Sequential compound exocytosis of large dense-core vesicles in PC12 cells studied with TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis

We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular polar tracers (TEP imaging) at the basal region of PC12 cells adjacent to the glass cover slip. TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis revealed that most exocytosis was mediated by large dense-core vesicles (LVs) with a mean diameter of 220 nm, and that exocytosis of LVs occurred slowly with a mean latency of ∼7 s even though exocytosis was induced with large increases in cytosolic Ca²⁺ concentration by uncaging of a caged-Ca²⁺ compound. We also found that 97% of exocytic LVs remained poised at the plasma membrane, 72% maintained their fusion pores in an open conformation for more than 30 s, and 76% triggered sequential compound exocytosis of vesicles that were located deeper in the cytosol. Sequential compound exocytosis by PC12 cells was confirmed by electron microscopic investigation with photoconversion of diaminobenzidine by FM1-43 (a polar membrane tracer). Our data suggest that pre-stimulus docking of LVs to the plasma membrane does not necessarily hasten the fusion reaction, while docking and resulting stability of exocytic LVs facilitates sequential compound exocytosis, and thereby allowing mobilization of deep vesicles.     

Characterization of enteropathogenic and enteroaggregative Escherichia coli isolated from diarrheal outbreaks.

Virulence characteristics of diarrheal outbreak-associated Escherichia coli O55:NM, O126:NM, and O111:NM were examined. The E. coli O55:NM strains were atypical enteropathogenic E. coli (EPEC), while the E. coli O126:NM and O111:NM strains should be classified as enteroaggregative E. coli (EAggEC). The contributions of EPEC and EAggEC to the human disease burden in Japan might be significantly greater than is currently appreciated.     

Normal development of the gut-associated lymphoid tissue except Peyer's patch in MyD88-deficient mice

MyD88 is a key adaptor molecule for signalling via Toll-like receptors (TLRs) and the response to gut commensal microbes. To investigate the role of TLRs/MyD88 pathway in the development of the gut-associated lymphoid tissue (GALT), we examined the development of Peyer's patches (PPs) and cryptopatch (CP), and also one of effector compartment, intraepithelial lymphocyte (IEL) in MyD88-/-, TLR2-/- and TLR4-/- mice. In MyD88-/- mice, the organogenesis of PPs was not disturbed. However, PPs in 2-week-old MyD88-/- mice were significantly smaller than those in MyD88+/- mice. Also, in 2-week-old TLR4-/-, but not TLR2-/- mice, PPs did not develop rapidly. The development of PPs in MyD88-/- and TLR4-/- mice was completely recovered in 10 weeks. PP cells from MyD88-/- mice showed significant decrease in proliferation when stimulated with lipopolysaccharide. The development of CP and IEL was also normal in 10-week-old MyD88-/- mice. These results suggest that the TLRs/MyD88 pathway might be involved in the development of PPs only at early postnatal stage, and TLRs/MyD88-independent signalling is critically involved in the development of GALT in adult mice.     

Strain combination-dependent genesis of necrotizing arteritis in anti-ICAM-1 antibody-perfused renal allografts in the rat

Rat kidneys were perfused with anti-intercellular adhesion molecule-1 (anti-ICAM-1) monoclonal antibody prior to allo-transplantation. In the two strain combinations examined, LEJ-to-WKAH transplants resulted in accelerated graft loss, and no prolongation of graft survival. The accelerated graft logs was the resut of frequent occurrence of necrotizing arterttis wlthln the grafts. In contrast, TO-to-WKAH transplants resulted in no change In graft survival and no arteritis. Necratidng vasculitis in the LEJ-to-WKAH grafts was characterlzed by flbrinoid necrosis, collection of cellular infiltrates and serum macromolecular protein entrapment. The F(ab')² form of anti-ICAM-1 antlbody partially preserved the antibody's capacity to accelerate graft loss. Therefore, although endothelial injury by Fc-mediated cytotoxicity may be involved in vascular damage, other mechanisms also come into play. The amount and distribution pattern of ICAM-1 antigen were identical in both TO and LEJ strains. Intravenous anti-CAM-1 antibody administration combined with lipopolysaccharide, Poly(1)-Poly(C), warm ischemia to the kidney, or subcutaneous immunization with allogeneic spleen cells, but without renal transplantation, did not generate necrotizing vasculitis or proteinuria. These observations plus our previous data on the rat liver transplantation model clearly show that graft perfusion with anti-ICAM-1 monoclonal antibody invokes extensive vascular damage within allografts by Fc-mediated and Fc-independent mechanisms, depending on the donor-to-host combination.     

