Isolation of amantadine-resistant influenza a viruses (H3N2) from patients following administration of amantadine in Japan

In Japan, the use of amantadine for treatment of influenza A virus infection was not accepted until November 1998, although it was widely used for treatment of Parkinsonism. Since then, we have monitored the emergence of amantadine-resistant viruses and isolated two viruses from patients on long-term treatment with amantadine.     

Direct in vivo protein transduction into a specific restricted brain area in rats

Attempts at protein transduction into specific restricted brain areas have remained unsuccessful. We attempted targeted, direct in vivo protein transduction by microinjecting beta-galactosidase (beta-gal) with hemagglutinating virus of Japan envelope (HVJ-E) vector into the rat nucleus tractus solitarius (NTS). The medulla oblongata including the NTS was removed 6h post-injection and cryostat sections were histochemically stained to detect beta-gal enzymatic activity. beta-gal-positive cells were present in these sections as was beta-gal activity determined by colorimetric analysis. beta-gal-positive cells were not present in the rats microinjected only beta-gal protein without HVJ-E vector. Our findings suggest that direct in vivo protein transduction into specific restricted brain areas is possible. The type of targeted delivery system we present may have wide applications in the administration of therapeutic proteins to the central nervous system.     

HISTOPATHOLOGICAL STUDIES ON THROMBOTIC MICROANGIOPATHY WITH SPECIAL REFERENCES TO THREE CASES ASSOCIATED WITH ACUTE PROMYELOCYTIC LEUKAEMIA

Five autopsy cases of thrombotic microangiopathy, including 3 cases associated with acute promyelocytic leukaemia, were examined macroscopically, light-and electronmicroscopically.
The so-called hyaline thrombi In thrombotic microangiopathy were composed of fibrin and its degenerative products. Thrombocytes and other blood cells were not seen in the thrombi.
At the site of the formation of a thrombus, there was no conspicuous change in the walls of the capillaries and arterioles. It was considered, therefore, that the intravascular deposition of fibrin was the primary event in the development of thrombotic microangiopathy.
In regard to the distribution and morphologic findings, there was no basic difference between the microthrombi in cases associated with acute promyelocytic leukaemia and those without it.
The bone marrow and some other organs in cases of thrombotic microangiopathy associated with acute promyelocytic leukaemia macroscopically revealed a green colour. Many thrombi composed of leukaemic cells and fibrin were found in the pulmonary arteries of these cases. Furthermore, prominent erythrophagocytosis in the bone marrow and lymph nodes was a common finding in these cases.     

Epitope mapping of the influenza A virus RNA polymerase PA using monoclonal antibodies

Summary.  To obtain reagents to functionally map the PA protein, we produced monoclonal antibodies specific to this protein. Twenty-two monoclonal antibodies reacting with PA protein in ELISA were divided into 10 groups on the basis of competitive binding patterns to this protein. Of these, seventeen monoclonal antibodies bound to PA polypeptide spanning amino acids 101–400 and three bound to that of amino acids 518–600, while the other two did not react with any PA polypeptides tested with the exception of full-length PA. Among these monoclonal antibodies, only five reacted with PA in A/PR/8/34 virus-infected cells in indirect immunofluorescence assay. Thus, we obtained monoclonal antibodies that recognize at least 10 distinct regions of the PA molecule. These monoclonal antibodies should be useful in dissecting functions of the PA protein. Received September 6, 1999/Accepted January 5, 2000     

Human vascular smooth muscle cells and endothelial cells lack catalase activity and are susceptible to hydrogen peroxide

⁵Cr release as lytic and cell detachment as nonlytic injury were employed to estimate neutrophil-mediated injury of cultured human vascular smooth muscle cells and endothelial cells. The reagents hydrogen peroxide or hypoxanthine-xanthine oxidase produced dose-dependent killing and nonlytic cell detachment, which were specifically inhibited by catalase but not by superoxide dismutase. The concentration of hydrogen peroxide or xanthine oxidase to induce cell detachment was less than lytic dose, suggesting that cell detachment was a much more sensitive assay of injury. Neutrophil-mediated cell lysis averaged 15% at most and was mostly dependent on hydrogen peroxide, while neutrophil-mediated cell detachment was nearly 100% and its dependency on hydrogen peroxide varied from 46% to 60%. These results suggest that vascular smooth muscle cells and endothelial cells in neutrophil-mediated events are destroyed by a hydrogen peroxide-dependent process, mainly via a nonlytic cell detachment mechanism. There was no striking difference of sensitivity to hydrogen peroxide between vascular smooth muscle cells and endothelial cells. Vascular smooth muscle ceils and endothelial cells contained fairly high concentrations of superoxide dismutase, but not catalase, activity. The sensitivity of these cells to hydrogen peroxide but not to superoxide may arise from the fact that these cells lack intracellular catalase activity. The injury of vascular cells, which constitute important components of blood vessels, may lead to vascular injury and subsequent tissue damage.     

