ABSTRACT
Insect haemocytes play significant roles in innate immunity. The silkworm, a lepidopteran species, is often selected as the model for studies into the functions of haemocytes in immunity; however, our understanding of the role of haemocytes remains limited because the lack of haemocyte promoters for transgene expression makes genetic manipulations difficult. In the present study, we aimed to establish transgenic silkworm strains expressing GAL4 in their haemocytes. First, we identified three genes with strong expression in haemocytes, namely, lp44, Haemocyte Protease 1 (HP1) and hemocytin. Transgenic silkworms expressing GAL4 under the control of the putative promoters of these genes were then established and expression was examined. Although GAL4 expression was not detected in haemocytes of HP1-GAL4 or hemocytin-GAL4 strains, lp44-GAL4 exhibited a high level of GAL4 expression, particularly in oenocytoids. GAL4 expression was also detected in the midgut but in no other tissues, indicating that GAL4 expression in this strain is mostly oenocytoid-specific. Thus, we have identified a promoter that enables oenocytoid expression of genes of interest. Additionally, the lp44-GAL4 strain could also be used for other types of research, such as the functional analysis of genes in oenocytoids, which would facilitate advances in our understanding of insect immunity.
Subject(s)
Bombyx/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation , Hemocytes/metabolism , Insect Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/genetics , Animals , Animals, Genetically Modified/genetics , Animals, Genetically Modified/immunology , Animals, Genetically Modified/metabolism , Bombyx/growth & development , Bombyx/immunology , Bombyx/metabolism , DNA-Binding Proteins/metabolism , Immunity, Innate , Insect Proteins/metabolism , Larva/genetics , Larva/immunology , Larva/metabolism , Lectins/genetics , Lectins/metabolism , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/metabolismABSTRACT
Sacrificing model animals is required for developing effective drugs before being used in human beings. In Japan today, at least 4,210,000 mice and other mammals are sacrificed to a total of 6,140,000 per year for the purpose of medical studies. All the animals treated in Japan, including test animals, are managed under control of "Act on Welfare and Management of Animals". Under the principle of this Act, no person shall kill, injure, or inflict cruelty on animals without due cause. "Animal" addressed in the Act can be defined as a "vertebrate animal". If we can make use of invertebrate animals in testing instead of vertebrate ones, that would be a remarkable solution for the issue of animal welfare. Furthermore, there are numerous advantages of using invertebrate animal models: less space and small equipment are enough for taking care of a large number of animals and thus are cost-effective, they can be easily handled, and many biological processes and genes are conserved between mammals and invertebrates. Today, many invertebrates have been used as animal models, but silkworms have many beneficial traits compared to mammals as well as other insects. In a Genome Pharmaceutical Institute's study, we were able to achieve a lot making use of silkworms as model animals. We would like to suggest that pharmaceutical companies and institutes consider the use of the silkworm as a model animal which is efficacious both for financial value by cost cutting and ethical aspects in animals' welfare.
Subject(s)
Animal Use Alternatives/ethics , Animal Use Alternatives/methods , Animal Welfare , Bombyx , Drug Evaluation, Preclinical/ethics , Drug Evaluation, Preclinical/methods , Animal Use Alternatives/economics , Animals , Bioethics , Drug Evaluation, Preclinical/economics , Drug Industry , Ethics, Research , JapanABSTRACT
In silkworm larvae, the mature form of paralytic peptide (PP), an insect cytokine, is produced from pro-PP in association with activation of innate immune responses, resulting in slow muscle contraction. We utilized this reaction, muscle contraction in silkworms coupled with innate immunity stimulation, to quantitatively measure the innate immune stimulating activity of various natural polysaccharides. ß-Glucan of Gyrophora esculenta (GE-3), fucoidan from sporophyll of Undaria pinnatifida, and curldan induced silkworm muscle contraction. We further demonstrated that GE-3 had therapeutic effects on silkworms infected by baculovirus. Based on these findings, we propose that the silkworm muscle contraction assay is useful for screening substances that stimulate innate immunity before evaluating therapeutic effectiveness in mammals.
Subject(s)
Drug Evaluation, Preclinical/methods , Immunity, Innate/drug effects , Muscle Contraction/drug effects , Polysaccharides/immunology , Polysaccharides/pharmacology , Animals , Bombyx , Hemolymph/metabolism , Larva/drug effects , Neuropeptides/metabolism , Nucleopolyhedroviruses/drug effects , beta-Glucans/pharmacologyABSTRACT
A polysaccharide was purified from a hot water extract of green tea leaves by measuring the immunostimulatory activity in silkworm larvae. Nuclear magnetic resonance and chemical analysis of acid hydrolysates revealed that the purified substance possessed a backbone containing polygalacturonic acids with methyl ester residues. Treatment with ß-glucanase attenuated the muscle contraction activity of the purified sample, suggesting that the ß-glucan structure, probably as a branched form, was required for its activity. The purified fraction stimulated the production of interleukin-6 by mouse peritoneal macrophages. These results suggest that measuring immunostimulation in silkworm larvae is useful for evaluating innate immunostimulants from various sources.
ABSTRACT
Sheep red blood cells (SRBCs) rapidly aggregated when injected into the blood (hemolymph) of living silkworms. SRBCs also rapidly aggregated when incubated with hemolymph in vitro. SRBCs did not aggregate when incubated with single hemolymph components, hemocytes and cell-free plasma separated by centrifugation, whereas incubation with the mixture of components induced SRBC aggregation, suggesting that both hemocytes and plasma are required for the reaction. Treatment of hemocytes with sodium azide inhibited SRBC aggregation. On the other hand, SRBCs pre-incubated with hemocytes aggregated in the plasma, even in the presence of sodium azide. SRBC aggregation was not observed when the SRBCs were physically separated from the hemocytes by a polycarbonate filter. These findings suggest that SRBCs are directly attacked by hemocytes and become sensitive to humoral factors that cause SRBC aggregation.
ABSTRACT
The therapeutic effect of dye compounds with antibacterial activity was evaluated in a silkworm model of Staphylococcus aureus infection. Among 13 chromogenic agents that show antibacterial activity against S. aureus (MIC = 0.02 to 19 µg/mL), rifampicin had a therapeutic effect. The ED(50) value in the silkworm model was consistent with that in a murine model. Other 12 dyes did not increase survival of the infected silkworms. We examined the reason for the lack of therapeutic efficacy. Amidol, pyronin G, and safranin were toxic to silkworms, which explained the lack of therapeutic effects. Fuchsin basic and methyl green disappeared quickly from the hemolymph after injection, suggesting that they are not stable in the hemolymph. Although coomassie brilliant blue R250/G250, cresyl blue, and nigrosin showed no toxic effects or instability in the hemolymph, they also did not have a therapeutic effect. The in vitro antibacterial actions of these dyes were inhibited by silkworm plasma or bovine serum albumin and filtration experiments demonstrated that cresyl blue bound to plasma proteins in the silkworm, suggesting that plasma protein binding inhibited the therapeutic efficacy of these four dyes. These findings indicate that drug screening using the silkworm infection model is useful for evaluating toxicity and pharmacokinetics of potential antibiotics.
Subject(s)
Bombyx , Staphylococcus aureus , Animals , Anti-Bacterial Agents/pharmacology , Bombyx/microbiology , Drug Evaluation, Preclinical , Hemolymph/metabolism , Staphylococcus aureus/drug effectsABSTRACT
SII-K1 is a member of the transcription elongation factor S-II family. In the mouse, SII-K1 is expressed exclusively in the liver, kidney, heart, and skeletal muscle. Here, we report that deletion of the SII-K1 gene in mice resulted in the downregulation of the synaptotagmin-like 1 (Sytl 1) gene in liver and of the coiled-coil domain-containing 21 (Ccdc21) gene in liver and kidney. Moreover, the induction of the metallothionein I (Mt I) gene in SII-K1-deficient mice liver was impaired in diethyl maleate-induced oxidative stress conditions. Our results suggest that SII-K1 regulates these genes in vivo.
Subject(s)
Metallothionein , Oxidative Stress , Animals , Kidney/metabolism , Liver/metabolism , Mice , Mice, KnockoutABSTRACT
Oseltamivir, an antiviral drug used for the treatment of influenza, contains the L-glutamic acid motif in its chemical structure. We focused on this structural characteristic of oseltamivir and examined the pharmacologic effects of the drug on the nervous system in invertebrate and vertebrate animal models. Injection of oseltamivir or L-glutamic acid into silkworm (Bombyx mori) larvae induced muscle relaxation. Oseltamivir and L-glutamic acid inhibited kainate-induced rapid muscle contraction, but neither drug affected insect cytokine paralytic peptide-induced slow muscle contraction. In the mammalian system, mice (Mus musculus) treated intracerebrally with oseltamivir developed convulsive seizures. Hydrolyzed oseltamivir, the active form containing a carboxylic acid, evoked epileptiform firing of hippocampal neurons in rat (Rattus norvegicus) organotypic hippocampal slice cultures. These results are the first to demonstrate that oseltamivir exerts pharmacologic effects on the nervous system in insects and mammals.
ABSTRACT
We constructed gene knockout mice lacking either the Duffy antigen (Dfy) or glycophorin A (GPA), major glycoproteins that are expressed on erythrocyte membranes, to examine the role of these proteins in malaria infection and erythrocyte. All of the rodent malarias examined proliferated in the erythrocytes of these knockout mice, indicating that neither the Duffy antigen nor GPA has an essential role as a receptor for malaria parasites. Duffy antigen knockout mice infected by Plasmodium yoelii 17XL exhibited autotherapy. At the early stage of the infection, the parasite proliferated exponentially, whereas at the late stage, parasitemia decreased to a level at which the mice were considered cured. The results of depletion experiments with anti-CD4 antibodies suggested that CD4-positive cells in the Duffy antigen knockout mice were responsible for the autotherapy effect. The Duffy antigen is a chemokine receptor. Compared to wild-type mice, chemokines which have affinities for the Duffy antigen injected intravenously more rapidly disappeared from the Duffy antigen knockout mice. Stimulation of the immune response by the increase of leukocytes might lead to the suppression of parasitemia in the Duffy antigen knockout mice. The absence of GPA decreased the amount of O-linked oligosaccharides on the erythrocyte membranes. The erythrocyte membranes of the GPA knockout mice decreased several O-linked glycoproteins and TER-119 protein. GPA has an essential role in the expression of O-linked antigens on erythrocyte membranes, but these proteins are not important for malaria parasite invasion of erythrocytes.
Subject(s)
Genome, Viral , Magnaporthe/virology , RNA Viruses/genetics , RNA, Viral/genetics , Base Sequence , Molecular Sequence Data , Open Reading Frames , Oryza/microbiology , Phylogeny , RNA Viruses/isolation & purification , RNA, Double-Stranded/genetics , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Viral Proteins/geneticsABSTRACT
Inhalation of airborne pollen causes irritative symptoms in humans, known as pollinosis. The changing global climate and increased pollution contribute to enhance the release of pollen, thereby increasing the number of people suffering from allergies. We examined the effect of spraying weak alkaline solutions onto cedar trees, the main allergenic culprit in Japan, on pollen release. Weak alkaline solutions were sprayed onto Japanese cedar blossoms to disrupt the external walls of the pollen, and to induce swelling of the cytosolic components containing the nucleus. This morphologic change of the pollen grains depended on the pH of the suspending solution, with a threshold pH of near 7.5. As the breakdown of the external walls and swelling of the cytosolic components are inhibited by high osmolarity, the influx of water triggered the morphologic changes. Weak alkaline solutions sprayed onto cedar blossoms decreased the amount of pollen released from the anthers in a pH dependent manner. The addition of detergent to the sodium bicarbonate solution facilitated this effect on cedar pollen release. We suggest that spraying cedar and cypress forests with a weak alkaline solution might prevent the scattering of pollen that causes allergies in humans.
ABSTRACT
We examined the pattern of the N gene-mediated systemic hypersensitive response (HR), which was induced by tobacco mosaic virus upon temperature shift, and analyzed the distribution of the coat protein and the virus-encoded 126 kDa replicase protein (126 K protein) by immunoblot analysis. In the middle- and lower-positioned leaves, HR occurred in the advancing edge of the infected area, where we detected both the coat protein and the 126 K protein. In the areas between the main vein and the advancing edge of these leaves, we observed no HR and did not detect 126 K protein, though virus was present in these areas. In the upper-positioned mosaic leaves, patterns of the HR were different depending on the leaf age. In these mosaic leaves, the mechanism preventing the virus from invading dark green tissue seemed to be broken down in 8-14 days old leaves, and HR was observed only in the tissue just invaded by the virus, where we detected the 126 K protein. Those results suggested that the viral 126 K protein was present when the viral replication was taking place, and easily degraded when the amount of the virus was saturated in the cells.
Subject(s)
Genes, Plant , Nicotiana/genetics , Nicotiana/virology , RNA-Dependent RNA Polymerase/metabolism , Tobacco Mosaic Virus/physiology , Capsid Proteins/analysis , Temperature , Virus ReplicationABSTRACT
An 81-year-old female found to have an aneurysm in the distal aortic arch was successfully treated with arch translocation method. Under selective cerebral perfusion, a stent graft bound to a nephrostomy balloon catheter with a chainstitch was inserted from the ascending aorta into the descending aorta and its proximal end was sutured together with the distal aortic stump. Then a 4-branched Dacron graft replaced the ascending aorta reconstructing cephalobracheal branches individually. Each proximal end of cephalobracheal branches was ligated. The patient had no neurological deficit except for recurrence nerve palsy that had already existed before the operation. Postoperative angiography revealed complete thrombocclusion of the aneurysm.
Subject(s)
Aortic Aneurysm, Thoracic/surgery , Stents , Aged , Aged, 80 and over , Female , Humans , Nephrostomy, Percutaneous/instrumentationABSTRACT
We have cloned a novel ABC transporter gene PMR5 from the phytopathogenic fungus Penicillium digitatum by RT-PCR using degenerate primers. The deduced amino acid sequence of PMR5 showed 37% identity to PMR1 from the same fungus, 71% identity to AtrB from Aspergillus nidulans, and 65% identity to BcatrB from Botrytis cinerea. Disruption mutants for PMR5 were generated in two independent P. digitatum strains and their phenotypes were characterized. These mutants displayed increased sensitivity to thiabendazole (a benzimidazole), benomyl (a benzimidazole), dithianon (a quinone), resveratrol (the phytoalexin of grape), and camptothecin (an alkaloid). Delta pmr1 disruption mutants were previously reported to show resistance to demethylation inhibitors (DMIs). These mutants were found also to display increased sensitivity to phloretin (the phytoanticipin of apples), camptothecin and oligomycin (an antibiotic). Transcription of PMR1 and PMR5 was strongly induced in response to several toxicants, including DMIs that specifically induced PMR1. In contrast, dithianon and resveratrol specifically induced PMR5 transcription. These findings indicate that expression of the two ABC transporter genes is regulated differently, and that they have complementary roles in multidrug resistance, with each having different substrate-specificities.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Bacterial Proteins , Drug Resistance, Multiple, Fungal/genetics , Genes, Fungal , Penicillium/genetics , Amino Acid Sequence , Antifungal Agents/pharmacology , Aspergillus nidulans/genetics , Base Sequence , Botrytis/genetics , Cloning, Molecular , DNA, Fungal/genetics , Gene Expression Regulation, Fungal/drug effects , Molecular Sequence Data , Mutation , Penicillium/drug effects , Penicillium/pathogenicity , Plants/microbiology , Transcriptional Activation/drug effectsABSTRACT
A remarkable effect of (C(6)F(5)O)(3)Al for promoting the rearrangement of epoxy acylates via stable cation intermediates was found, and new methods for constructing chiral benzylic, vinylic, and acetylenic quaternary carbon centers were developed. During the study, the importance of the ionic nature of the O-metal bond in the intermediates of such epoxides was addressed. This method was applied to the asymmetric total synthesis of (S)-(+)-sporochnol A.
Subject(s)
Alkenes/chemical synthesis , Aluminum , Epoxy Compounds/chemistry , Organometallic Compounds/chemistry , Phenols/chemical synthesis , Alkenes/chemistry , Cations , Eukaryota/chemistry , Indicators and Reagents , Phenols/chemistry , StereoisomerismABSTRACT
OBJECTIVES: We sought to determine the electrocardiographic (ECG) features associated with acute left main coronary artery (LMCA) obstruction. BACKGROUND: Prediction of LMCA obstruction is important with regard to selecting the appropriate treatment strategy, because acute LMCA obstruction usually causes severe hemodynamic deterioration, resulting in a less favorable prognosis. METHODS: We studied the admission 12-lead ECGs in 16 consecutive patients with acute LMCA obstruction (LMCA group), 46 patients with acute left anterior descending coronary artery (LAD) obstruction (LAD group) and 24 patients with acute right coronary artery (RCA) obstruction (RCA group). RESULTS: Lead aVR ST segment elevation (>0.05 mV) occurred with a significantly higher incidence in the LMCA group (88% [14/16]) than in the LAD (43% [20/46]) or RCA (8% [2/24]) groups. Lead aVR ST segment elevation was significantly higher in the LMCA group (0.16 +/- 0.13 mV) than in the LAD group (0.04 +/- 0.10 mV). Lead V(1) ST segment elevation was lower in the LMCA group (0.00 +/- 0.21 mV) than in the LAD group (0.14 +/- 0.11 mV). The finding of lead aVR ST segment elevation greater than or equal to lead V(1) ST segment elevation distinguished the LMCA group from the LAD group, with 81% sensitivity, 80% specificity and 81% accuracy. A ST segment shift in lead aVR and the inferior leads distinguished the LMCA group from the RCA group. In acute LMCA obstruction, death occurred more frequently in patients with higher ST segment elevation in lead aVR than in those with less severe elevation. CONCLUSIONS: Lead aVR ST segment elevation with less ST segment elevation in lead V(1) is an important predictor of acute LMCA obstruction. In acute LMCA obstruction, lead aVR ST segment elevation also contributes to predicting a patient's clinical outcome.
Subject(s)
Coronary Stenosis/diagnosis , Coronary Vessels , Electrocardiography/methods , Acute Disease , Adult , Aged , Analysis of Variance , Collateral Circulation , Coronary Angiography , Coronary Stenosis/classification , Coronary Stenosis/mortality , Coronary Stenosis/physiopathology , Coronary Stenosis/therapy , Discriminant Analysis , Electrocardiography/instrumentation , Electrocardiography/standards , Female , Hemodynamics , Humans , Linear Models , Male , Middle Aged , Patient Selection , Predictive Value of Tests , Prognosis , Retrospective Studies , Sensitivity and Specificity , Treatment OutcomeABSTRACT
A simple method for detecting sterol demethylation inhibitor (DMI)-resistant strains of the citrus green mould pathogen, Penicillium digitatum, has been developed. The method involves detection of a tandem repeat of a transcriptional enhancer in the promoter region of PdCYP51, which encodes the target enzyme of DMIs, by PCR, using conidia as template. The presence of the tandem repeat leads to overexpression of this gene and confers DMI resistance to the fungus. We examined the relationship between the presence of the tandem repeat and DMI resistance in 39 strains of P digitatum. The results suggested that the DMI resistance mechanism based on the tandem repeat is common in this fungus. Using this method, the presence of DMI resistance of this fungus can be detected within 5-6 h.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Fungicides, Industrial/pharmacology , Oxidoreductases/genetics , Penicillium/drug effects , Polymerase Chain Reaction/methods , Tandem Repeat Sequences/genetics , Cytochrome P-450 Enzyme System/metabolism , DNA, Fungal/genetics , Drug Resistance, Microbial , Enhancer Elements, Genetic/genetics , Mycelium/genetics , Oxidoreductases/metabolism , Penicillium/genetics , Penicillium/metabolism , Promoter Regions, Genetic/genetics , Spores/genetics , Sterol 14-Demethylase , Transcription, GeneticABSTRACT
We treated a rare case of malignant fibrous histiocytoma (MFH) of soft tissue that produced granulocyte colony-stimulating factor (G-CSF). The patient, an 80-year-old woman, was admitted because of a feeling of abdominal fullness and leg edema. An elastic, hard, tender tumor was palpated in the right thigh. Histopathological examinations of the tumor revealed atypical and prominent pleomorphic spindle-shaped cells with funicular arrangement, which were compatible with the diagnosis of MFH. Prominent leukocytosis (up to 84,300/microl), a high serum G-CSF concentration (82 pg/ml) and positive immunohistochemical staining of the tumor tissue for G-CSF indicated that G-CSF was produced by the MFH.
Subject(s)
Granulocyte Colony-Stimulating Factor/biosynthesis , Histiocytoma, Benign Fibrous/metabolism , Liver Neoplasms/metabolism , Soft Tissue Neoplasms/metabolism , Aged , Aged, 80 and over , Female , Histiocytoma, Benign Fibrous/secondary , Humans , Leg , Liver Neoplasms/pathology , Soft Tissue Neoplasms/secondaryABSTRACT
The athlete's heart is commonly characterized by an increase in left ventricular mass because of an increase in the left ventricular diastolic cavity dimensions or wall thickness or both. Endurance exercise also induces numerous cardiovascular adaptations, including increased vagal tone. However, the sympathetic function has not yet been precisely elucidated, so the present study evaluated cardiac sympathetic nerve function from metaiodobenzylguanidine (MIBG) images obtained 15 and 180 min after the injection of 123I-MIBG at a dose of 111 MBq. The ratio of heart/mediastinum count (H/M) and the washout rates of 123I-MIBG (WR) were calculated in 25 consecutive patients who were athletes (aged 52+/-13 years) and 23 normal subjects. There was a significant difference in the H/M between the athletic and normal hearts (2.3+/-0.3 vs 2.6+/-0.3, p<0.01, Scheffe's test). An increased WR was observed in the athletes group when compared with the normal group (34+/-4 vs 28+/-3, p<0.01), and there was a significant correlation between WR and the left ventricular mass index (r=0.578, p<0.01). Prolonged exercise training may alter cardiac sympathetic nerve function, which can be detected by MIBG imaging.
Subject(s)
Heart/physiopathology , 3-Iodobenzylguanidine , Adult , Aged , Female , Heart/diagnostic imaging , Heart/innervation , Heart Diseases/physiopathology , Humans , Male , Middle Aged , Radionuclide Imaging , Sports , Sympathetic Nervous System/physiopathologyABSTRACT
Severe mitral regurgitation was associated with cardiogenic shock in five (0.8%) of 623 patients with acute myocardial infarction who were urgently admitted to the authors' hospitals between 1994 and 1996. The infarct was located in the inferior wall in four patients and in the inferoposterior wall in one patient. Severe mitral valve regurgitation occurred concurrently with cardiogenic shock between one and six days after the onset of myocardial infarction. A mitral regurgitant murmur was not audible in four of five patients. Similarly, mitral regurgitant Doppler signals were not detected in four patients by transthoracic echocardiographic examination, while transesophageal echocardiographic examination detected mitral regurgitant signals clearly in all patients. Thus, when cardiogenic shock is unexpectedly associated with inferior or inferoposterior wall acute myocardial infarction, severe mitral regurgitation should be suspected, even when a mitral regurgitant murmur is not audible. Furthermore, mitral regurgitant flow signals may not always be detected by transthoracic echocardiography. Thus, examination for mitral regurgitation by transesophageal echocardiography should be considered.