Intra cytoplasmic and Extracytoplasmic Lumina with a Peripheral Halo and Spikes (Spicular Bodies) Found in Epithelial Tumors

Intracytoplasmic lumina, each with a peripheral halo and spikes, a hitherto undescribed histological structure, were found in several human carcinomas. Similar structures found at the free surfaces of carcinoma cells, or those composed of several individual carcinoma cells, were designated extracytoplasmic lumina with a peripheral halo and spikes. Haloes were seen along the cytoplasm around the lumina, and the spikes penetrated into the cytoplasmic haloes. Transmission electron microscopy revealed that these spicular bodies were lumina equipped with microvilli containing an actin core. Spicular bodies were found in the following neoplasms; 22% of hepatocellular carcinomas (11/50), 35% of colonic adenocarcinomas (7/20), 31% of gastric adenocarcinomas (16/51). In contrast, no or only very occasional spicular bodies were found in thyroid (0/20) and mammary (1/22) adenocarcinomas. Although the diagnostic value of this structure is unknown at the present time, its identification in a carcinoma may be a histologic indicator of its endodermal nature. Acta Pathol Jpn 40: 815-819, 1990.     

A missense mutation in the proteolipid protein gene responsible for Pelizaeus--Merzbacher disease in a Japanese family

We investigated the proteolipid protein (PLP) gene of two boysin a Japanese family with Pelizaeus—Merzbacher disease(PMD), an X-linked neurologic disorder characterized by dysmyelinationin the central nervous system (CNS). The patients showed similarclinical signs from birth and autopsy on the elder brother confirmeda connatal type of PMD. Direct sequencing of the PLP gene andPLP mRNAs from the brain of the PMD patient revealed a G toT transition in exon V of the PLP gene, which leads to a glycineto cystein substitution at residue 220. Allele-specific oligonucleotidehybridization revealed that this mutation was also present inhis brother, but was absent in 100 X chromosomes of normal Japaneseindividuals. Northern blot analysis showed that the mRNA levelsof PLP and myelin basic protein, two major myelin proteins producedby oligodendrocytes, were much reduced in the PMD brain, hence,there was a specific loss of oligodendrocytes. It seems likelythat the substitution is responsible for PMD (connatal type)in this particular family and causes oligodendrocytes deathin the CNS.     

Correlation between mutated genes and forearm deformity in patients with multiple osteochondroma

BackgroundsExostosin-1 (EXT1) and exostosin-2 (EXT2) cause multiple osteochondromas (MO). In this study, we investigated the correlation between forearm deformity and mutant EXTs in Japanese families with MO.MethodsWe evaluated 112 patients in 71 families with MO. Genomic DNA was isolated from peripheral blood leucocytes. Of these, 28 patients were selected and underwent radiography for their forearms since they had gross forearm deformities. We measured the radial articular angle (RAA), ulna variance (UV), carpal slip (CS), and percentage of radial bowing (%RB) to compare between patients with mutant EXT1 or EXT2 and those with missense or other mutations using Student's t-test.ResultsTwenty-two (78.6%) and 6 (11.4%) out of 28 patients had mutations in EXT1 and EXT2, respectively. Nine (32.1%) and 19 (67.9%) of the 28 patients had missense and other mutations, respectively. The mean age of patients with EXT1 and EXT2 were 25.9 ± 20.3 and 33.5 ± 25.4 years, respectively and those with missense mutation and other mutations were 28.7 ± 27.0 and 24.6 ± 17.0 years, respectively. There were no significant differences in RAA, UV, and RB between patients harbouring mutant EXT1 or EXT2 (RAA, 40.1 ± 8.7 and 31.5 ± 13.9°; UV, ?2.7 ± 5.7 and ?3.1 ± 3.7 mm; %RB, 8.6 ± 1.5 and 8.3 ± 2.0%). CS was significantly greater in patients with mutant EXT1 than that in those with mutant EXT2 (EXT1, 44.1 ± 16.8%; EXT2, 18.6 ± 14.0%). There were no significant differences in RAA, UV, CS and %RB between patients with missense and other mutations.ConclusionsPatients with mutant EXT1 displayed greater CS than patients with mutant EXT2, indicating that patients with MO harbouring EXT1 mutations sustain more severe ulnar drift deformities than those with EXT2 mutations.     

