Installation and application of an intense 7Li(p,n) neutron source for 20-90 MeV region.

An intense (7)Li(p,n) neutron source was installed with the K = 110-MeV azimuthally varying field (AVF) cyclotron at the Cyclotron and Radioisotope Center (CYRIC), Tohoku University, by employing a special arrangement to allow a short target-sample distance down to 1 m. The source can deliver a neutron flux around 1.5 x 10(6) n cm(-2) s(-1) with a lithium target of thickness equivalent to 2-MeV energy loss and 3-microA proton beam, which is the highest intensity in the world. The source is successfully used for (1) measurement of neutron cross sections relevant to radiation safety and radiation effect and (2) semiconductor irradiation test for single-event effect and (3) characterisation of neutron detectors and dosemeters.

Identification and characterization of presenilin I-467, I-463 and I-374.

We cloned a novel isoform of presenilin I (presenilin I-374) besides previously published presenilin I-467 and I-463 in human lymphocytes. Presenilin I-463 was identical to presenilin I-467 except a 12 bp nucleotides deletion in its amino terminal region. Another isoform, presenilin I-374 was produced by an alternative splicing with an additional exon consisting of 92 bp nucleotides (exon 11), which resulted in the frame shift with a stop codon to generate a truncated presenilin consisting of 374 amino acids. The transcripts for presenilin I-467/463 was ubiquitously detected while that for presenilin I-374 was selectively detected in liver, spleen, kidney. Abnormal behavior of presenilin I on gel electrophoresis was found with affinity-purified antibodies against presenilin I.

Severe aplastic anemia associated with chronic natural killer cell lymphocytosis.

Chronic natural killer (NK) lymphocytosis involves a persistent increase in CD56+ large granular lymphocytes (LGLs) that is sometimes associated with immune-mediated complications, such as anemia and neutropenia. However, aplastic anemia (AA) is a rare complication. Here we describe 2 patients with severe AA who presented with persistent increases in NK cells. Their LGLs were positive for CD56, CD16, and intracellular interferon (IFN)-gamma but negative for CD3, Fas-ligand, and T-cell receptor rearrangement, findings that are compatible with NK cells. Not only the number of NK cells, but NK activity as well, was increased in both patients. The number of NK cells changed according to hematologic recovery and relapse in 1 case. Thus, there seemed to be a close relationship between NK cells and the progression of AA, at least in this instance. Further investigation of the clinical course of similar cases and the characteristics of NK cells is necessary.

Binary expression of olfactory bulb-protein tyrosine phosphatase in rat central nervous system: developmental gene regulation in neonate cerebral cortex and constitutive expression in olfactory-rhinencephalon.

Olfactory bulb-protein tyrosine phosphatase (OB-PTP) is a receptor type PTPase dominantly expressed in olfactory bulb. Previously, we isolated and molecularly cloned a rat OB-PTP cDNA from an embryonal brain cDNA library. In the present study, we investigated its temporal and spatial gene expression by Northern blot and in situ hybridization analysis. The expression of OB-PTP gene was firstly detected in day 16 post coitum embryo and significantly increased during the late-gestational stage, attaining the highest level in the first week of neonate. The OB-PTP transcript was then down-regulated postnatally and was detected barely in an adult brain. In situ hybridization analysis showed that the transcript was characteristically localized in the postmitotic neurons of cerebral cortex and subcortical structures, and was down-regulated by day 28 when the cortical and subcortical structures have been organized. In the olfactory-rhinencephalon system including olfactory bulb and piriform cortex, the OB-PTP was preferentially expressed in the postmitotic neurons, and in contrast continuously expressed in the matured brain. Based on the evidence that DPTP10D, the Drosophila homolog of OB-PTP, is localized in the axons of specific pioneer neurons in Drosophila embryo, the OB-PTP is presumably involved in the axonogenesis of cortical and subcortical neurons as well as olfactory neurons in mammalian central nervous system. The biological significance of transcriptional regulation in olfactory system is discussed in terms of continuous axonal connections by regenerating olfactory neurons.

[Posterior subtemporal transtentorial approach for a lower basilar trunk aneurysm (author's transl)].

A lower basilar trunk aneurysm is rare and it has been difficult to operate on this kind of aneurysm which is located in so-called "no man's land". We have recently operated on the aneurysm which was located between the vertebrobasilar junction and the origin of AICA. The aneurysm was approached posterior-subtemporal-transtentorially and wrapped with a muscle piece because of its broad neck. After the operation the patient developed amnestic aphasia which, however, disappeared 4 months postoperatively. The advantage of this approach is that it enables a better visualization of the lower basilar trunk, the lateroventral portion of the pons, the distal part (5 mm) of both vertebral arteries and the upper portion of the medulla oblongata than any other approaches hitherto reported. The retraction of the temporal lobe and subsequent brain damage may be minimized by using intraoperative ventricular drainage and microtechnical maneuver.

