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Charles Bonnet syndrome is a condition characterized by visual hallucinations. These simple or complex visual hallucinations are more common in elderly individuals with impaired peripheral vision. The current report describes a case of transient Charles Bonnet syndrome appearing after the removal of a meningioma. The patient was a 61-year-old man who already had impaired visual acuity due to diabetic retinopathy. Brain MRI revealed a cystic tumor severely compressing the right occipital lobe. Starting on day 2 postoperatively, the patient was troubled by recurring visual hallucinations involving people, flowers, pictures, and familiar settings(the train and a coffee shop). These continued for 3.5 months. This period roughly coincided with the time for the occipital lobe to recover from the compression caused by the tumor, a fact that was confirmed by several MRI scans. ¹²³I-IMP SPECT performed 1 month after the surgical operation showed an area of hypoperfusion in the right parieto-occipital lobe. Based on the patient's clinical course and MRI findings, the mechanism of onset of visual hallucinations in this patient was put forward. The release of pressure in the brain by tumor removal and subsequent recovery changed the blood flow to the brain. This triggered visual hallucinations in the patient, who was already predisposed to developing Charles Bonnet syndrome because of diabetic retinopathy. This case is interesting since it indicates that central neurological factors, as well as visual deficits, may induce the appearance of visual hallucinations in Charles Bonnet syndrome.

Quantitative crossed cerebellar diaschisis (CCD) and the correlation with a reduction in supratentorial regional cerebral blood flow (rCBF) and cerebrovascular reserve capacity (CVR) were investigated in clinically stable patients with major cerebral artery occlusion by the iodine-123-N-isopropyl-p-iodoamphetamine (I-123 IMP) single photon emission computed tomography (SPECT) method. Thirty patients with major cerebral artery occlusion underwent SPECT by the I-123 IMP autoradiographic method. Regional CBF was measured in the cerebral hemisphere, frontal and parietal lobes, temporo-parietal lobe, and cerebellum both at rest and after administration of acetazolamide. Eighteen of 30 patients (60%) had CCD. CCD was significantly related to magnetic resonance imaging evidence of infarction. Quantitative CCD was 17% and the CVR in the cerebellum was preserved in patients with CCD. There was a significant difference in CBF and CVR between the affected and normal sides in all regions of interest in the patients without CCD [CBF (ml/100 g/min): hemisphere (H), normal side (N): 31.4 +/- 6.8, affected side (A): 27.5 +/- 7.4; p0.05. CVR: H, N: 0.56 +/- 0.38, A: 0.42 +/- 0.18; p0.01]. CCD is common in patients with major cerebral artery occlusion, and quantitative I-123 IMP SPECT is helpful in detecting CCD in clinically stable patients with occlusion of major cerebral arteries.

Elevation of the intracellular calcium concentration is necessary for cell growth and the activation of several lymphokine genes. The immunosuppressive drugs cyclosporin A and FK506 profoundly inhibit the calcium-dependent signaling pathway in T lymphocytes by interfering with the activity of the calcium/calmodulin (CaM)-dependent serine/threonine phosphatase, calcineurin (CN). Little is known, however, about how activation of CN enzyme activity or interaction with its substrate, nuclear factor of activated T cell (NF-AT), is regulated. We show here that the binding of CaM to CN may affect the conformation of CN at both the CaM-binding and the autoinhibitory (AI) domains and that this is critical for activation of CN to dephosphorylate NF-AT. Dissociation of the AI domain from the enzyme active site on CN leads to expose a binding site for NF-AT at the N terminus of CN and allows the CN binding to NF-AT. Since the cytoplasmic form of NF-AT can interact with CN mutants lacking enzyme activity, the interaction of the two molecules is independent of CN enzyme activity and occurs prior to the dephosphorylation of NF-AT. Dephosphorylation converts NF-AT from the cytoplasmic to the nuclear form by exposing the DNA-binding domain on NF-AT, and the nuclear form of NF-AT brings CN together into the nuclei. We therefore propose that the activation of CN by the CaM binding independently regulates the interaction with NF-AT and the dephosphorylation of NF-AT.

The human ST2 gene can be specifically induced by growth stimulation in fibroblastic cells, and can also be induced by antigen stimulation in Th2 cells. The gene encodes a soluble secreted protein, ST2, and a transmembrane protein, ST2L, which are closely related to the interleukin-1 receptor. To gain insight into the biological roles of the ST2 gene, three monoclonal antibodies (MAbs) against human ST2 gene products were obtained. To obtain these antibodies, immunization was carried out using two different immunogens: purified soluble human ST2 protein (hST2), and COS7 cells, which express the extracellular portion of human ST2L. 2A5 and FB9 MAbs were derived from the immunization with soluble hST2, and HB12 was derived from the COS7 cell immunization. All three antibodies were shown to detect native forms of the human ST2 gene products by immunoprecipitation, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). In the competitive ELISA using biotinylated and nonlabelled MAbs, neither FB9 nor HB12 affected the binding of 2A5 to ST2 gene products. Based on this result, we constructed a sandwich ELISA system using 2A5 and FB9 to measure the concentration of soluble hST2 in sera. The ELISA, combined with the flow cytometry using these antibodies, will be a useful tool for elucidating the functions of human ST2 gene products in individuals.

Donation after cardiac death (DCD) is increasing in importance as a potential source of vital organs for clinical transplantation. In Japan, the method of kidney procurement from DCD is one of the critical factors in successful deceased renal transplantation. The efforts to shorten the warm ischemic time (WIT), which is defined as the time from cardiac arrest to initiation of cooling in situ, or maintaining adequate renal blood flow during WIT by consistent chest compression are essential. We experienced one case of successful kidney transplantation using a non-invasive load-distributing-band chest compression device using AutoPulse (ZOLL Circulation, Sunnyvale, California), which is designed for use as an adjunct to cardiopulmonary resuscitation (CPR) to maintain adequate aortic pressure. This is the first report of our experiences of successful kidney transplantation from DCD using a load-distributing-band chest compression device for maintaining renal blood flow during long WIT. We can speculate that this mechanical CPR device can become a bridge to deceased organ transplantation.

