Preparation and optimization of epitaxial growth of transparent ZnO nanotip thin films by hydrothermal method.

Zinc oxide (ZnO) nanotip thin films were prepared on ZnO coated nanocrystalline ITO/glass substrates by hydrothermal method. In order to obtain the ZnO nanotip arrays with high aspect ratio, the experimental conditions were optimized. The scanning electron microscope images showed that the surface morphology of ZnO thin films could be easily manipulated by changing the seed layer thickness and growth time. The ZnO nanotip thin films were grown epitaxially on ZnO seed layer coated ITO/glass substrates. The surface morphology of ZnO thin films on ITO/glass substrate changed from nanorods with a flat-top end to nanotips as the growth time was increased from 3 to 15 h. The ZnO thin films prepared under these deposition conditions were highly oriented along (002) direction. The as-prepared sample (15 h) was annealed at different temperatures (30, 100, 150, and 270 degrees C). The surface morphologies of annealed ZnO thin films did not show any remarkable change and the best crystallinity was observed at 100 degrees C. The photoluminescence spectra showed that the near band edge emission shifted to shorter wavelength as the annealing temperature was increased from 30 to 270 degrees C, it was due to the intrinsic stress in the films. This was confirmed by X-ray diffraction analyses. NPB thin films were prepared on ITO/clay and ITO/glass substrates by thermal evaporation method. The electrical properties of Ag/NPB/ITO/Clay showed the Ohmic characteristics (J proportional V(1.0)). The J-V characteristic of Ag/NPB/PMMA/ZnO/ITO/Glass showed good rectification behaviour with a diode-ideality factor of 1.36.

Development of water vapor transmission rate measuring device using a quadrupole mass spectrometer and standard gas barrier films down to the 10-6 g m-2 day-1 level.

Water vapor transmission rate (WVTR) measuring devices with a quadrupole mass spectrometer (QMS) have an advantage in measuring low WVTRs because measurements are taken under an extremely low background of water vapor by realizing ultrahigh vacuum conditions. Here, the reliability of the QMS measurements was improved by including a porous plug with known molecular conductance in the device to generate a reference molar flux for in situ QMS calibration. Then, standard gas barrier (SGB) films made from a clay-polyimide nanocomposite film were also developed and used to validate the measurement. The measurement results for the SGB films were on the extrapolated calibration curve obtained with the porous plug down to WVTR at the 10-6 g m-2 day-1 level within the estimated measurement uncertainty.

Inhibition of formation of microtubular paracrystals in HeLa-S3 cells by neocarzinostatin.

The effect of an antitumor antibiotic, neocarzinostatin (NCS), on the formation of microtubular paracrystals (PC) induced by vinblastine sulfate, 10 mug/ml, in HeLa-S3 cells was examined by phase-contrast microscopy. The pretreatment of HeLa-S3 cells with NCS, 5 to 50 mug/ml, for 4 hr prevented the PC formation, and there was a dose response of NCS to the degree of inhibition. When the same inhibitory effect on PC formation was examined with other antitumor agents at high doses (50 mug/ml), colchicine was found to be one of the most effective agents, like NCS. Puromycin, antimycin, adriamycin, cytochalasin B, and cycloheximide revealed moderate activity, and the other antibiotics, such as mitomycin C, bleomycin, and rifampicin, did not show any effect at all. NCS was a unique antibiotic that inhibited PC formation among inhibitors of DNA synthesis. It was suggested that NCS affects the microfibrillar-microtubular proteins system in vivo, resulting in the inhibitions of organization of spindle fibers from microtubules at the G2 phase in HeLa cells.

Effects of caffeine on neocarzinostatin-induced inhibition of cell cycle traverse in HeLa-S3 cells.