[Case of teratoma with intraventricular free fat].

Intracranial fat-containing congenital tumors are characterized by negative absorption values on computed tomography (CT). We are reporting a case of teratoma with intraventricular free fat diagnosed preoperatively by CT. The case is a 19-year-old female who was admitted to our hospital because of continuous severe headache, nausea and vomiting. At the time of admission, her physical and neurological examination was negative except for bilateral papilledema. CT demonstrated marked enlargement of the right lateral ventricle. In addition, there was negative absorption value (-90 H.U.), suggesting free fat, within right frontal horn layering above the CSF with a fluid level. metrizamide ventriculography demonstrated complete obstruction and revealed an irregular shadow defect at the right foramen of Monro. At surgery, yellowish cheese-like material, white hair was found on the surface of the CSF. Tumor arose from the floor of the right foramen of Monro and extended upward. The patient made an uneventful recovery and was discharged 17 days after surgery. Intraventricular free fat is likely that to be released from the teratoma cyst ruptured spontaneously when the patient complained of severe headache 40 days prior to admission. There have been several published reports of the CT appearances of intracranial fat-containing tumors, however, teratoma with intraventricular free fat is very rare. It was concluded that fat-containing tumors should be highly suspected, when negative absorption values were found on CT.

[The role of daunorubicin in induction therapy for adult acute myeloid leukemia].

The relationship between the total dose of daunorubicin (DNR) in induction therapy and the treatment outcome were evaluated based upon individualized doses of DNR during induction therapy for patients with acute myeloid leukemia(AML). Ninety-two previously untreated adult AML patients admitted to our hospital were analyzed for the dose of DNR required for complete remission (CR), the CR rate, disease-free survival (DFS) and overall survival (OS). The induction therapy consisted of DNR (40 mg/m2/d, i.v., from D 1 until the marrow was hypoplastic), Ara-C, prednisolone, and/or 6-thioguanine. Eighty-three out of 92 patients were assessable. Sixty-three patients entered CR (76%), of whom 52 attained CR with the first course of induction therapy. The 10-year DFS and OS rates were 31.2% and 42.3%, respectively. The median total dose of DNR in the induction therapy was 280 mg/m2 (120-480 mg/m2), which was not influenced by initial WBC count, or FAB type. These results indicate that when the dose is linked to the observed tumor response, the optimal dose of DNR in the induction therapy is around 280 mg/m2 (40 mg/m2 x 7 times), which is higher than the conventional dose of 40-60 mg/m2 for 3 days. The higher dose of DNR in the induction therapy for adult AML should be selected when the feasibility of a new drug is evaluated in a randomized trial.

Identification of HLA class II antigens as the targets of effector clones which may cause transfusion-associated graft-versus-host disease.

We established T cell clones, which were considered to be the possible cause of transfusion-associated graft-versus-host disease (TA-GVHD), from the peripheral blood lymphocytes (PBLs) of two patients. In both cases, several CD4+ cytotoxic T-cell (CTL) clones were established. In case I, the target antigen of the established CD4+ clones was a DRB1*0403-related antigen serologically typed as HLA DR4, which was one of the patient HLA antigens. In case II, the target of four out of five established CD4+ CTL was a DRB1*1302-related antigen. One CD4+ CTL clone showed cytotoxicity against cells carrying A*2402, B*4403, Cw*1403 and DPB1*0401. A monoclonal antibody (mAb) blocking study showed only anti-DP mAb inhibited the cytotoxicity of this clone. Thus, it might be considered that this clone recognizes HLA-DP with its binding peptides derived from either A*2402, B*4403, Cw*1403 or DRB1*1302. Our findings indicate that CD4+ CTLs may play important roles in the aetiology of TA-GVHD and that the antigens of patients recognized by donor-derived effector cells may not always recognize a single HLA antigen.

Characterization of T-cell clones derived from peripheral blood lymphocytes of a patient with transfusion-associated graft-versus-host disease: Fas-mediated killing by CD4+ and CD8+ cytotoxic T-cell clones and tumor necrosis factor beta production by CD4+ T-cell clones.

