Expression and characterization of mouse angiotensin II type 1a receptor tagging hemagglutinin epitope in cultured cells

The octapeptide angiotensin II mediates the physiological actions of the renin-angiotensin system through activation of several angiotensin II receptor (AT) subtypes, in particular AT1 (AT1a and AT1b in the case of rodents). Although we and others have generated mutant mice in which the AT1a gene was disrupted, the function of mouse AT1 remains to be fully elucidated, due to the lack of effective tools involving antibodies against AT1 for detecting biological responses in cellular conditions. To avoid these problems, we constructed the hemagglutinin (HA)-tagged mouse AT1a, and stably introduced this recombinant receptor into human embryonic kidney 293-T cells. Radioligand binding of [(125)I] angiotensin II to AT1a was specific, saturable, and reversible. Scatchard analysis demonstrated that the transfected receptor had a dissociation constant of 1.7 nM with a density of 1.2 x 10(5) sites/cells. Angiotensin II stimulated a rapid increase in cytosolic free calcium, and angiotensin II-induced phosphorylation of extracellular signal-regulated kinases (Erk) was found in a dose-dependent manner. After solubilization, Western blot analysis showed specific interactions between an anti-HA antibody and HA-tagged mouse AT1a. Furthermore, a significant proportion of HA-tagged mouse AT1a was specifically immunoprecipitated with this antibody. In the immunocytochemical and electronmicroscopic studies, treatment of this cell line with angiotensin II resulted in decrease in signals of the surface receptors. Based on these results, the cell line established here provides an excellent tool for studying angiotensin II actions mediated through mouse AT1a, at sub-nanomolar concentrations.     

Hyperplastic epithelial foci in honeycomb lesions in idiopathic pulmonary fibrosis

Seventy-two cases of idiopathic pulmonary fibrosis (IPF) were examined from 2856 consecutive autopsy cases at the Japanese Red Cross Medical Center in Tokyo from 1973-1996. Primary lung cancer had arisen in 31 of 72 cases of IPF (43%), significantly higher than the incidence in cases without IPF (8.1%) and in the cases with non-IPF chronic lung diseases (11.9%). Hyperplastic epithelial foci in the honeycomb lesions of IPF cases were significantly more prominent in the lower than in the upper lobe, in cases with or without lung cancer, and they were more prominent in the lower lobe of IPF with than in those without cancer. The length of hyperplastic epithelial foci in the lower lobe of IPF cases was longer than that in interstitial pneumonia-associated with collagen vascular diseases. There was a higher PCNA labeling index of hyperplastic epithelial foci in IPF cases than in cases of interstitial pneumonia-associated with collagen vascular diseases. The PCNA labeling index was almost the same between smokers and nonsmokers with IPF. Overexpression of p53 was observed in hyperplastic epithelial foci in honeycomb lesion of IPF. DNA ploidy analysis of hyperplastic epithelial foci in the paraffin sections of 12 IPF cases revealed aneuploidy patterns in eight cases. These results strongly suggest that accelerated cell proliferation occurs in the honeycomb lesion of IPF, and that regenerative epithelia becomes susceptible to carcinogenic agents in addition to the smoking effect.     

Molecular characterisation of glutamate dehydrogenase gene defects in Japanese patients with congenital hyperinsulinism/hyperammonaemia

Congenital hyperinsulinism and hyperammonaemia (CHH) is caused by dysregulation of glutamate dehydrogenase (GDH). We characterised the GDH gene in two Japanese patients with CHH. Patient 1 showed late-onset and mild hypoglycaemic episodes and mild hyperammonaemia, compared with patient 2. In GDH activity of lymphoblasts, patient 1 showed twofold higher basal GDH activity than control subjects and mild insensitivity for GTP inhibition. Patient 2 showed severe insensitivity for GTP inhibition, and similar allosteric stimulation by ADP in the controls. Genetic studies identified heterozygous and de novo L413V and G446D mutations in patients 1 and 2, respectively. COS cell expression study confirmed that both mutations were disease-causing gene. The insensitivity for GTP inhibition in L413V and G446D was emphasised in COS cell expression system as a result of the dosage effect of mutant GDH gene. L413V showed less impairment of GDH than G446D based on biochemical and genetic results, which was consistent with the clinical phenotype. Based on the structure of bovine GDH, G446D was located in GTP binding site of pivot helix and its surroundings, while L413V was located in alpha-helix of antenna-like structure. These different locations of mutations gave different effects on GDH enzyme. The antenna-like structure plays an important role in GDH activity.     

Three Kinds of Foamy Cells in the Spleen: Comparative Histochemical and Ultrastructural Studies

By light and electron microscopy, we observed foamy cells in the spleens from a patient with hemolytic anemia due to red cell adenosine deaminase (ADA) overproduction, a patient with rheumatoid arthritis (RA) treated with gold, and patients with idiopathic thrombocytopenic purpura (ITP)

The foamy cells associated with red cell ADA overproduction were essentially similar to Gaucher-like cells described in patients with thalassemia, and it was suggested that the accelerated destruction of red cells was one of the factors responsible for the development of foamy cells. Foamy cells in ITP and RA were closely associated with an increased destruction of platelets in the spleen. Morphologic transitions between phagocytosed platelets and myelinlike materials were traced in these disorders. In RA, however, foamy cells were heterogeneous from an ultrastructural standpoint, with different cytoplasmic inclusions. In addition to myelinlike materials, dense bodies, vacuoles with flocculent materials, and gold were noted in most of foamy cells. As gold compounds are known to inhibit lysosomal enzymes, we surmise that an acquired disturbance in lysosomal digestion is partially responsible for the accumulation of intermediate metabolites.