Antitumor effects of oral administration of an interferon-inducing pyrimidinone,Bropirimine, on murine renal-cell carcinoma

Bropirimine [2-amino-5-bromo-6-phenyl-4-(3H)-pyrimidinone] is a low-molecular-weight compound that acts as an inducer of interferon in several animal species. Experiments were designed to explore the possibility of using this drug for the treatment of renal-cell carcinoma (RCC). Euthymic BALB/c mice were inoculated with murine RCC (Renca) cells and given graded doses of Bropirimine p.o. for 5 consecutive days beginning on day 1 following tumor inoculation. These mice were killed and tumors were excised on day 21. Bropirimine significantly (P<0.01) inhibited the tumor growth at a daily dose of 1,000 or 2,000 mg/kg. No adverse effect or toxicity was noted at 1,000 mg/kg, and at 2,000 mg/kg there was only a marginal body-weight reduction without any other appreciable side effect. In addition to the inhibition of tumor growth, there was a small yet significant (P<0.05) increase in the duration of survival (in days) in the Bropirimine-treated animals. When the treatment was delayed to begin on day 6 following tumor inoculation, Bropirimine did not suppress tumor growth in euthymic mice, pointing to the importance of the timing of the treatment. In athymic nude BALB/c mice lacking T-cells or T-cell function, Bropirimine also inhibited tumor growth (P<0.01). The antitumor effect of this drug was abolished by pretreatment with anti-asialo GM1 serum, which eliminated natural killer (NK) activity in euthymic mice. In vivo treatment with Bropirimine augmented the cytotoxicity of lymphocytes isolated from the spleens or lungs of the tumor-bearing mice, which were active against Renca and YAC-1 cells in vitro. This activity was NK-cell-dependent as judged on the basis of the results of the in vitro complement-dependent cytotoxicity assay. Since Bropirimine induced interferon (IFN)-/ production, significantly (P<0.05) elevating its serum concentration, and since this drug mimics the effects of IFN-/, it seemed likely that the Bropirimine-induced NK cell augmentation we found was mediated by IFN-/. These results suggest that Bropirimine, a booster of NK activity, may have potential as an adjunct to other therapeutic modalities in the treatment of human RCC.     

Activation of neutrophil function by recombinant human granulocyte colony-stimulating factor improves the survival of rats with peritonitis

We examined the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and cefmetazole sodium on survival, neutrophil count, and neutrophil function in rats with peritonitis produced by cecal ligation and puncture. Rats with peritonitis received either rhG-CSF (50 or 100 g/kg) with or without cefmetazole (50 mg/kg) for 3 days, cefmetazole alone, or no treatment and were evaluated as controls. The mortality rate of all treated rats was significantly lower than that of the untreated rats. The survival rate was 57.1% for the rats given both rhG-CSF and cefmetazole, but there was no significant improvement of survival as compared with cefmetazole therapy alone. Treatment with rhG-CSF at 100 |Gmg/kg caused the circulating neutrophil count to increase significantly. The phagocytic activity for latex beads and neutrophil H₂O₂ production showed a greater enhancement by phorbol myristate acetate (PMA) in the untreated rats, thus indicating that neutrophils from treated rats were more activated. These findings show that rhG-CSF can improve survival and neutrophil function in rats with peritonitis, while combined therapy with cefmetazole was also found to be beneficial.     

Recombinant human-type SOD attenuates circulatory disorders after reperfusion of splanchnic organs in rats

Oxygen free radicals (OFRs) have been reported to play pivotal roles in the pathogenesis of cell damage induced by ischemia and reperfusion. The efficacy of recombinant human superoxide dismutase (rh-SOD) in the treatment of circulatory disorders after reperfusion of the splanchnic area was investigated in rats. All rats died within 3 hours after release of 60-min superior mesenteric artery occlusion (SMAO) when no treatment was given. Animals which received rh-SOD, 2mg·100g^–1BW, at reperfusion followed by a continuous infusion of rh-SOD 0.67mg·100g^–1BW·hr^–1, exhibited prolonged survival times compared with no treatment rats (231 ± 35min and 149 ± 43min, respectively). Mean blood pressure in rats treated with rh-SOD was higher than in controls after reperfusion, and was concomitant with improvement in splanchnic perfusion. The results suggest excessive activity of OFRs in reperfused organs and a possible scavenging effect of rh-SOD as a means of eliminating them.(Bitoh H: Recombinant human-type SOD attenuates circulatory disorders after reperfusion of splanchnic organs in rats. J Anesth 6: 247–254, 1992)