Transfusion-associated graft-versus-host disease (TA-GVHD) is one of the most serious adverse effects of blood transfusion. It is generally thought to be caused by the infused lymphocytes. Donor-derived cytotoxic T lymphocytes (CTLs) directed against the recipient's HLAs, which have escaped the recipient's immune system and are proliferating, are considered to attack recipient organs and tissues. Despite the seriousness of the disease, the precise mechanism of its development remains unclear and no definitive treatment has been developed. With the aim of developing an effective treatment, we established and characterized T-cell clones from peripheral blood lymphocytes (PBLs) of a TA-GVHD patient. Three types of clones were established. Type I clones were CD8+ and specifically lyse cells that express HLA B52. Type II clones were CD4+, specifically lysed cells that express HLA DR15, and proliferated in response to stimulation with cells that express DR15. Type III clones were also CD4+, showed no cytotoxic activity toward any HLA-expressing cells, and proliferated in response to stimulation with cells that express DR15. Furthermore, we found that the Fas/Fas-ligand (Fas-L) system is involved in the cytotoxicity of the type I and II clones and that the type III clones produce and secrete a large amount of tumor necrosis factor beta (TNFbeta) after antigen stimulation. Based on our results, these three types of clones can be classified into two categories: those that have the ability to induce GVHD directly by cytolysis and that show no cytotoxic activity and those that have the ability to cause GVHD indirectly through secretion of cytotoxic lymphokines.

Identification of a receptor-type protein tyrosine phosphatase expressed in postmitotic maturing neurons: its structure and expression in the central nervous system.

We have isolated a rat cDNA encoding a receptor-type protein-tyrosine-phosphatase (RTP) expressed in brain and kidney (RPTP-BK) and characterized its expression in the developing central nervous system. RPTP-BK has seven fibronectin type III-like repeats in the extracellular region and a unique catalytic phosphatase domain in the cytoplasmic region. Bacterial expression of its phosphatase domain showed that the dephosphorylation of phosphotyrosine residues was mediated by the cytoplasmic catalytic domain. Sequence comparison revealed that RPTP-BK is homologous with GLEPP1, a rabbit PTP expressed in renal glomerular epithelia, and has the same phosphatase domain as murine PTPphi expressed in macrophages. RPTP-BK has also significant homology with Drosophila DPTP10D in the phosphatase domain, whose expression is localized exclusively in growth cones of the embryonal brains. The gene for RPTP-BK is well conserved among other species, and the expression in the brain but not in the kidney is developmentally regulated during the neonatal stage. Hybridization in situ showed that RPTP-BK is highly expressed in the postmitotic maturing neurons of the olfactory bulb, developing neocortex, hippocampus and thalamus. Because the expression of RPTP-BK in the developing neocortex is correlated with the stage of axonogenesis in cortical neurons, RPTP-BK might be crucial in neural cell development of the mammalian central nervous system.

Protein tyrosine kinases expressed in glomeruli and cultured glomerular cells: Flt-1 and VEGF expression in renal mesangial cells.

Protein tyrosine kinases play an important role in cellular proliferation and differentiation of various cell types. To identify potential tyrosine kinases involved in glomerular functions we have utilized the polymerase chain reaction (PCR) and degenerate oligonucleotides for isolation of such genes from isolated glomeruli, cultured mesangial cell, and glomerular endothelial cells. Sequence analysis of PCR-amplified cDNAs resulted in the isolation of 24 tyrosine kinases. Here we describe for the first time the constitutive expression of 15 tyrosine kinases, tyro-1, tyro-4, tyro-6, hyk, Ptk-3, Ryk, tie, yes, lyn, tec, Jak1, Jak2, Jak3, c-abl, and flk, in renal glomeruli. In addition, Flt-1, an endothelial cell-specific receptor for vascular endothelial growth factor (VEGF), is expressed in renal mesangial cells and its gene expression is up-regulated upon the stimulation of platelet-derived growth factor (PDGF) with the concomitant up-regulation of VEGF. These data suggest that possible involvement of VEGF/Flt-1 system in cytokine-induced mesangial cell proliferations.

Abnormal gel-electrophoretic behavior of presenilin 1 and it's fragment.

Presenilin 1 (PS-1) is the main causal gene of familial Alzheimer's disease. In this report, we describe the abnormal behavior of PS-1 in gel electrophoresis in the presence of SDS. Freshly in vitro synthesized PS-1 was identified as a single molecule with the molecular size of 43,000 on SDS gels but was found to disappear after incubation at 37 degrees C for 24 hr due to the formation of aggregates. Intermediate aggregates with M(r) 74,000 and 100,000 were formed before the final aggregate which was retained at the top of the gel. Thus the amount of 43,000-protein species of PS-1 was found to decrease on gels with a concomitant increase in the amount of 74,000/100,000 proteins. Similar abnormality was seen in PS-1 expressed in COS cells transfected with PS-1 cDNA. Moreover, cellular PS-1 was strongly suggested to be cleaved into the fragments with M(r) approximately 20,000 in COS and CHO cells. Fragmentation of cellular PS-1 was not affected by the missense point mutation of Ala260Val on PS-1 which was identified in a pedigree with familial Alzheimer's disease.