Secreted Reelin molecules form homodimers

During mammalian brain development, neurons are generated along the ventricle, migrate radially, and become aligned in defined patterns. These precise patterns of neuronal alignment are regulated by an extracellular matrix protein Reelin, and binding of Reelin to its receptors induces tyrosine phosphorylation of the intracellular adaptor protein disabled 1 (Dab1). We recently reported that Reelin molecules assemble to form a homomeric protein complex. Although the number of molecules in the full-length complex is unknown, recombinant N-terminal fragments, which contain the epitope for the function-blocking CR-50 antibody, assembled to form a complex of more than 40 monomers. When the N-terminus was deleted from Reelin, the truncated protein did not form a stable complex. To further characterize the Reelin assembly, we performed biochemical analysis of the full-length Reelin assembly in this study. Here, we report that a full-length Reelin forms a disulfide-linked homodimer. A chemical crosslinking experiment on secreted Reelin confirmed that only dimers are formed by the full-length protein. However, interestingly, chemical crosslinking of the N-terminus-truncated Reelin resulted in the formation of larger complexes, in addition to dimers, suggesting that the tertiary structure required for the proper and stable assembly/dimerization was altered by the truncation. The truncated protein did not induce efficient tyrosine phosphorylation of Dab1, although it bound well to the receptors. These findings demonstrate the functional importance of the N-terminal region of Reelin for proper dimerization and signaling. Proper but not simple extracellular crosslinking of the receptors by these dimers may be important for Reelin signaling to occur.     

Distinct progression pathways involving the dysfunction of DUSP6/MKP-3 in pancreatic intraepithelial neoplasia and intraductal papillary-mucinous neoplasms of the pancreas.

DUSP6/MKP-3 is identified as a candidate tumor suppressor gene for pancreatic cancer. The aim of this study was to elucidate the roles of DUSP6 in the pancreatic carcinogenesis through the pancreatic intraepithelial neoplasia and/or intraductal papillary-mucinous neoplasms, both of which are considered to be precursor lesions of invasive carcinoma of the pancreas, by comparing with involvements of other major tumor suppressive pathways. Expressions of DUSP6, CDKN2A, TP53, and SMAD4 were investigated by immunohistochemistry in a total of 206 lesions of dysplastic ductal precursors and carcinomas retrieved from 52 pancreata with invasive ductal carcinomas and 51 of those with intraductal papillary-mucinous neoplasms. The intensity of staining was evaluated in lesions at different atypical grades and statistically compared among them. Mutations of KRAS2 were analyzed by methods of the allele-specific oligonucleotide hybridization and nucleotide sequencing. In pancreata with invasive ductal carcinomas, expressions of DUSP6 were abrogated exclusively in the invasive carcinoma cells in contrast to its fairly preserved expressions in pancreatic intraepithelial neoplasia. In pancreata with intraductal papillary-mucinous neoplasms, abrogated expressions of DUSP6 were observed in a relatively small fraction of intraductal adenoma/borderlines and intraductal carcinomas. Most of the intraductal adenoma/borderline lesions with abrogation of DUSP6 harbored mutations of KRAS2. None of the molecules was associated with each other in any grade of lesions. Morphological variations of papillae of the intraductal papillary-mucinous neoplasms were evaluated and analyzed for their associations with abrogations of the molecules, which resulted in finding of no significant associations. Our results suggest that the abrogation of DUSP6 is associated exclusively with progression from pancreatic intraepithelial neoplasia to the invasive ductal carcinoma while it is potentially associated with initiation of intraductal papillary-mucinous neoplasms with mutated KRAS2, which is independent of other major tumor suppressive pathways in both types of neoplasms.     

Role of macrophage migration inhibitory factor in otitis media with effusion in adults

Otitis media with effusion (OME) is one of the most common ear diseases. Bacterial endotoxins and several inflammatory cytokines appear to be involved in the pathogenesis of OME in children; however, little is known of the immunological aspects of the onset of OME in adults. We sought to determine the presence of macrophage migration inhibitory factor (MIF) as well as interleukin 1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), RANTES (regulated upon activation, normal T-cell expressed and presumably secreted), and endotoxin in middle ear effusions (MEEs) from adult patients with OME. In addition, the levels of MIF in MEEs from adults and children were compared. MEE was obtained from 95 adults and 11 children. The levels of MIF, IL-1beta, TNF-alpha, and RANTES were determined by enzyme-linked immunosorbent assay, and the concentrations of endotoxin and total protein were determined by the Endospec assay and bicinchoninic acid assay, respectively. MIF was detected in 97.9% of the MEEs from adults, while endotoxin, IL-1beta, TNF-alpha, and RANTES were detected in 96.8, 12.6, 5.3, and 43.9%, respectively. In addition, the level of MIF was significantly higher than those of endotoxin, IL-1beta, and TNF-alpha. A positive correlation between the levels of MIF and endotoxin was observed. MIF and endotoxin were detected in 81.8 and 72.7%, respectively, of the MEEs from the children. The level of MIF was significantly higher in the children, and conversely that of endotoxin was significantly higher in the adults. These results suggest that the interaction between MIF and endotoxin may promote fluid collection in the middle ear, particularly in adults.     

Ghrelin Induces Fasted Motor Activity of the Gastrointestinal Tract in Conscious Fed Rats

Ghrelin is a newly discovered orexigenic peptide originating from the stomach. However, its action in regulating the fed and fasted motor activity of the digestive tract is not fully understood. In the present study, we examined the effects of intracerebroventricular ( i.c.v. ) and intravenous ( i.v. ) injection of ghrelin on the physiological fed and fasted motor activities in the stomach and duodenum of freely moving conscious rats. i.c.v. and i.v. injection of ghrelin induced fasted motor activity in the duodenum in normal fed rats, while i.v. injection of ghrelin induced fasted motor activity in both the stomach and duodenum in vagotomized rats. The effects of i.c.v. and i.v. injected ghrelin were blocked by growth hormone secretagogue receptor (GHS-R) antagonist given by the same route and also blocked by immunoneutralization of neuropeptide Y (NPY) in the brain. The effects of i.v. injected ghrelin were not altered by i.c.v. injection of GHS-R antagonist in vagotomized rats. Injection of GHS-R antagonist blocked the fasted motor activity in both the stomach and duodenum in vagotomized rats but did not affect the fasted motor activity in normal rats. Low intragastric pH inhibited the effect of ghrelin. The present results indicate that ghrelin is involved in regulation of fasted motor activity in the stomach and duodenum. Peripheral ghrelin may induce the fasted motor activity by activating the NPY neurons in the brain, probably through ghrelin receptors on vagal afferent neurons. Once the brain mechanism is eliminated by truncal vagotomy, ghrelin might be primarily involved in the regulation of fasted motor activity through ghrelin receptors on the stomach and duodenum. The action of ghrelin to induce fasted motor activity is strongly affected by intragastric pH; low pH inhibits the action.     

Bilateral diffuse nephroblastomatosis, pancortical type. A case report with immunohistochemical investigations

A variation of Perlman's syndrome of the pancortical type is reported in a male neonate whose parents were cousins. The patient was the product of a 35-week pregnancy, the Apgar score was 3, and the patient died of respiratory failure one hour and 12 minutes after delivery. Autopsy revealed bilateral diffuse nephroblastomatosis, pancortical type, associated with malformations (usually facial), congenital anomalies of the heart, hepatosplenomegaly, pancreatic islet cell hyperplasia, bilateral cryptorchidism, and hyperflexibility of the left knee joint. Renal immunohistochemical investigations revealed positive bindings with peanut and soybean agglutinins and epithelial membrane antigen along the luminal surface of the epithelium in the moderately differentiated tubules, but not in blastoma or primitive epithelium.