Monoclonal antibodies were generated against the adenosine A1 receptor (A1R) purified from rat brain. In immunoblot analyses of purified or partially purified A1R preparations from rat brain, these antibodies recognized a solitary band, the size of which corresponded to that expected for A1R. These antibodies recognized not only the native form of A1R but also the deglycosylated form of A1R. Immunocytochemical analysis of Chinese hamster ovarian cells that were transfected stably with rat A1R cDNA showed that their cell bodies were stained intensely by these antibodies, whereas nontransfected Chinese hamster ovarian cells were not. These antibodies detected the A1R naturally present in the DDT(1)( )MF-2 smooth muscle cells. One of these antibodies (the 511CA antibody) was then used to examine the immunohistochemical distribution of A1Rs in rat forebrain. On light microscopy, A1R immunoreactivity was observed in the cerebral cortex, septum, basal ganglia, hippocampal formation, and thalamus. However, in some regions of the forebrain, regional differences in staining intensity were found as follows: In the cerebral cortex, the strongest immunoreactivity was found in the large pyramidal neurons of layer V. This immunoreactivity was detected in the pyramidal cell bodies, dendrites, and axon initial segments. In the hippocampus, A1R immunoreactivity was detected mainly in the stratum pyramidale. The pyramidal cells in fields CA2-CA3 of the hippocampus were stained more intensely or more clearly than those in field CA1 or the dentate gyrus. More intense A1R immunoreactivity of the apical dendrites was detected in field CA2 compared with other hippocampal fields and the dentate gyrus. Many interneurons of the hippocampus were stained by the 511CA antibody. The subcellular distribution of A1Rs in the forebrain was examined by using a digital deconvolution system and electron microscopy. In the cerebral cortex, the view obtained by removing the background haze by deconvolution revealed that the immunofluoresence-labeled A1Rs were distributed on the surfaces of the cell bodies and dendrites and in the cytoplasm of layer V neurons as small spots. In field CA1, immunoreactivity was detected in the areas surrounding pyramidal cells. Electron microscopy revealed the presence of A1R-immunoreactive products in both the presynaptic terminals and the postsynaptic structures. The specific cellular distribution of A1Rs is consistent with the physiological premise that endogeneously released adenosine exerts control over the excitability of forebrain neurons at both presynaptic and postsynaptic sites through A1Rs.

Embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF). LIF acts through a receptor complex composed of a low affinity LIF receptor (LIFRbeta) and gp130. We reported that the intracellular domain of gp130 plays an important role in self-renewal of ES cells. In the present study, we examined the signaling pathway through which gp130 contributes to the self-renewal of ES cells. Mutational analysis of the cytoplasmic domain of gp130 revealed that the tyrosine residue of gp130 responsible for STAT3 activation is necessary for self-renewal of ES cells, while that required for SHP2 and MAP kinase activation was dispensable. Next, we constructed a fusion protein composed of the entire coding region of STAT3 and the ligand binding domain of the estrogen receptor. This construction (STAT3ER) induced expression of junB (one of the targets of STAT3) in ES cells in the presence of the synthetic ligand 4-hydroxytamoxifen (4HT), thereby indicating that STAT3ER is a conditionally active form. ES cells transfected with STAT3ER cultured in the presence of 4HT maintained an undifferentiated state. Taken together, these results strongly suggest that STAT3 activation is required and sufficient to maintain the undifferentiated state of ES cells.

Early dynamic and late 123 I-MIBG SPET studies were performed to investigate several parameters used to distinguish the characteristics of various cardiac disorders. Forty-six individuals (34 non-diabetic, 12 diabetic) with or without heart disease were included in the study. Early dynamic and late static SPET images were acquired using a triple-headed gamma camera. After selecting mid-sections from vertical (VLA) and horizontal (HLA) long-axis images, regions of interest were created over the apex, whole heart and anterior, inferior, septal and lateral walls of the heart. Various uptake ratios at 3, 11 and 19 min and 4 h after injection (HU3, HU11, HU19, DUP) and clearances (K se : between HU3 and HU11; K e : between HU11 and HU19; K d : between HU19 and DUP) were calculated. There were significant differences among various cardiac pathologies on the delayed images. Cardiomyopathy patients showed the lowest uptake on the delayed images. When all segments in normal patients and all involved segments in myocardial infarcted patients were compared, there was significantly lower uptake of MIBG in infarcted segments at all time points. K d showed the lowest value compared with K se and K e . In cardiomyopathy patients, K se , K e and K d were significantly different from each other. Both K se and K e were significantly higher in cardiomyopathy patients than in normal patients. In conclusion, the results of this study are in line with published data and precise measurement of uptake and clearance was possible when excluding background and blood pool activity.

To gain insights into the different forms of modified amyloid beta peptides (A beta) in the Alzheimer disease (AD) and Down syndrome (DS) brain, we used two-site ELISAs with antibodies specific for isomerized (i.e. A beta with L-isoaspartate at positions 1 and 7) and pyroglutamate-modified (i.e. A beta beginning with pyroglutamate at position 3) forms of A beta to quantitate the levels of these different A beta peptides in formic acid extracts of AD and DS frontal cortex. Despite variations in the proportions of distinct forms of A beta in AD and DS frontal cortex, the major species of A beta in these samples were A betaN3(pyroGlu)-42 as well as A beta x-42 (where x is a residue at position 2 or less in A beta), whereas isomerized A beta was a minor species. Further, the levels of isomerized and pyroglutamate-modified forms of A beta terminating at amino acid 42 were higher than those ending at amino acid 40. The abundance of the distinct forms of A beta reported here in formic acid extracts of AD and DS frontal cortex suggests that these A beta species could play important roles in the deposition of A beta in AD and DS brains.

