia.