Interaction of Arc with CaM kinase II and stimulation of neurite extension by Arc in neuroblastoma cells expressing CaM kinase II

We investigated the relationship between Arc (activity-regulated cytoskeleton-associated protein) and Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). Arc and CaM kinase II were concentrated in the postsynaptic density. These proteins were accumulated after electroconvulsive treatment. Arc increased about 2.5-fold within 30 min and was maintained at this level for 8h after the stimulation. CaM kinase II also increased within 30 min and remained at this level for at least 24h. The interaction of Arc with CaM kinase II was demonstrated using GST-Arc fusion protein, and confirmed in neuroblastoma cells by immunoprecipitation. We examined the function of Arc by introducing Arc cDNA into neuroblastoma cells expressing CaM kinase II. The cells expressing both Arc and CaM kinase II had longer neurites than those expressing CaM kinase II alone. Arc itself did not promote neurite outgrowth. The growth of neurites by Arc was completely blocked by treatment with KN62, an inhibitor of CaM kinases. These results indicated that Arc potentiated the action of CaM kinase II for neurite extension.     

Simplified Quantitative Assay System for Measuring Activities of Drugs against Intracellular Legionella pneumophila

We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-α agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumophila were determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-α broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples.     

Afterdepolarization mechanism in the in vitro, cesium-loaded, sympathetic preganglionic neuron of the cat

Intracellular recordings were performed in Cs-loaded sympathetic preganglionic neurons (SPNs) of the intermediolateral nucleus, identified by antidromic stimulation, in the slice of the T2 or T3 segment of the cat spinal cord. Loading the neurons with Cs resulted in broadening of the action potential, depression of the fast component of the afterhyperpolarization (AHP), and appearance of an afterdepolarization (ADP). A typical ADP in a Cs-loaded neuron had time to peak of 45-110 ms, half-decay time of 70-250 ms, and amplitude of 2-10 mV at membrane potentials between -60 and -70 mV and at a Ca and K concentration of 2.5 and 3.6 mM, respectively, in the superfusion medium. The ADP was associated with a decrease in neuron input resistance and increased in magnitude with hyperpolarization of the cell membrane. The relation between peak ADP amplitude and membrane potential was linear within the range of membrane potentials from -60 to -100 mV. The ADP was reversibly suppressed by the Ca-channel blocker cobalt (2 mM) or by low Ca Krebs solution (0.25 mM). Superfusion with BaCl2 (1.0 mM) or tetraethylammonium (TEA) (10-20 mM) caused an increase in amplitude of the ADP and an increase in action potential duration. Hyperpolarizing pulses, delivered during the course of the spike shoulder, resulted in a decrease of spike duration and ADP amplitude. The ADP was not affected by tetrodotoxin, at a dose blocking the Na-spike, and was enhanced, in association with an increase in action potential duration, when NaCl in the Krebs solution was replaced with choline chloride. Increasing intracellular Cl concentration or decreasing extracellular Cl concentration had no effect on the ADP. Changes in external K concentration from 3.6 to 10 or 0.36 mM increased and decreased, respectively, the amplitude of the ADP. In the absence of Cs, and ADP, with similar time course to that recorded in Cs-loaded SPNs, was recorded when CaCl2 was replaced by BaCl or NaCl was replaced by TEAC1. It is concluded that the SPN afterpotential includes a Ca-dependent inward current, in addition to the already described fast and slow outward K currents of the AHP.     

Construction and characterization of a fimA mutant of Porphyromonas gingivalis.

