ABSTRACT
We propose a three-step strategy that uses structural and physicochemical properties of chemicals to predict their 72 h algal growth inhibition toxicities against Pseudokirchneriella subcapitata. In Step 1, using a log D-based criterion and structural alerts, we produced an interspecies QSAR between algal and acute daphnid toxicities for initial screening of chemicals. In Step 2, we categorized chemicals according to the Verhaar scheme for aquatic toxicity, and we developed QSARs for toxicities of Class 1 (non-polar narcotic) and Class 2 (polar narcotic) chemicals by means of simple regression with a hydrophobicity descriptor and multiple regression with a hydrophobicity descriptor and a quantum chemical descriptor. Using the algal toxicities of the Class 1 chemicals, we proposed a baseline QSAR for calculating their excess toxicities. In Step 3, we used structural profiles to predict toxicity either quantitatively or qualitatively and to assign chemicals to the following categories: Pesticide, Reactive, Toxic, Toxic low and Uncategorized. Although this three-step strategy cannot be used to estimate the algal toxicities of all chemicals, it is useful for chemicals within its domain. The strategy is also applicable as a component of Integrated Approaches to Testing and Assessment.
Subject(s)
Chlorophyta/drug effects , Water Pollutants, Chemical/chemistry , Water Pollutants, Chemical/toxicity , Animals , Chemical Phenomena , Chlorophyta/growth & development , Daphnia/drug effects , Hydrophobic and Hydrophilic Interactions , Narcotics/chemistry , Narcotics/toxicity , Pesticides/chemistry , Pesticides/toxicity , Quantitative Structure-Activity Relationship , Quantum TheoryABSTRACT
In addition to molecular structure profiles, descriptors based on physicochemical properties are useful for explaining the eco-toxicities of chemicals. In a previous study we reported that a criterion based on the difference between the partition coefficient (log POW) and distribution coefficient (log D) values of chemicals enabled us to identify aromatic amines and phenols for which interspecies relationships with strong correlations could be developed for fish-daphnid and algal-daphnid toxicities. The chemicals that met the log D-based criterion were expected to have similar toxicity mechanisms (related to membrane penetration). Here, we investigated the applicability of log D-based criteria to the eco-toxicity of other kinds of chemicals, including aliphatic compounds. At pH 10, use of a log POW - log D > 0 criterion and omission of outliers resulted in the selection of more than 100 chemicals whose acute fish toxicities or algal growth inhibition toxicities were almost equal to their acute daphnid toxicities. The advantage of log D-based criteria is that they allow for simple, rapid screening and prioritizing of chemicals. However, inorganic molecules and chemicals containing certain structural elements cannot be evaluated, because calculated log D values are unavailable.
Subject(s)
Chlorophyta/drug effects , Daphnia/drug effects , Oryzias/physiology , Quantitative Structure-Activity Relationship , Water Pollutants, Chemical/toxicity , Animals , Chlorophyta/growth & development , Hydrogen-Ion Concentration , Organic Chemicals/chemistry , Organic Chemicals/toxicity , Species Specificity , Toxicity Tests , Water Pollutants, Chemical/chemistryABSTRACT
We propose interspecies quantitative structure-activity-activity relationships (QSAARs), that is, QSARs with descriptors, to estimate species-specific acute aquatic toxicity. Using training datasets consisting of more than 100 aromatic amines and phenols, we found that the descriptors that predicted acute toxicities to fish (Oryzias latipes) and algae were daphnia toxicity, molecular weight (an indicator of molecular size and uptake) and selected indicator variables that discriminated between the absence or presence of various substructures. Molecular weight and the selected indicator variables improved the goodness-of-fit of the fish and algae toxicity prediction models. External validations of the QSAARs proved that algae toxicity could be predicted within 1.0 log unit and revealed structural profiles of outlier chemicals with respect to fish toxicity. In addition, applicability domains based on leverage values provided structural alerts for the predicted fish toxicity of chemicals with more than one hydroxyl or amino group attached to an aromatic ring, but not for fluoroanilines, which were not included in the training dataset. Although these simple QSAARs have limitations, their applicability is defined so clearly that they may be practical for screening chemicals with molecular weights of ≤364.9.
Subject(s)
Amines/chemistry , Chlorophyta/drug effects , Daphnia/drug effects , Oryzias/physiology , Phenols/chemistry , Quantitative Structure-Activity Relationship , Water Pollutants, Chemical/chemistry , Amines/toxicity , Animals , Chlorophyta/growth & development , Models, Biological , Phenols/toxicity , Species Specificity , Toxicity Tests, Acute , Water Pollutants, Chemical/toxicityABSTRACT
The validity of chemical reaction mechanistic domains defined by skin sensitisation in the Quantitative Structure-Activity Relationship (QSAR) ecotoxicity system, KAshinhou Tools for Ecotoxicity (KATE), March 2009 version, has been assessed and an external validation of the current KATE system carried out. In the case of the fish end-point, the group of chemicals with substructures reactive to skin sensitisation always exhibited higher root mean square errors (RMSEs) than chemicals without reactive substructures under identical C- or log P-judgements in KATE. However, in the case of the Daphnia end-point this was not so, and the group of chemicals with reactive substructures did not always have higher RMSEs: the Schiff base mechanism did not function as a high error detector. In addition to the RMSE findings, the presence of outliers suggested that the KATE classification rules needs to be reconsidered, particularly for the amine group. Examination of the dependency of the organism on the toxic action of chemicals in fish and Daphnia revealed that some of the reactive substructures could be applied to the improvement of the KATE system. It was concluded that the reaction mechanistic domains of toxic action for skin sensitisation could provide useful complementary information in predicting acute aquatic ecotoxicity, especially at the fish end-point.
Subject(s)
Environmental Pollutants/toxicity , Models, Chemical , Quantitative Structure-Activity Relationship , Animals , Daphnia/drug effects , Ecotoxicology , Fishes , Risk Assessment , Toxicity Tests/methodsABSTRACT
A 66-year-old man was admitted to our hospital because of induction therapy of interferon for chronic hepatitis due to hepatitis C virus. On routine investigation, a mass-like lesion was detected at the level of the hilum of the left lung on the chest plain radiograph. On the thoracic CT, the heterogeneous tumor, including calcification, was 6 cm in diameter in the posterior mediastinal area, and the rib and vertebra had been damaged by it. The tumor extended to the descending aorta and the left main bronchus. A small nodule in the left S1 + 2 was noted on another slice section of this thoracic CT. The possible diagnoses, based on these radiological findings, for this tumor were primary lung cancer, posterior mediastinal tumor and malignant tumor originating from the chest wall. The diagnosis of chondrosarcoma was made based on the histological findings of the specimen obtained from the tumor. This tumor could not be resected in this case, because it had extensively damaged the rib and vertebra, and the nodular lesion in the left S1 + 2 was regarded as a metastasis from it. Although reports of chondrosarcoma originating from a rib and presenting as a posterior mediastinal tumor-like shadow are rare, chondrosarcoma should be considered as a possible diagnosis for such tumors-posterior mediastinal masses with characteristic findings such as a large tumor in contact with the chest wall, and containing calcification, with destruction of bone and dissection of the rib as shown in our CT findings.
Subject(s)
Bone Neoplasms/diagnosis , Chondrosarcoma/diagnosis , Ribs/pathology , Aged , Bone Neoplasms/pathology , Chondrosarcoma/pathology , Diagnosis, Differential , Humans , Lung Neoplasms/secondary , Male , Mediastinal Neoplasms , Spine/pathologyABSTRACT
In Neurospora crassa, the phosphorylation of nucleoside diphosphate kinase (NDK)-1 is rapidly enhanced after blue light irradiation. We have investigated the function of NDK-1 in the blue light signal transduction pathway. A mutant called psp (phosphorylation of small protein) shows undetectable phosphorylation of NDK-1 and is defective in light-responsive regulation of perithecial polarity. Sequencing analysis of ndk-1 cDNA by reverse transcription-polymerase chain reaction revealed that proline 72 of ndk-1 was replaced with histidine in psp. The mutation ndk-1(P72H) resulted in accumulation of normal levels of mRNA and of about 25% of NDK-1(P72H) protein compared with that of wild type as determined by Western blot analysis. The ectopic expression of cDNA and introduction of genomic DNA of wild type ndk-1 in psp (ndk-1(P72H)) suppressed the reduction in accumulation and phosphorylation of NDK-1 and the light-insensitive phenotype. These findings demonstrated that the phenotype of psp was caused by the ndk-1(P72H) mutation. Biochemical analysis using recombinant NDK-1 and NDK-1(P72H) indicated that the P72H substitution in NDK-1 was responsible for the decrease in phosphotransfer activities, 5% of autophosphorylation activity, and 2% of V(max) for protein kinase activity phosphorylating myelin basic protein, compared with those of wild type NDK-1, respectively.
Subject(s)
Cell Polarity/physiology , Neurospora crassa/cytology , Neurospora crassa/enzymology , Nucleoside-Diphosphate Kinase/genetics , Nucleoside-Diphosphate Kinase/metabolism , Point Mutation , Amino Acid Substitution , Cell Polarity/radiation effects , Darkness , Glutathione Transferase/metabolism , Histidine , Light , Neurospora crassa/radiation effects , Nucleoside-Diphosphate Kinase/chemistry , Phosphorylation , Proline , RNA, Messenger/genetics , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription, GeneticABSTRACT
A novel dominant mutant designated 'dwarf in light 1' (dfl1-D) was isolated from screening around 1200 Arabidopsis activation-tagged lines. dfl1-D has a shorter hypocotyl under blue, red and far-red light, but not in darkness. Inhibition of cell elongation in shoots caused an exaggerated dwarf phenotype in the adult plant. The lateral root growth of dfl1-D was inhibited without any reduction of primary root length. The genomic DNA adjacent to the right border of T-DNA was cloned by plasmid rescue. The rescued genomic DNA contained a gene encoding a GH3 homologue. The transcript was highly accumulated in the dfl1-D. The dfl1-D phenotype was confirmed by over-expression of the gene in the wild-type plant. The dfl1-D showed resistance to exogenous auxin treatment. Moreover, over-expression of antisense DFL1 resulted in larger shoots and an increase in the number of lateral roots. These results indicate that the gene product of DFL1 is involved in auxin signal transduction, and inhibits shoot and hypocotyl cell elongation and lateral root cell differentiation in light.
Subject(s)
Arabidopsis Proteins , Indoleacetic Acids/pharmacology , Light , Plant Growth Regulators , Plant Proteins/physiology , Plant Roots/growth & development , Plant Shoots/growth & development , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/growth & development , Base Sequence , DNA Primers , DNA, Bacterial , Gene Expression Regulation, Plant/drug effects , Molecular Sequence Data , Mutation , Phenotype , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Roots/cytology , Sequence Homology, Amino Acid , Signal Transduction/physiologyABSTRACT
A 56-year-old man was admitted for further evaluation of mediastinal lymphadenopathy and left pleural effusion. A blood test revealed polyclonal hypergammaglobulinemia with an elevated erythrocyte sedimentation rate and level of C-reactive protein. Chest computed tomography disclosed bilateral hilar and multiple mediastinal lymph-node swelling and bilateral pleural thickening accompanied by a moderate amount of left pleural effusion. Biopsy specimens from mediastinal lymph nodes and the right pleura were obtained under video-assisted thoracoscopy. Histology on microscopic examination demonstrated mature plasma cells and lymphocyte infiltration in the conserved lymph-node structure and the pleura, suggesting a diagnosis of idiopathic plasmacytic lymphadenopathy (IPL) with polyclonal hyperimmunoglobulinemia. Interestingly, IL-6 was elevated in the pleural effusion but normal in serum. This was a rare and instructive case of IPL accompanied by pleural effusion that was considered to be an important determinant of the clinical spectrum of the disease.
Subject(s)
Hypergammaglobulinemia/complications , Lymphatic Diseases/complications , Mediastinal Diseases/complications , Plasma Cells/pathology , Pleural Effusion/etiology , Biomarkers/analysis , Humans , Lymphatic Diseases/pathology , Male , Mediastinal Diseases/pathology , Middle AgedABSTRACT
A 67-year-old woman presented in September 1985 with productive cough, bloody sputum, and dyspnea on exertion. Productive cough and bloody sputum had developed when the patient was 55 years old. Sputum culture and radiologic findings yielded a diagnosis of nontuberculous mycobacteriosis (NTM). Antituberculous therapy with INH, RFP, and EB was initiated in November 1987 because of the development of a cavity in the right upper lobe, and led to resolution of the lesion and clinical symptoms. Despite progression of bronchiectatic changes in both lungs and a relapse of her clinical symptoms during the following 10 years, the patient retained enough pulmonary function to be able to maintain an active daily life until she died of advanced gastric cancer at the age of 79. Autopsy revealed cystic bronchiectasis accompanied by bronchial wall thickening in both lungs, with some granuloma and acid-fast-bacteria observed in lung tissue. In this report, we concluded that patients with NTM usually experience a gradual progression of symptoms and radiographic changes during their clinical course, and that their pulmonary function may be conserved well enough to maintain an active daily life.
Subject(s)
Mycobacterium Infections, Nontuberculous , Mycobacterium avium Complex/isolation & purification , Mycobacterium avium-intracellulare Infection , Pneumonia, Bacterial/microbiology , Aged , Bronchi/pathology , Female , Follow-Up Studies , Humans , Lung/pathology , Mycobacterium chelonae/isolation & purification , Mycobacterium fortuitum/isolation & purification , Pneumonia, Bacterial/complications , Pneumonia, Bacterial/pathology , Sputum/microbiology , Stomach Neoplasms/complications , Time FactorsABSTRACT
We have previously reported that phosphorylation of a 15-kDa protein increased after blue-light irradiation in Neurospora crassa. In this study, the 15-kDa protein was purified using four columns; DEAE-cellulose, Blue-Sepharose, SP-Sepharose and Mono Q. The 15-kDa protein was shown to be homologous with nucleoside diphosphate kinase by amino acid sequencing and was also shown to possess nucleoside diphosphate kinase activity. A gene encoding N. crassa nucleoside diphosphate kinase, ndk-1, was isolated from the mycelial cDNA and genomic libraries. The deduced amino acid sequence of NDK-1 was identical to that of the 15-kDa protein. Northern blot analysis suggested that WC-1 and WC-2, the key factors of blue-light signal transduction in N. crassa, did not regulate NDK-1 at the transcriptional level. NDK-1 also showed rapid autophosphorylation activity and protein kinase activity against myelin basic protein with a Km value of 0.36 mM. These results suggest that NDK-1 acts as a signal transducer by phosphorylating proteins.
Subject(s)
Neurospora crassa/enzymology , Nucleoside-Diphosphate Kinase/isolation & purification , Amino Acid Sequence , Animals , Cloning, Molecular , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Fungal , Genes, Fungal , Humans , Kinetics , Light , Molecular Sequence Data , Molecular Weight , Neurospora crassa/genetics , Neurospora crassa/radiation effects , Nucleoside-Diphosphate Kinase/genetics , Nucleoside-Diphosphate Kinase/metabolism , Phosphorylation , Sequence Homology, Amino Acid , Signal TransductionABSTRACT
A 44-year-old woman was admitted to our hospital for further evaluation of a consolidated shadow in the left lower lobe and the evaluation of serum tumor markers (CEA 46.3 ng/ml, CA 19-9 1911 U/ml, and CA 125 103 U/ml). Chest computed tomography revealed an irregular shaped, low density mass shadow in the left S10 region, suggesting the diagnosis of pulmonary sequestration or bronchial atresia. However digital subtraction angiography failed to demonstrate an anomalous feeding artery. We could not rule out the possibility that a malignant lesion was included in the consolidated shadow. A left thoracotomy revealed an intralobar pulmonary sequestration of the left lower lobe. Hyphae of aspergillus were found in the lumen of the cystic bronchus of the resected lung. Immunohistochemical studies showed strong expression of CEA, CA 19-9, and CA 125 by bronchial epithelia in the pulmonary sequenstration. The serum values of tumor markers returned to their normal ranges after surgery.