The aim of this study was to characterize lung lesions by means of the uptake, retention and washout of 201 Tl, and to quantify the sensitivity of 201 Tl SPET (single photon emission tomography) in detecting mediastinal and hilar lymph nodes. A total of 68 patients with lung lesions (tumours and inflammation) underwent both helical computed tomography (CT) and 201 Tl SPET examinations. Uptake, retention and washout parameters were calculated from early and delayed 201 Tl SPET by drawing regions of interest over the primary lesion and the background of the lung. The findings on 201 Tl SPET were compared with those on CT and surgical findings. A significant difference in the retention index (RI) was observed between primary malignant and benign lesions. In detecting lymph nodes, the sensitivity, specificity and negative predictive value were 83%, 60% and 90% respectively for early 201 Tl SPET, and 50%, 80% and 80% respectively for delayed 201 Tl SPET. 201 Tl SPET with semi-quantitative analysis, especially RI, helps to differentiate between malignant and benign lung lesions. Lymph nodes larger than 1 cm on CT but not showing 201 Tl uptake could be considered non-metastatic lesions.

A recombinant human neutrophil chemotactic protein LECT2 (rhLECT2) was purified as a 16-kDa protein from the culture fluids of stable transfectants derived from CHO cells (clone C1D8-1) and L929 cells (clone L2E4-1). The N-terminal amino acid sequence of the protein secreted by both clones were homologous to the previously described bovine LECT2. We produced polyclonal and monoclonal antibodies against rhLECT2 and investigated secretion of LECT2 protein in six human hepatoma cell lines, which express LECT2 mRNA, and in hepatocytes of normal human livers by a sandwich enzyme-linked immunosorbent assay and by immunostaining using the antibodies, respectively. We revealed that five of six hepatoma cell lines secreted LECT2 into culture fluids at concentrations of 30-135 ng/mg. We also demonstrated that the cytoplasm of human hepatocytes was diffusely stained, although periportal hepatocytes tended to be weakly and granularly stained by immunostaining. These results indicated that the novel protein was expressed in hepatocytes and suggested an important role of LECT2 in the cells in addition to the activation of neutrophils.

High mobility group 2 (HMG2) protein is ubiquitously distributed in the nucleus of higher eukaryotic cells. Accumulation of an HMG2-beta-galactosidase fusion protein expressed in COS-7 cells suggested active transport of HMG2 protein into the nucleus after translation in the cytoplasm. Deletion analysis of the HMG2 sequence in the HMG2-beta-galactosidase fusion protein indicated that basic regions interspaced with the long DNA-binding sequence in HMG2, called the HMG1/2 box, are necessary for the nuclear accumulation of HMG2. The close configuration of basic regions at both ends of the DNA-binding sequence in the tertiary structure may function as the nuclear localization signal. This novel nuclear localization signal structure is different from typical ones such as the single or bipartite basic cluster in many nuclear proteins. A portion of the HMG2 molecule remained in the cytoplasm after translation. Interspecies heterokaryon assay demonstrated that the acidic carboxyl terminus of HMG2 was necessary for retention of the protein in the nucleus.

The kinetics, intracellular distribution and effects of ion transport inhibitors on the uptake of 99 Tc m -tetrofosmin, 99Tcm-methoxyisobutyl isonitrile ( 99 Tc m -MIBI) and 201 Tl were investigated in a tumour cell line. Both uptake and washout of the tracers were measured at specific intervals. Cells were treated with ouabain, dimethyl amiloride (DMA) and bumetanide to observe the effects on uptake, while carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used to study the release of the tracers. The tracers showed similar uptake and washout kinetics. Ouabain inhibited 55-67% of 201 Tl, 16-22% of 99 Tc m -tetrofosmin and 8-14% of 99 Tc m -MIBI. DMA inhibited the uptake of both 99 Tc m -tetrofosmin (30%) and 99 Tc m -MIBI (21%). Bumetanide stimulated the uptake of 99 Tc m -tetrofosmin and 99 Tc m -MIBI but inhibited that of 201 Tl (37%). When cells were pretreated with various combinations of these ion transport inhibitors, the uptake of 201 Tl was related to Na + ,K + pump, Na + ,K + ,2Cl - co-transport systems, whereas the uptake of both 99 Tc m -tetrofosmin and 99 Tc m -MIBI was related to the Na + ,K + pump, Na + /H + antiport systems. Addition of CCCP released 55% and 90% of accumulated 99 Tc m -tetrofosmin and 99 Tc m -MIST respectively, indicating that the percentage released was related to mitochondrial uptake.

The effects of monoclonal anti-tubulin antibodies on the motility of demembranated and reactivated sea urchin spermatozoa were investigated. Two out of ten antibodies examined significantly reduced the motility of spermatozoa, both in motile rate and swimming speed. The binding patterns of the two antibodies YL1/2 and TUB2.1 to the axoneme were studied by immunoblot, immunofluorescence, and immuno-electron microscopy. YL1/2 bound to the axoneme in a specific pattern; signals were very intense in the tail, rich in the proximal portion, and scarce in the middle part of the axoneme. Because the inhibitory effects of the antibody on the motility of spermatozoa with fully long flagella and short flagella were similar, the inhibition was probably due to the binding of the antibody to the proximal portion of the flagellum. TUB2.1 evenly bound to the axoneme by immunofluorescence and immunoelectron microscopy. On the other hand, the eight antibodies which did not affect sperm motility, did not bind to unfixed axonemes, although epitopes for these antibodies were detected abundantly in the axoneme.

The characteristics of a new ionization gauge for pressure measurement in XHV were examined by experiments and computer simulations. The new gauge (axial-symmetric transmission gauge : AT gauge) has a Bessel-Box type energy filter between a B-A gauge type ionizer and a secondary electron multiplier as an ion collector to eliminate the limiting factors of pressure measurement in XHV, such as the X-ray effect and electron stimulated desorption (ESD) ions. The lower limit of the pressure measurement at present is estimated to be about 10-12 Pa. The sensitivity factor of the AT gauge, which was further corrected for the effect of outgassing by the conductance modulation method (CMM), is almost constant in the pressure range from 10-10 to 10-6 Pa and is (2.3±0.1) ×10-3 Pa-1 for hydrogen.

Background Intracytoplasmic inclusions composed of filamentous tau proteins are defining characteristics of neurodegenerative tauopathies, but it remains unclear why different tau isoforms accumulate in different diseases and how they induce abnormal filamentous structures and pathologies. Two tau isoform-specific antibodies, RD3 and RD4, are widely used for immunohistochemical and biochemical studies of tau species in diseased brains. Results Here, we show that extensive irreversible post-translational deamidation takes place at asparagine residue 279 ( N279) in the RD4 epitope of tau in Alzheimer’s disease (AD), but not corticobasal degeneration (CBD) or progressive supranuclear palsy (PSP), and this modification abrogates the immunoreactivity to RD4. An antiserum raised against deamidated RD4 peptide specifically recognized 4R tau isoforms, regardless of deamidation, and strongly stained tau in AD brain. We also found that mutant tau with N279D substitution showed reduced ability to bind to microtubules and to promote microtubule assembly. Conclusion The biochemical and structural differences of tau in AD from that in 4R tauopathies found in this study may therefore have implications for prion-like propagation of tau.

The human epithermoid carcinoma-derived cell line MA1, established by introduction of the adenovirus E1A 12 S cDNA linked to the mouse mammary tumor virus long terminal repeat, elicits apoptosis after induction of E1A12S in response to dexamethasone. The level of topoisomerase IIalpha begins to decrease steeply within 36 h preceding the onset of DNA fragmentation, whereas its mRNA level is unchanged (Nakajima, T., Ohi, N., Arai, T., Nozaki, N., Kikuchi, A., and Oda, K. (1995) Oncogene 10, 651-662). Topoisomerase IIalpha prepared by immunoprecipitation or extraction of the nuclear matrix was degraded much more efficiently in the S10 extract prepared from MA1 cells treated with dexamethasone for 42 h (the 42-h extract) than in the extract from untreated MA1 cells (the 0-h extract) in an ATP- and ubiquitin-dependent manner. The proteolytic activity for degradation of topoisomerase IIalpha was suppressed specifically by inhibitors for the proteasome and was much reduced in the 42-h extract prepared from MA1-derivative cell lines expressing E1B19k or Bcl-2. The proteolytic activity was lost after fractionation of the 42-h S10 extract into the S70 and P70 fractions by centrifugation at 70,000 x g for 6 h but partially recovered when these fractions were combined. Polyubiquitinated forms of topoisomerase IIalpha could be detected by incubating it in the S70 or S100 extract, which lacks most of the proteasome activity. The ubiquitination activity in S70 prepared from the 42-h extract was 4- to 5-fold higher than that prepared from the 0-h extract. These results suggest that a component(s) in the ubiquitin proteolysis pathway, responsible for ubiquitination and degradation of topoisomerase IIalpha, is activated or induced during the latent phase of E1A-induced apoptosis.

We report scan findings of Tl-201 in a case of histologically proved Warthin’s tumor. Dual isotopes acquisition was performed in a case of left parotid Warthin’ s tumor. Both early and delayed Tc-99m-pertechnetate (Tc-99m) images showed increased uptake at the tumor. Tl-201 scan also showed an increase of uptake on the early image and retention on the delayed image. The uptake ratios of Tl-201 were 3.0 (early) and 2.3 (delayed). Scan findings of Tl-201 in a case of Warthin’s tumor were similar to those of Tc-99m, and the interpretation of Tl-201 images should be accompanied with Tc-99m images.

Glycosylphosphatidylinositol-anchored protein Thy-1 is abundantly expressed on the cell surface of neurons and T lymphocytes in rodents. Although Thy-1 is known to bind integrins as a ligand and to mediate neurite outgrowth and immune responses, its precise function is not fully understood. Previously we produced several anti rat Thy-1 monoclonal antibodies and identified one, 2E11, which induces PC12 cell neurite outgrowth. Here we screened antibodies that inhibit 2E11-induced neurite outgrowth and stimulate or inhibit rat thymocyte aggregation. Since Thy-1 lacks an intracellular region, it requires other membrane-bound molecules for the signal transduction. Hence these antibodies are hypothesized to play key roles in the interaction between Thy-1 and signaling molecules. To elucidate the mechanisms of antibody-induced Thy-1 functions, antibody characterization and epitope determination were carried out. Thy-1 cleavage and mutation revealed that the antibodies recognize not only amino acid sequences, but also the three-dimensional structures consisting of immunoglobulin-like domains. Two antibodies were suggested to bind spatially close to the integrin binding site and crosslink Thy-1 molecules, while a third antibody is believed to inhibit Thy-1 crosslinking and subsequent Thy-1 signaling. The antibodies reported here may therefore function as crosslinkers, agonists, or antagonists that modify Thy-1 signaling.

Pulmonary metastases of benign meningiomas are extremely rare. The case of a 34-year-old man with bilateral parasagittal meningioma who developed pulmonary metastases is described. The meningioma was an enormous hypervascular tumor with invasion of the superior sagittal sinus. The tumor was resected completely and histologically diagnosed as transitional meningioma. The Ki-67 labeling index was 5%. Four months after operation, the patient subsequently developed bilateral multiple lung lesions later identified as metastases. The lung lesions were partially removed surgically and histologically diagnosed as meningothelial meningioma WHO grade I. The Ki-67 labeling index was 2%. The histological findings demonstrated that the tumor occupied the arterial lumen and the perivascular space, suggesting that pulmonary tumors might metastasize via the vascular route. The histopathological features and mechanisms of metastasizing meningiomas are reviewed and discussed.

A total of 7 (4 males and 3 females) patients were included in this retrospective study to determine the sensitivity of radioimmunoscintigraphy with I-131 labeled anti CEA/CA 19-9 monoclonal antibodies. Out of 7 patients 2 had ascending colon cancer, one had sigmoid colon cancer, one had rectal cancer and one had adenocarcinoma in the CBD and the remaining two had metastatic tumor (one in the lungs and the other in the liver). Whole body as well as spot images showed a 72% (5/7) positive scan. But post operative specimen counts and imaging showed a high tumor to non-tumor ratio and a good tumor to non-tumor contrast of activity of I-131 labeled monoclonal antibody. We did not find any relation between CEA/CA 19-9 levels and scan findings. A case of liver metastasis was also detected by this radioimmunoscintigraphy.