Although fimbriae of Porphyromonas gingivalis have been implicated as playing a major role in adherence to gingival tissue surfaces, no conclusive genetic evidence has yet been obtained. The fimA gene, the determinant for the major fimbrial subunit protein, was cloned and sequenced (D. P. Dickinson, M. A. Kubiniec, F. Yoshimura, and R. J. Genco, J. Bacteriol. 170:1658-1665, 1988). We undertook to inactivate the fimA gene by a homologous recombination technique and examined the fimA mutant for changes in surface properties, including production of fimbriae, adherence to human gingival fibroblasts and epithelial cells, hemagglutinating activity, and surface hydrophobicity. To inactivate the fimA gene, we disrupted a fimA clone by insertion of a DNA segment containing an erythromycin resistance (Emr) gene. This was then delivered into P. gingivalis ATCC 33277 from an Escherichia coli K-12 strain, SM10 lambda pir, by using a mobilizable suicide vector, pGP704; recombination at the fimA locus led to the isolation of a fimA mutant. Disruption of the fimA locus and disappearance of FimA production were confirmed by Southern hybridization with a fimA-specific DNA probe and Western immunoblotting with a monoclonal antibody against the FimA protein, respectively. The fimA mutant constructed failed to express long (0.5- to 1.0-micron) fimbriae from the bacterial surface and had a diminished adhesive capacity to tissue-cultured human gingival fibroblasts and epithelial cells. Observation of the bacteria adhering to human gingival fibroblasts by scanning electron microscopy revealed that the wild-type strain had dramatic local changes in the appearance of the microvilli at the point of contact with large bacterial clumps, whereas the fimA mutant did not. In contrast, neither the hemagglutinating activity nor the surface hydrophobicity was changed in the fimA mutant. These data thus constitute the first direct genetic evidence demonstrating that the FimA protein of P. gingivalis is essential for the interaction of the organism with human gingival tissue cells through a function(s) encoded by the fimA gene.     

Dna ploidy of pheochromocytoma on cytology specimen by image analysis

Pheochromocytoma usually shows prominent nuclear atypia, but the presence of such atypical cells is known to be an unreliable predictor of malignancy. DNA ploidy of pheochromocytomas has been analyzed by flow cytometry or photospectrometry on paraffinem-bedded tissue, but the results were controversial. We performed DNA analysis on cytology specimens of 11 pheochromocytomas using an image analysis system. All tumors had a mixed pattern of a large population of diploid cells and a small population of polyploid cells. DNA content correlated with nuclear size, and larger cells had more DNA content. Such larger tumor cells had polyploid nuclei, such as 4 C, 8 C, 16 C, and 32 C, in both malignant and benign pheochromocytomas. The larger polyploid nuclei may result from difficulty of duplication at the mitotic phase of the cell cycle.     

Wegener's granulomatosis. Associated with diffuse pulmonary hemorrhage.

The authors report a case of Wegener's granulomatosis with the unusual manifestation of diffuse pulmonary hemorrhage. A 58-year-old man complained of bloody sputum and fever. Chest X-ray films showed multiple nodular shadows in both lung fields. He was diagnosed as having Wegener's granulomatosis by transbronchial lung biopsy, which revealed necrotizing granulomatous inflammation with necrotizing vasculitis. Despite treatment with cyclophosphamide and prednisolone, his condition rapidly deteriorated. An extensive diffuse alveolar shadow appeared in both lung fields in chest X-ray films, anemia became worse, and he died of respiratory failure. Autopsy revealed diffuse alveolar hemorrhage with necrotizing capillaritis in addition to the typical pathological findings in Wegener's granulomatosis. The capillaritis was characterized by neutrophilic infiltration of alveolar septa, and fibrin thrombi in alveolar capillaries. Diffuse pulmonary hemorrhage is uncommon in Wegener's granulomatosis. However, once diffuse pulmonary hemorrhage occurs, the respiratory condition rapidly deteriorates and is life-threatening. Therefore, accurate diagnosis and appropriate treatment are required.     

Regulation of IgE receptor expression on human peripheral blood lymphocytes by lymphocytosis promoting factor (LPF), lectins and dexamethasone.