Subject(s)
Aspergillosis/complications , Biomarkers, Tumor/blood , Bronchopulmonary Sequestration/diagnosis , CA-125 Antigen/blood , CA-19-9 Antigen/blood , Carcinoembryonic Antigen/blood , Lung Diseases, Fungal/complications , Adult , Bronchopulmonary Sequestration/complications , Bronchopulmonary Sequestration/surgery , Diagnosis, Differential , Female , HumansABSTRACT
The molecular mechanism of light signal perception was analyzed using stem sections of etiolated rice (Oryza sativa L.) seedlings irradiated with red light from a fluorescent lamp. The membrane and cytosol fractions were labeled by 40 nM [gamma-32P]ATP for 10 s at 0 degrees C and proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Phosphorylation of three proteins with molecular weights of 16, 17 and 18 kDa in the rice increased with the intensity of red light irradiation (50 micromol/m2/s) for 16 min. Most of the phosphorylation activity was present in the cytosol fraction. The three proteins cross-reacted with the anti-nucleoside diphosphate (NDP) kinase antibody. Phosphorylation of these proteins was correlated with changes in the activity of NDP kinase. These proteins phosphorylated histone III-S, a substrate for measuring the protein kinase activity. By phospho-amino acid analysis, phosphoserine was found present in the phosphorylated proteins. These rapidly phosphorylated proteins would thus appear to have the features of NDP kinase.
Subject(s)
Oryza/radiation effects , Plant Proteins/metabolism , Amino Acid Sequence , Blotting, Western , Electrophoresis, Gel, Two-Dimensional , Light , Molecular Sequence Data , Nucleoside-Diphosphate Kinase/metabolism , Oryza/enzymology , Oryza/metabolism , Phosphorylation , Plant Stems/enzymology , Plant Stems/metabolismABSTRACT
To clarify the molecular mechanism for the transduction of light signals in plants, we have established an in vitro system that uses crude membrane and soluble fractions of stem sections of etiolated Pisum sativum L. cv. Alaska after irradiation by red light, or sequential application of red and far-red light to the stem section. In a previous report (T. Hamada et al., J. Photochem. Photobiol. B: Biol. 33 (1996) 143-151) the labelling of proteins in membrane fraction by [gamma-32P] ATP at 0 degree C for 15 s and subsequent separation of proteins by two-dimensional electrophoresis allowed unambiguous identification of a heavily phosphorylated protein spot at 18 kDa (p18). In the present study we have confirmed the former results in the membrane fraction, and obtained the result that an increase in the phosphorylation of p18 by red-light irradiation is observed in the soluble fraction. Further, we have provided evidence that the p18 in the soluble fraction is purified and identified as nucleoside diphosphate (NDP) kinase by Western blotting, immuno-precipitation, amino acid sequencing and cDNA analysis. Purified p18 shows autophosphorylation activity and strong phosphorylating activity against myelin basic protein (MBP), a substrate of MAP (mitogen activated protein) kinase. The results show that phytochrome-mediated light signals are transduced to NDP kinase, which may elicit signals by providing high concentrations of, for example, GTP from GDT and ATP, by the autophosphorylation and by the protein kinase activity similar to MAP kinase.
Subject(s)
Nucleoside-Diphosphate Kinase/metabolism , Pisum sativum/enzymology , Signal Transduction , Alaska , Amino Acid Sequence , Membrane Proteins/analysis , Molecular Sequence Data , Phosphoamino Acids/analysis , Phosphorylation , Phytochrome , Plant Proteins/metabolismSubject(s)
Light Signal Transduction , Photoreceptor Cells, Invertebrate , Plant Physiological Phenomena , Amino Acid Sequence , Circadian Rhythm , Cryptochromes , Flavoproteins/physiology , Molecular Sequence Data , Nucleoside-Diphosphate Kinase/chemistry , Nucleoside-Diphosphate Kinase/physiology , Phosphorylation , Phytochrome/physiology , Plants , Receptors, G-Protein-CoupledABSTRACT
A 57-year-old-man was admitted because of persistent cough and progressive dyspnea over a period of 2 months. Examination revealed wheezing in both lungs, severe hypoxemia, and marked obstructive impairment of pulmonary function. A chest CT scan showed diffuse small nodular lesions with patchy air space opacifications. The patient was initially given a diagnosis of bronchiolitis of unknown cause. He showed marked improvement after treatment with oral prednisolone, but developed fever and dyspnea after returning home. This episode indicated hypersensitivity pneumonitis, a diagnosis supported by findings of increased CD 8 positive T-lymphocytes in bronchoalveolar lavage fluid, and a high titer of serum anti-Trichosporon antibody. Lung biopsy samples obtained under video-assisted thoracoscopy disclosed noncaseating granulomas in terminal and respiratory bronchioles, which resulted in marked narrowing of the lumen. The pathologic changes seemed to be consistent with obstructive impairment of pulmonary function in this patient.