Jak3 expression in glomerular epithelia of IgA nephropathy (IgA-N) patients.

Jak3 is a member of the Janus kinase family which plays an important role in cytokine signal transduction. Jak3 associates the gamma(c) chain of receptors for IL-2, IL-4, IL-7, IL-9 and IL-15, and is essential for the signal transduction of these cytokines. We have isolated Jak3 kinase from renal mesangial cells and demonstrated the constitutive expression of Jak3 in glomeruli in vivo. To investigate the physiological and pathological role of Jak3 in glomeruli, we prepared anti-Jak3 antibody and analysed the localization of Jak3 in glomeruli of renal biopsy samples from various nephritis patients and normal subjects. Among 61 nephritis patients and four normal subjects investigated in the present study, Jak3 was selectively localized to glomerular epithelia of IgA-N patients (14/34 cases) and focal glomerulosclerosis patients (1/5 cases), but not detected in minimal changes (n = 6), membranous glomerulonephropathy (n = 7), crescentic glomerulonephritis (n = 4), lupus nephritis patients (n = 5), and normal subjects (n = 4). The intense immunoreactivity for Jak3 is significantly associated with the decrease in creatinine clearance (81.5 +/- 10.4 ml/min versus 104.3 +/- 29.6 ml/min; P < 0.05, Student's t-test) and the increase in level of serum creatinine (1.13 +/- 0.33 mg/dl versus 0.75 +/- 0.23 mg/dl; P < 0.01, Student's t-test) in IgA-N patients. Furthermore, gamma(c) chain was concomitantly expressed with Jak3 in glomerular epithelia in vivo and in vitro, suggesting that signal transduction via gamma(c)-Jak3 cascade may be involved in the pathogenesis of glomerular injury of IgA-N. Taken together with the recent findings that IL-4-secreting T lymphocytes in affected glomeruli injure glomerular epithelium, the responsiveness of glomerular epithelium for IL-4 may be pathologically enhanced in IgA-N.

Transforming growth factor-beta is a survival factor for neonate cortical neurons: coincident expression of type I receptors in developing cerebral cortices.

Transforming growth factor-beta (TGF-beta) is a multifunctional polypeptide which plays a crucial role in the regulation of cell proliferation, differentiation, and organogenesis. In the present study, we investigated the expression of signaling receptors for TGF-beta in developing mice by in situ hybridization, revealing a significant difference in the expression of TGF-beta type I and type II receptors. Unexpectedly, the TGF-beta type I receptors were exclusively expressed without any detectable expression of the TGF-beta type II receptors in developing cerebral cortices. In primary cortical neurons, a neutralizing antibody for TGF-beta significantly reduced the expression of bcl-2 and subsequently induced neuronal cell death, indicating that TGF-beta functions as a survival factor for cortical neurons in vitro. Consistent with the result of in situ hybridization, the TGF-beta, type I but not type II receptors were detected in primary cortical neurons by affinity crosslink and RT-PCR analyses. The concomitant expression of TGF-beta2 and the TGF-beta type I receptors in developing cerebral cortices suggests that the TGF-beta signaling system plays a pivotal role in neuronal differentiation and that unidentified components may be involved in TGF-beta signaling in the development of the central nervous system.

Deficiency in protein L-isoaspartyl methyltransferase results in a fatal progressive epilepsy.

Protein L-isoaspartyl methyltransferase (PIMT) is suggested to play a role in the repair of aged protein spontaneously incorporated with isoaspartyl residues. We generated PIMT-deficient mice by targeted disruption of the PIMT gene to elucidate the biological role of the gene in vivo. PIMT-deficient mice died from progressive epileptic seizures with grand mal and myoclonus between 4 and 12 weeks of age. An anticonvulsive drug, dipropylacetic acid (DPA), improved their survival but failed to cure the fatal outcome. L-Isoaspartatate, the putative substrate for PIMT, was increased ninefold in the brains of PIMT-deficient mice. The brains of PIMT-deficient mice started to enlarge after 4 weeks of age when the apical dendrites of pyramidal neurons in cerebral cortices showed aberrant arborizations with disorganized microtubules. We conclude that methylation of modified proteins with isoaspartyl residues is essential for the maintenance of a mature CNS and that a deficiency in PIMT results in fatal progressive epilepsy in mice.