The effect on fixation on the reactivities of mitotic microtubules with monoclonal anti-tubulin antibodies was investigated by the indirect immunofluorescence procedure. All of the seven antibodies used intensely stained mitotic microtubules in sea urchin eggs lysed and fixed with methanol at −20°C, whereas only two of them stained the stabilized microtubules in the lysed eggs before the fixation. The other five did not stain the mitotic microtubules even after microtubule components other than tubulin were removed by treating the lysed eggs with 0.4 M KCl solution containing taxol. These results exclude the possibility that the fixation affects proteins, which interact with microtubules including microtubule-associated proteins (MAPs) and interfere with the binding of monoclonal antibodies with tubulin, and strongly suggest that the fixation directly affects the three-dimensional conformation of tubulin Furthermore, microinjection of these antibodies indicated the results as follows [combining the results reported previously; Oka et al., 1990: Cell Struct. Funct. 15: 373–378]: The antibodies which stained mitotic microtubules stabilized in the lysed eggs induced disassembly of native mitotic microtubules in the living eggs, but those which did not stain the stabilized microtubules did not disassemble the native microtubules. From these results, it is suggested that the monoclonal antibodies which stain microtubules in the eggs lysed but not fixed are useful for microinjection experiments. © 1994 Wiley-Liss, Inc.

Presence of cytoplasmic tau aggregates is a hallmark of brains in patients with tauopathies such as Alzheimer's disease. However, the mechanism underlying formation of these insoluble tau aggregates remains elusive. In this study, we investigated the impact of prolonged nitric oxide (NO) exposure on neuronal SH-SY5Y cells overexpressing human tau. Treatment with the NO donor DETA NONOate for up to 48h resulted in an increase in S-nitrosation of cellular proteins, inactivation of proteasome, and impairment of respiration. Western blot analysis of Triton X-soluble fractions of NO-treated cells revealed that persistent NO treatment increased heterogeneity in tau molecule size, as a result of dephosphorylation, and induced the formation of sodium dodecyl sulfate (SDS)-stable oligomeric tau aggregates, stabilized by disulfide bonds. Moreover, further NO treatment induced the formation of SDS-stable insoluble tau mega-aggregates that were composed of dephosphorylated full-length tau molecules and other proteins, and were stabilized through disulfide bonds. Evaluation of the role of these tau aggregates as potential seeds for tau fibrillization and elucidation of their formation mechanism in our model, could lead to better understanding of the pathogenesis of tauopathies.

A 70-year-old man presented with a rare case of paranasal osteoma with secondary mucocele extending intracranially, manifesting as a generalized convulsion. Computed tomography showed a large calcified tumor adjacent to the cystic mass in the left frontal lobe. He underwent left frontal craniotomy, and the cystic lesion was totally removed. Histological examination confirmed the diagnosis of osteoma and mucocele. The giant paranasal sinus osteoma prevented growth of the mucocele into orbital recess and extension into the orbital space and paranasal sinus. The mucocele disrupted the dura in the anterior cranial fossa, resulting in a giant cystic intracranial lesion. Frontal osteoplastic craniotomy was effective for exposing both lesions and plastic repair of the dural perforation to prevent cerebrospinal fluid leakage and secondary infection.

The incidence of brain abscess remains high, despite the development of novel antibiotics. Vancomycin or carbapenems, which are third-generation cephems, are recommended as standard therapy for bacterial meningitis or brain abscess. The effectiveness of the high-dose meropenem therapy on brain abscess has occasionally been reported. We experienced 2 consecutive cases of brain abscess in adults. The first patient was a 67-year-old man with diplopia, dizziness, and dysesthesia on the left upper and lower extremities. Images of T1-weighted magnetic resonance imaging (MRI) with contrast medium and diffusion-weighted MRI showed a ring enhancing cystic lesion and a high intensity lesion, respectively, in the right pons. The second patient was a 37-year-old man who complained of right hemiparesis. MRI revealed a ring-enhancing cystic mass in the left thalamus. On the basis of MRI findings, patients were diagnosed with brain abscess and were given high-dose meropenem (6g/day) continuously for 2 months. The abscess resolved completely after treatment with meropenem administered intravenously. Further, neurological deficits caused by abscess successfully improved. High-dose meropenem therapy should be considered as an effective treatment for brain abscess, even in the brain stem and basal ganglia, where it is quite difficult to achieve surgical access.

Genetically-encoded Forster resonance energy transfer (FRET) biosensors enable us to visualize a variety of signaling events, such as protein phosphorylation and G protein activation in living cells (Miyawaki, 2003). Using FRET-based biosensors we can obtain spatiotemporal information on the changes in activity of signaling molecules in living cells. From this viewpoint, FRET imaging of signaling molecules that regulate membrane traffic is one of the most suitable applications of this technique. The Rab family GTPases constitute the largest branch of the Ras GTPase superfamily. Rab GTPases use the guanine nucleotidedependent switch mechanism common to the Ras superfamily to regulate each of the four major steps in membrane trafficking: vesicle budding, vesicle delivery, vesicle tethering, and fusion of the vesicle membrane with that of the target compartment (Zerial and McBride, 2001; Grosshans et al., 2006; Stenmark, 2009). Recently, we developed a FRET sensor for Rab5, and demonstrated that live-cell imaging with FRET sensors enables us to pinpoint the activation and inactivation of Rab5, and thereby to understand its relationship with other events linked to vesicle transport (Kitano et al., 2008). In the first half of this chapter, we describe step-by-step strategies to develop unimoleculartype FRET biosensors for Rab family GTPases. We use the development of a Rab35 sensor as an example. Although improvements to FRET sensors are still made on a trial-and-error basis, we provide practical tips for their optimization. In the second half of this chapter, we introduce FRET imaging with total internal reflection fluorescence (TIRF) microscopy. TIRF microscopy is particularly well suited to visualize the dynamics of molecules and events near the plasma membrane (Mattheyses et al., 2010). We have used FRET imaging with TIRF microscopy to show that the activity of TC10, a Rho family GTPase, at tethered vesicles drops immediately before vesicle fusion in HeLa cells stimulated with epidermal growth factor (EGF) (Kawase et al., 2006). We describe how to set up and use TIRF-FRET to visualize local changes in GTPase activity on vesicles during membrane fusion.