Subject(s)
Alveolitis, Extrinsic Allergic/complications , Lung Diseases, Obstructive/etiology , Alveolitis, Extrinsic Allergic/diagnosis , Antibodies, Fungal/analysis , CD8-Positive T-Lymphocytes , Humans , Lung Diseases, Obstructive/pathology , Lymphocyte Count , Male , Middle Aged , Trichosporon/immunologyABSTRACT
We have previously demonstrated that blue light induces the phosphorylation of a 15-kDa protein in crude membrane fractions of Neurospora crassa mycelia. Here we report the isolation and characterization of a mutant (psp; phosphorylation of small proteins that is completely defective for phosphorylation of that protein, as assayed in both crude membrane and soluble fractions. This mutation defines a unique locus that maps to linkage group VR between al-3 and his-6. To elucidate the photobiological significance of the phosphorylation of the protein, we analyzed known photobiological phenomena and discovered that the positioning of beaks on the perithecia, defined as perithecial polarity, was light-dependent in the wild type. In the psp mutant, beaks were phototropic as in the wild type, but their position was random. In a wc-l mutant, however, beaks were positioned at random and were not phototropic. Thus light-induced perithecial polarity and phototropism of perithecial beaks are controlled differently. A psp; wc-l double mutant showed the same phenotype as that of wc-l with respect to these two photomorphogenetic characters. These results indicate that the wc-l gene is epistatic to psp in the light-signal transduction pathway that controls both phototropism and perithecial polarity.
Subject(s)
Fungal Proteins/physiology , Genes, Fungal , Neurospora crassa/genetics , Phosphoproteins/physiology , Phototropism/genetics , Protein Processing, Post-Translational/radiation effects , Signal Transduction/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Fungal Proteins/genetics , Morphogenesis/radiation effects , Neurospora crassa/growth & development , Neurospora crassa/radiation effects , Neurospora crassa/ultrastructure , Phosphoproteins/genetics , Phosphorylation/drug effects , Signal Transduction/radiation effects , Transcription Factors/genetics , Transcription Factors/physiologyABSTRACT
A 53-year-old woman with a smoking history of 20 pack-years was admitted to the hospital because of dry coughing and exertional dyspnea that had progressed over the previous 3 months. A chest X-ray film taken one month before the onset of symptoms was almost normal. Diffuse small nodular shadows with infiltration were evident one month after the onset of symptoms, and increased rapidly until admission to the hospital. Eosinophilic granuloma was diagnosed histologically after thoracoscopy. The symptoms, chest X-ray findings and pulmonary-function data improved spontaneously after admission to the hospital and the nodular shadows had a almost completely disappeared by 5 months later; the patient continued to smoke. The present case is unique and valuable because it shows the natural course of eosinophilic granuloma from the onset of symptoms to remission.
Subject(s)
Eosinophilic Granuloma/diagnostic imaging , Lung Diseases/diagnostic imaging , Eosinophilic Granuloma/physiopathology , Female , Humans , Lung Diseases/physiopathology , Middle Aged , Radiography, ThoracicABSTRACT
Soluble fractions prepared from the mycelia of wild type (74-OR23-1A) and band (bd) exhibited an increase in the rate of the ADP ribosylation of a 38 kDa protein from nicotinamide adenine [32P]dinucleotide ([32P]NAD) in the presence of 10(-7) M riboflavin caused by blue light irradiation in vitro. The soluble fraction was mixed with a reaction mixture containing 5 microCi [32P]NAD at 0 degree C for 20 s and then it was irradiated with blue light (420 nm, 42 mumol m-2 s-1) for 12.5, 25, 50, 100, 200 or 400 s at 0 degree C or for 100 s with photon irradiance of 0.42, 4.2, 6.4 or 42 mumol m-2 s-1. Immediately after irradiation, the reaction was stopped and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. An increase in the ADP ribosylation of the 38 kDa protein could be detected within 100 s of irradiation, and the enhancement in the rate of ADP ribosylation of the 38 kDa protein was proportional to the increase in the photon irradiance. By the irradiation with blue light for 200 or 400 s, the ADP ribosylation of a 56 kDa protein could also be detected. Analysis by two-dimensional gel electrophoresis of proteins after ADP ribosylation of them revealed that the 38 kDa proteins displayed at least four radioactive protein spots and the 56 kDa protein a single radioactive protein spot. Soluble fractions of mycelia prepared from blind mutants wc-1, wc-2, delta ps15-1, lis-1, lis-2 and lis-3 exhibited also the enhancement of the ADP ribosylation of the 38 kDa protein by blue light irradiation, and at least wc-1, delta ps15-1, lis-1 and lis-2 displayed a similar blue light response in the 56 kDa protein.