Using a monoclonal anti-human Fc epsilon R antibody (H107), we found that lymphocytosis promoting factor (LPF), phytohaemagglutinin (PHA-P) and Concanavalin A (Con A) could induce Fc epsilon R, detected by immunofluorescence study, on normal human peripheral blood lymphocytes without IgE. The number of Fc epsilon R bearing lymphocytes was increased by stimulation with 3, 10 and 10 micrograms/ml of LPF, PHA-P and Con A, respectively, from 6.0 +/- 3.0/1000 cells to 26.0 +/- 7.9, 54.0 +/- 6.7 and 24.8 +/- 7.1/1000 cells, respectively. Although the induction of Fc epsilon R occurred neither in the separated T-enriched fraction (TEF) nor the T-depleted fraction (TDF), it recovered when the two fractions were mixed. The cell free supernatants from TEF stimulated with LPF or PHA-P could increase Fc epsilon R(+) cells in TDF, whereas those from TDF failed to increase them in TEF. The results suggest that the induction of Fc epsilon R occurs mainly on B lymphocytes by the soluble factor(s) formed by T cells stimulated with LPF or PHA-P. The induction of Fc epsilon R by stimulants was completely inhibited by 10(-6) M dexamethasone. It was demonstrated that the effects of dexamethasone on lymphocytes were dual: one was on B cells to inhibit responsive increases of Fc epsilon R, and the other was on T cells to suppress the formation of the soluble factor(s) which induced Fc epsilon R on B cells.     

Molecular targeted treatment--new treatment strategy for patients with chronic myeloid leukemia

Imatinib mesylate is a new drug that can inhibit the tyrosine kinase activity of Bcr-Abl, the receptors for platelet-derived growth factor receptor(PDGF) and stem cell factor, or c-kit. Chronic myeloid leukemia (CML) is distinguished by the presence of a reciprocal translocation between chromosomes 9 and 22 that results in a shortened chromosome 22, termed the Philadelphia(Ph) chromosome. As a result of the translocation, a fusion gene called the Bcr-Abl gene is created from two normal cellular genes, encoding a chimeric Bcr-Abl protein with a deregulated tyrosine kinase activity. The expression of Bcr-Abl tyrosine kinase has been shown to be necessary and sufficient for the transformed phenotype of CML cells. Imatinib can block the kinase activity of Bcr-Abl, thus inhibiting the proliferation of Ph-positive progenitors, and has shown activity against all phases of CML, though responses are most substantial and durable in patients in the chronic phase. An international phase III study which compared the efficacy of imatinib with that of interferon alpha combined with low-dose cytarabine in newly diagnosed chronic-phase CML showed the rate of major cytogenetic response at 24 months was 90%, including 82% of complete cytogenetic response. These results indicated that imatinib was superior to interferon-containing treatment as a first-line therapy. More than 10,000 patients worldwide, including those in Japan, have been treated with imatinib in clinical trials, and a lot of information has been accumulated on the use of this drug. The aim of this article is to review the use of this drug and the practical management of patients with chronic myeloid leukemia.     

Anticoagulants preventing pseudo-thrombocytopenia]

It is well known that some anticoagulants, especially EDTA (ethylene diamine tetraacetic acid), sometimes lead to pseudo-thrombocytopenia due to the aggregation of thrombocytes. We evaluated appropriate anticoagulants that prevent pseudo-thrombocytopenia without affecting other hematological data. In this study, 10 mg EDTA-2K and 1 mg citric acid were added to 1 ml of blood as anticoagulants. Using these anticoagulants (EC method), an aggregation of thrombocytes was clearly inhibited and the platelet counts remained stable in 10 cases with pseudo-thrombocytopenia. This method did not affect the other blood cell counts, the stainability and the morphology of the cells. Since the addition of citric acid to EDTA could prevent the cell volume change induced by the high concentration of EDTA, the micro-hematocrit values were remained unchanged. Hematological data in 20 cases which did not show any pseudo-thrombocytopenia, correlated significantly between the EDTA method and the EC method, and did not change significantly 24 hours after blood sampling. It is concluded that EDTA and citric acid may be a useful combination of anticoagulants for the prevention of pseudo-thrombocytopenia.