In the pathogenesis of foamy cells associated with blood cell dyscrasia, the accelerated destruction of blood cells and/or acquired disorders in catabolic pathways within the macrophages are suggested to be the underlying mechanism of an intralysosomal accumulation of incompletely degraded cellular debris.     

Polyovular follicles in the ovary of immature mice exposed prenatally to diethylstilbestrol

Summary Polyovular follicles (PF) occur in the ovary of 30-day-old offspring of ICR/JCL mice given 4 daily subcutaneous injections of 20–2,000 g diethylstilbestrol (DES)/day from days 15 to 18 of gestation. PF containing 2–9 oocytes per follicle in the prenatally DES-exposed mice are increased 33- to 112-fold as compared to controls. In 5- to 25-day-old offspring of mothers given injections of 2,000 g DES/day, PF are observed 17–65 times more frequent than in controls.     

Investigation of optimal drug in moderate bronchial asthma]

BACKGROUND: Many drugs and the combinations of drugs are recommended for each treatment step in bronchial asthma. However, there are few issues examined about the optimal drug and combination of drugs in a long term prognosis. In this study, we investigated the optimal drugs and combinations of drugs from a point of view of prognosis. METHODS: One hundred and ninety four patients who visited our hospital for treatment from November, 2003 to October, 2004 and were managed according to GINA guideline were surveyed retrospectively. We compared the rate of step up and the frequency of urgent visit and urgent hospitalization in one year between drug groups in each treatment step. RESULTS: The rate of step up was significantly higher in leukotriene receptor antagonist (LTRA) group than in inhalation corticosteroid (ICS) group and theophylline group in Step 2. The frequency of urgent visit and urgent hospitalization was significantly higher in ICS+LTRA group than in ICS+theophylline group and ICS+long-acting beta 2-agonist (LABA) group in Step 3. CONCLUSION: There is a possibility that the prognosis becomes bad when we use LTRA in the practical treatment according to GINA guideline.     

Molecular and biochemical mechanisms ofPasteurella haemolyticaleukotoxin-induced cell death

Pasteurella haemolyticaleukotoxin (LKT) is a member of the RTX family of pore-forming toxins that kill bovine immune cells. Several studies have suggested that RTX toxins kill target cells by the induction of apoptosis. In the present study, BL3 bovine leukaemia cells were exposed to LKT and assessed by molecular and flow cytometric techniques that measure different aspects of apoptotic cell death. The intoxicated cells demonstrated morphological, light scatter and Hoechst 33258 staining characteristics consistent with cells undergoing apoptosis. The cells also exhibited internucleosomal DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage, both indicators of apoptosis. LKT-treated cells bound annexin-V-FITC indicating that phosphatidylserine groups were translocated from the inner to the outer leaflet of the cell membrane. The effect of LKT on cells was dose dependent and inhibitable by incubation with anti-LKT monoclonal antibody. Finally, an early step for induction of apoptosis appears to be the binding of LKT to a β2 integrin since pre-incubating cells with anti-β2 integrin antibodies inhibited LKT-induced apoptosis. This study provides new insights into understanding the pathogenesis of bovine pasteurellosis and could lead to the development of both preventative and therapeutic strategies for disease management.     

Heparin-induced thrombocytopenia (HIT): pathogenesis, laboratory test, and treatment

Heparin-induced thrombocytopenia(HIT) due to immunological mechanisms is known as an important adverse reaction to heparin treatment, and heparin treatment should be applied while keeping in mind the risk of onset of HIT 5-14 days after the initiation of heparin. The presence of HIT had not been fully recognized in clinical practice in Japan despite the management of HIT being well confirmed in Western countries. Recognition of HIT has increased since argatroban, a direct thrombin inhibitor, obtained the approval of the FDA for prevention and treatment of HIT. Although the incidence of HIT in Japan has not yet been clarified, there is some evidence that HIT is encountered in critically ill patients undergoing heparin anticoagulation. Clinical diagnosis of HIT is performed by means of thrombocytopenia of a drop of 50% or 100 x 10(30/microl for 5 -14 days after starting heparin treatment. Confirmatory laboratory tests examine whether the patients have antibodies against heparin/PF4 complexes or not. Two assay tests for detecting heparin/PF4 complex antibodies are available in Japan. As a functional test, the heparin-induced platelet aggregation method is easily performed and the result is obtained in a short time. The result of the test has, however, been misleading due to the selection of donors. Low platelet activity of the donors on the addition of heparin induces a negative response in spite of positive antibodies in the sample. Before testing samples, it is important to check heparin reactivity of the donor's platelets. Enzyme immunoassay detecting the antibodies is available as a commercial kit. Sensitivity obtained by enzyme immunoassay is very high and often introduces false-positives. Careful attention to interpretation of the result is required. Treatment of HIT should be started at the time of recognition of thrombocytopenia while antibody testing for HIT is performed. As an alternative anticoagulant to heparin, argatroban should immediately be applied to avoid complication of thrombosis. Thrombocytopenia and hypercoagulability quickly recover to the preheparin level by the appropriate use of argatroban.