Subject(s)
Adenosine Diphosphate Ribose/metabolism , Fungal Proteins/metabolism , Light , Molecular Weight , Neurospora crassa , Time FactorsABSTRACT
A rebound phenomenon (severe arterial desaturation and pulmonary vasoconstriction) has been observed in some patients with pulmonary hypertension after sudden discontinuation of nitric oxide (NO) inhalation therapy. The mechanism responsible for this phenomenon is unknown. It has recently been reported that NO synthase (NOS) can be inhibited by NO as a negative feedback mechanism. We therefore hypothesized that this rebound phenomenon might be attributable to reduced endogenous NO production due to inhibition of pulmonary endothelial NOS (eNOS) activity by inhaled NO. To test this hypothesis, vasoreactivities were compared in isolated perfused lungs from rats exposed to 40 ppm NO gas and room air for 2 days. The pressor responses to angiotensin II and hypoxia of lungs from rats exposed to NO were markedly potentiated and their depressor response to bradykinin was considerably impaired, whereas that to sodium nitroprusside was preserved, compared with the responses of those exposed to room air. This altered pulmonary vasoreactivity was also induced by lower concentrations of inhaled NO (as low as 1 ppm) and was completely reversed by discontinuation of NO inhalation for several (< 8) hours. The expression of eNOS mRNA by lungs isolated from rats exposed to 40 ppm NO and room air for 2 days did not differ. These results suggest that pulmonary endogenous NO production is reversibly reduced after relatively short-term NO inhalation, which probably inhibits eNOS activity directly. We speculate that reduced production of pulmonary endogenous NO by exogenous (inhaled) NO could be the mechanism responsible for this rebound phenomenon.
Subject(s)
Nitric Oxide/administration & dosage , Pulmonary Circulation/drug effects , Administration, Inhalation , Animals , In Vitro Techniques , Male , Molecular Sequence Data , Pulmonary Circulation/physiology , Rats , Rats, Sprague-Dawley , Vasoconstriction/drug effectsABSTRACT
The molecular mechanism of light signal perception via phytochrome was analysed using the third internodes of etiolated pea seedlings irradiated with red or red followed by far-red light. A crude membrane fraction prepared from the tissue was labelled by [gamma-32P]ATP at 4 x 10(-8) M for 15 s at 0 degree C, and the proteins were separated by two-dimensional gel electrophoresis. The phosphorylation of a protein with a molecular mass of about 15 kDa in the crude membrane fraction increased with an increase in the intensity of red light irradiation (10, 50 and 100 mumol m-2 s-1) for 20 s. Successive irradiation with red light (100 mumol m-2 s-1 for 20 s) and subsequent irradiation with far-red light reduced the phosphorylation of the protein, depending on the intensity of the far-red light (from 0.1 to 8 mumol m-2 s-1 for 180 s). A plasma membrane purified from the crude membrane fraction from red light irradiated tissue showed a rapid phosphorylation of the 15 kDa protein by 4 x 10(-8) M [gamma-32P]ATP at 0 degree C for 7 s, and subsequent addition of ATP, GTP, ADP or GDP at 10(-5) or 10(-6) M efficiently removed the phosphoryl group of the 15 kDa protein. The 15 kDa protein was autophosphorylated in the gel following separation by sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis. The partially purified 15 kDa protein included nucleoside diphosphate kinase (NDP kinase) activity, as well as cross-reactivity with an antibody specific to rat NDP kinase as assayed by immunostaining and crossreactivity with an antibody specific to ricet NDP kinase as assayed by immunoprecipitation.