Trigeminal neuralgia is lancinating pain of a few seconds duration triggered by minor sensory stimuli such as speaking, chewing or even a breeze on the face. Vascular compression of the trigeminal nerve at the root entry zone and other vessels has been implicated in its cause. Despite the initial success of medical treatment in some cases, however, many patients become refractory over time and eventually require surgical intervention. This report describes a case involving a 62-year-old woman who presented with right orbital pain provoked by, among others, exercise and cold. Medication proved to be ineffective and, after magnetic resonance imaging, microvascular decompression and surgical observation, the diagnosis became clearer. The case highlights the importance of preoperative imaging and careful intraoperative findings to determine whether variant arteries are responsible for trigeminal neuralgia.A patient who presented with trigeminal neuralgia associated with a persistent primitive trigeminal artery (PPTA) is presented. A 62-year-old woman suffering from right orbital pain was admitted to the hospital. Medical treatment for three months was ineffective, and her neuralgia had deteriorated and gradually spread in the maxillary division. Magnetic resonance imaging demonstrated the flow void signal attached to the right trigeminal nerve. Thus, microvascular decompression was performed. The superior cerebellar artery was the responsible artery, and it was transposed to decompress the trigeminal nerve. After this manoeuvre, an artery was identified running parallel to the trigeminal nerve toward Meckel’s cave. The artery, which turned out to be a PPTA, communicated with the basilar artery. The PPTA was carefully observed, and it was found not to be the artery causing the neuralgia because it did not compress the nerve at surgical observation. No additional procedure between the PPTA and the trigeminal nerve was performed. The patient’s symptom improved dramatically following surgery, and her postoperative course was uneventful. Postoperative three-dimensional computed tomography showed the PPTA. The findings in the present case suggest that transposition of the responsible artery effectively decompresses the root entry zone and assists in determining whether the PPTA is affecting the trigeminal nerve.

The mechanism of protein S-nitrosation in cells is not fully understood. Using rat 3Y1 cells, we addressed this issue. Among S-nitrosothiols and NO donors tested, only S-nitrosocysteine (CysNO) induced S-nitrosation when exposed in Hanks' balanced salt solution (HBSS) and not in serum-containing general culture medium. In HBSS, NO release from CysNO was almost completely abolished by sequestering metal ions with a metal chelator without affecting cellular S-nitrosation. In contrast, L-leucine, a substrate of L-type amino acid transporters (LATs), significantly inhibited S-nitrosation. The absence of S-nitrosation with CysNO in general culture medium resulted not only from a competition with amino acids in the medium for LATs but also from transnitrosation of cysteine residues in serum albumin. Collectively, these results suggest that in simple buffered saline, CysNO-dependent S-nitrosation occurs through a cellular incorporation-dependent mechanism, but if it occurs in general culture media, it may be through an NO-dependent mechanism.

Two monoclonal antibodies C5 and M4 raised against Sarkosyl-insoluble paired helical filaments (PHF) specifically labeled fetal tau, but hardly labeled normal adult tau. C5 immunoreactivity was eliminated by alkaline phosphatase treatment at 37 degrees C, whereas M4 reactivity could be removed only by the treatment at 67 degrees C. Epitope analysis showed that C5 and M4 recognition sites are in residues 386-406 and 198-250, respectively, according to the numbering of the longest human tau isoform. Thus, the phosphorylation sites are located in the amino- and carboxyl-terminal portions of the microtubule-binding region. These two well-characterized monoclonals should be valuable in the identification of a protein kinase(s) that converts normal tau into PHF-tau.

A 5-year old boy presented with an arachnoid cyst in the middle cranial fossa with mild midline shift manifesting as headache and loss of activity. Computed tomography (CT) showed subdural hygroma. Burr-hole drainage was carried out and symptoms were improved postoperatively. However, recollection of subdural hygroma was found on follow-up CT 3 weeks after subdural drainage. He underwent craniotomy, and tearing of the outer wall of the arachnoid cyst was observed. The ruptured cyst wall was tightly closed by arachnoidplasty to prevent cerebrospinal fluid leakage. Arachnoidplasty was effective for traumatic subdural hygroma with arachnoid cyst for reconstruction.

A 61-year-old man presented with left lower quadrianopsia caused by cerebral infarction in the right occpital lobe. Cerebral angiography revealed occlusion of right transverse sinus and Sylvian–middle fossa dural arteriovenous fistula (d-AVF) draining into the Sylvian vein and dilation of basal vein of Rosenthal. Surgical operation with right frontotemporal craniotomy was carried out to obliterate the fistula point and resection of the dura mater containing vasculature networks. Histologically, the thickening of walls of dural arteries and veins lacking internal elastica lamina were observed. Interestingly, the dura mater involving d-AVF was hyalinized and lacked collagen fibers, resembling local hypoxia and suggesting the possible role of dural hypoxia with pathogenesis of d-AVF. The present case indicates that open surgery can be effective for Sylvian–middle fossa d-AVF for the purpose of obliteration of fistula point and resection of the dura for histopathologic analyses.

The purpose of our study was to evaluate the application of thermoreversible gelation polymer (TGP) as a local drug delivery system for malignant glioma.Polymeric microspheres or liposomes loaded with doxorubicin (sphere-dox or lipo-dox) were combined with TGP to provide continuous drug delivery of doxorubicin (dox) for kinetic release studies and cell viability assays on glioma cell lines in vitro. For in vivo studies, TGP loaded with dox alone (TGP-dox) was combined with sphere-dox or lipo-dox. Their antitumor effects on subcutaneous human glioma xenografts were evaluated in nude mice.In vitro, TGP combined with sphere-dox or lipo-dox released dox for up to 30 days. In vivo, TGP-dox combined with sphere-dox or lipo-dox inhibited subcutaneous glioma tumor growth until day 32 and day 38, respectively.TGP in combination with microspheres or liposomes successfully prolonged the release of dox and its antitumor effects.

A 50-year-old woman presented with nuchal pain, clumsiness in both hands, and gait disturbance. Cervical magnetic resonance (MR) imaging showed cervical canal stenosis at C3-C7 levels associated with a syrinx and cerebellar tonsillar herniation. The patient underwent C3-C7 laminoplasty. Her symptoms improved completely, and the patient was discharged 10 days after operation. Postoperative MR imaging showed disappearance of the syrinx. This case suggests that cerebellar tonsillar herniation may not cause syringomyelia. Posterior decompression, such as laminectomy and laminoplasty, without foramen magnum decompression may be an appropriate surgical strategy for diffuse cervical canal stenosis associated with syringomyelia below the narrow canal, even in the presence of co-existing Chiari malformation.

Several parameters calculated with a new functional imaging agent for the liver, Tc-99m DTPA-galactosyl human serum albumin, were evaluated in 9 patients with liver cirrhosis, one with hepatocellular carcinoma, and five with both liver cirrhosis and hepatocellular carcinoma. LU3, which represents the cumulative uptake of the tracer from 3 to 4 minutes after injection, showed a strong correlation (r = 0.858, p = 0.0001) with LHL15, which represents the count ratio for the liver to sum for the liver and heart 15 minutes after injection of the tracer. It also showed a strong correlation (r = -0.896, p = 0.0001) with the indocyanine green retention rate (ICGR15). Regional ICGR15 is therefore calculable from the regional LU3. GSAR15, which represents the radioactivity of the tracer retained in the blood 15 minutes after injection, showed a strong correlation (r = 0.878, p = 0.0001) with HH15, which represents the count ratio for the heart 15 minutes after injection of the tracer divided by the count for the heart 3 minutes after injection. In conclusion, LU3 and GSAR15 are interesting and promising parameters for assessing liver function.

Vital-organ transplantation has become acceptable as the treatment of choice for end-stage organ failure. If the patient, facing the end of life, wishes to donate organs after cardiac arrest (CA), donation after cardiac death (DCD) is increasingly important for the realization of the patient's desires after CA. In Japan, kidney transplantation from uncontrolled DCD donors, who are identified in modified Maastricht categories II or V, is one of the critical factors in expanding the donor pool. However, according to the forensic code for post-mortems and the requirement of legal consent for transplantation, the time required to meet all procedural requirements has sometimes prohibited organ procurement from uncontrolled DCD donors. We have therefore attempted to use an automated cardiopulmonary resuscitation (CPR) device and maintain arterial pressure for uncontrolled DCD donors during all interim procedures after sudden CA. Comparing kidneys procured from standard DCD donors (n = 10) and uncontrolled DCD donors (n = 4), significant differences were seen in warm ischemic time (WIT), defined as the time from CA to initiation of cooling in situ. However, our early experience showed good tolerance and viability of uncontrolled DCD kidneys. Immediate availability of an automated CPR device might provide a bridge to kidney procurement from uncontrolled DCD donors.

The authors describe a case of rhabdoid meningioma (RM) in a 17-year-old boy that was determined by measuring the tumor volume during preoperative follow-up. The volume of the tumor located in the left occipital lobe, was measured every 1-5 months, using an image analysis software. The tumor volume doubling time (Td) ranged from 1.0 to 4.9 years in the first 11 months, but became 0.3 years in the last two months. The tumor grew rapidly in the last two months at which time surgery was performed. Pathological examination of the surgical specimen showed that the tumor contained rhabdoid cells (RCs). RCs were heterogeneously distributed in the tumor admixed with spindle-shaped cells. The areas where RCs were predominant had malignant histological features, with necrosis and high proliferation indices, whereas the areas with few RCs lacked the malignant features. The tumor grew slowly in the initial phase, possibly because components with low proliferation rates occupied most of the tumor. The tumor began to grow rapidly when the malignant component containing abundant RCs became predominant. To the authors' knowledge, this is the first report monitoring the volumetric change of RM periodically. Our investigation indicated that volumetric analysis is useful to decide surgical intervention of the meningiomas with potential malignancy.

To test the hypothesis that tau-immunoreactive curly fibers represent a regenerative attempt of cortical neurons in the brain affected with Alzheimer's disease (AD), we established 2 monoclonal antibodies to isolated neurofibrillary tangles. Both antibodies extensively stained curly fibers and specifically labeled a novel protein of Mr 70,000 (p70), which was abundantly expressed in fetal brain, but undetectable in adult brain. Furthermore, the antibodies visualized curly fibers in newborn rat brain, which were very similar to those in the AD brain, and also intensely stained extending neurites of NGF-primed PC 12 cells. These observations suggest that P70 is a fetal antigen associated with growing neurites, and thus support the sprouting hypothesis.

This paper describes a depth recording technique for commercial VTR in which the digital audio signal and the video signal are recorded on the depth-multiplexed layer of a magnetic tape. Digital audio signals with a data rate of 2.6Mbps were recorded with video signals. To avoid crosstalk, a digital modulation method has been studied which gives less low-frequency components. The 0-QDPSK method was most suitable to magnetic recording. A data error rate of less than 10-4 was obtained and interference was eliminated.

We synthesized a series of neoechinulin A derivatives and examined the structure-activity relationships in terms of their anti-nitration and anti-oxidant activities as well as their cytoprotective activity against peroxynitrite from SIN-1 (3-(4-morpholinyl)sydnonimine hydrochloride) using PC12 cells. Our results showed that the C-8/C-9 double bond, which constitutes a conjugate system with indole and diketopiperazine moieties of neoechinulin A is essential for anti-nitration and anti-oxidant activities as well as protection against SIN-1 cytotoxicity. The presence of an intact diketopiperazine moiety is an additional requirement for anti-nitration activity but not for the cytoprotective action. Our results suggest that the antioxidant activity or electrophilic nature of the C-8 carbon, both of which are afforded by the C-8/C-9 double bond, may play a role in the cytoprotective properties of this alkaloid.

A major end product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE), is an electrophilic alkenal and produces Michael adducts with cellular proteins. It is known that exposure of cultured cells to HNE causes rapid disappearance of microtubule networks. In this study we addressed the mechanism. Immunochemical studies revealed that HNE preferentially modified alpha-tubulin in rat primary neuronal cells, PC12 cells, and rat fibroblast cell line 3Y1 cells. This was morphologically associated with the disappearance of microtubule structures in those cells. In a purified rat brain microtubule fraction, HNE modified unpolymerized tubulin and impaired its polymerizability, with a concomitant increase in insolubilized tubulin. Nevertheless, HNE had a marginal effect on the stability of pre-polymerized microtubules. These results suggest that disruption of microtubule assembly as a result of HNE modification of unpolymerized tubulin, rather than destruction of assembled microtubules, is responsible for the disappearance of microtubule structures in cells exposed to HNE.

p94/calpain 3 is a skeletal muscle-specific Ca(2+)-regulated cysteine protease (calpain), and genetic loss of p94 protease activity causes muscular dystrophy (calpainopathy). In addition, a small in-frame deletion in the N2A region of connectin/titin that impairs p94-connectin interaction causes a severe muscular dystrophy (mdm) in mice. Since p94 via its interaction with the N2A and M-line regions of connectin becomes part of the connectin filament system that serves as a molecular scaffold for the myofibril, it has been proposed that structural and functional integrity of the p94-connectin complex is essential for health and maintenance of myocytes. In this study, we have surveyed the interactions made by p94 and connectin N2A inside COS7 cells. This revealed that p94 binds to connectin at multiple sites, including newly identified loci in the N2A and PEVK regions of connectin. Functionally, p94-N2A interactions suppress p94 autolysis and protected connectin from proteolysis. The connectin N2A region also contains a binding site for the muscle ankyrin repeat proteins (MARPs), a protein family involved in the cellular stress responses. MARP2/Ankrd2 competed with p94 for binding to connectin and was also proteolyzed by p94. Intriguingly, a connectin N2A fragment with the mdm deletion possessed enhanced resistance to proteases, including p94, and its interaction with MARPs was weakened. Our data support a model in which MARP2-p94 signaling converges within the N2A connectin segment and the mdm deletion disrupts their coordination. These results also implicate the dynamic nature of connectin molecule as a regulatory scaffold of p94 functions.

The heterogeneity of mitotic microtubules in dividing sea urchin eggs was investigated by indirect immunofluorescence using five anti-α-tubulin (YL1/2, DM1A, E3B8, D2D6, and 6-11B-1) and two anti-β-tubulin (E6B6 and DM1B) antibodies. These antibodies were divided into four classes in regard to the different immunofluorescent staining patterns: class I, which strongly stained both the spindle and aster (YL1/2, DM1A, E3B8 and E6B6); class II, which strongly stained the spindle but weakly stained the aster (D2D6); class III, which stained only the aster (DM1B); and class IV, which did not stain the mitotic apparatus (6-11B-1). These results suggest that tubulin isotypes are distributed differently in the sea urchin mitotic microtubules and that α-tubulin isotype(s) recognized by D2D6 is (are) localized mainly in spindle microtubules, whereas β-tubulin isotype(s) recognized by DM1B is (are) found only in astral microtubules.

Two monoclonal antibodies against alpha-tubulin (YL1/2 and D2D6) were microinjected into the egg of the sand dollar Clypeaster japonicus, and their effects on cleavage of the egg were investigated. They had already been shown by immunoblotting to react specifically with egg tubulin and by immunofluorescence to stain the mitotic apparatus [OKA et al., (1990). Cell Motil. Cytoskel. 16:239-250]. Injection of YL1/2 prevented chromosome movement and cleavage, although the cleavage furrow developed in some cases. In all eggs injected at prometaphase, metaphase, or anaphase, the birefringence of the mitotic apparatus disappeared immediately after injection. Injection of D2D6 had no significant effect on mitosis or cleavage of whole eggs injected after nuclear disappearance, although it prevented the disappearance of the nuclear envelope in 54% of the eggs injected before the disappearance. FITC-conjugated D2D6 did not accumulate in the spindle when injected into the dividing sand dollar egg. These results indicate that YL1/2 disassembled microtubules, whereas D2D6 did not bind to microtubules in the living cell.

Fez-like (Fezl), the forebrain embryonic zinc finger-like protein, is a transcriptional repressor selectively expressed in the deep layers of the developing cortex. We examined the thalamocortical and corticofugal pathways in Fezl-deficient fetal mice by using immunohistochemistry and by axonal labeling with the lipophilic dyes DiI and DiA, with special attention to the spatiotemporal relation between thalamocortical and corticofugal axons. In normal mice, thalamic and cortical axons meet in the internal capsule between embryonic day (E) 13.5 and E14.5 and fasciculate with each other as they extend to their targets, the cortex and thalamus, respectively. In Fezl-deficient mice, most of the thalamic and cortical axons stop in the internal capsule and at the pallial-subpallial boundary at E14.5, respectively. This abnormality is transient, and the thalamic and cortical axons reach their targets at E15.5, although the number of thalamic axons is remarkably reduced in the cortical anlage. Double labeling with DiI and DiA demonstrated close apposition of the thalamic and cortical axons in the subpallium and pallium as well as in the external capsule of this mutant after E15.5. Because the expression of genes that define the pallial-subpallial boundary and guidance molecules of thalamocortical axons did not show remarkable changes in Fezl-deficient mice, abnormal formation of thalamocortical pathway in this mutant may be caused by the defect of axons of cortical efferent neurons that express Fezl. J. Comp. Neurol. 503:454–465, 2007. © 2007 Wiley-Liss, Inc.