Caffeine was found to suppress the cell cycle effects of the cancer chemotherapeutic agent neocarzinostatin (NCS). When caffeine was added together with NCS to the culture of HeLa-S3 cells, NCS-induced inhibition of DNA synthesis and of mitosis was markedly reduced in the presence of caffeine. Theophylline was also effective, but N6, O2'-dibutyryl cyclic adenosine 3':5'-monophosphate was not. The caffeine-caused reduction of cell cycle effects was also observed in several other cancer chemotherapeutic agents, including bleomycin and Adriamycin. In contrast, the single-strand scission of cellular DNA and the final cell lethality induced by NCS were not affected by caffeine. These results suggest that the mechanism by which NCS inhibits the cell cycle traverse involves a kind of cell damage which is repairable in a manner promoted by caffeine and hence is different from single-strand scission of DNA. Such a mechanism might be common to the cell cycle effects of X-irradiation and several cancer chemotherapeutic agents including NCS.

Inhibition of surface immunoglobulin centeral capping of Daudi cells and cell spreading of HeLa-S3 cells by neocarzinostatin.

The effects of an antitumor antibiotic, neocarzinostatin (NCS), on the surface immunoglobulin central capping induced by anti-immunoglobulin M antibody on Daudi cells and on the cell spreading of trypsinized HeLa-S3 cells were examined. Pretreatment of Daudi cells and HeLa-S3 cells with NCS, 5 to 30 micrograms/ml, for 4 hr inhibited cap formation and cell spreading, respectively. It was shown that there is a direct relationship between the dose and the degree of inhibition. Inhibitors of DNA synthesis such as bleomycin, mitomycin C, and 1-beta-D-arabinofuranosylcytosine showed no inhibitory effect on cap formation or cell spreading. However, known microtubule-acting agents such as colchicine and vinblastine sulfate completely inhibited both capping and cell spreading at a dose of 10 micrograms/ml. In view of the fact that 10 micorograms NCS per ml also inhibit the formation of microtubular paracrystals induced by vinblastine sulfate in HeLa-S3 cells and that other agents known to influence microtubule function such as local anesthetics and calcium ionophores also inhibit both paracrystal formation and cap formation, these new observations add further support to our hypothesis that NCS affects microtubular proteins transmembranously in vivo.

In vitro interleukin 12 activation of peripheral blood CD3(+)CD56(+) and CD3(+)CD56(-) gammadelta T cells from glioblastoma patients.

Interleukin (IL)-12 has recently been shown to be directly involved in the activation of natural killer and alphabeta T cells via an IL-2-independent pathway. We show here that another type of human cytotoxic cell, gammadelta T cells activated by solid-phase anti-CD3 antibody and expanded using IL-2, obtained, in this case, from the peripheral blood of glioblastoma patients, displays significant tumoricidal activity. In addition, its cytotoxic activity against K562 or Daudi cells or against autologous glioblastoma targets (but not lymphocytes) is significantly enhanced when costimulated with IL-2 and IL-12. To study this synergistic activation by the two interleukins of the patients' gammadelta T cells, we screened the cells for the presence of the IL-2 receptor (IL-2R) and IL-12 receptor (IL-12R) using both flow cytometric analysis and PCR. The patients' gammadelta T cells constitutively expressed the high-affinity IL-2R; when stimulated with IL-12 plus IL-2, the levels of IL-2Ralpha and IL-2Rbeta increased, whereas that of IL-2gamma did not. They also expressed marginal levels of low-affinity IL-12R both immediately after IL-2 expansion and after 24-h incubation, and significantly higher levels after 72-h incubation, consistent with the level of gammadelta T-cell activation. IL-12 alone induced little proliferation of patients' gammadelta T cells in a 24-h assay and none in a 72-h assay; however, it caused a marked inhibition of the IL-2-induced proliferative response in the 72-h assay. The synergistic action of IL-2 and IL-12 was completely abolished by combined pretreatment with anti-IL-2alpha, beta, and gamma mAbs. IL-12-mediated enhancement of gammadelta T cell cytotoxic activity was inhibited by anti-IL-2Rbeta mAb in a dose-dependent manner but not by anti-IL-2Ralpha or anti-IL-2Rgamma mAbs. Thus, the increased expression of the IL-2Rbeta is critical for the synergistic activation of gammadelta T cells by IL-12 plus IL-2; it is also probable that at least the low-affinity IL-12R contributes to the activation of gammadelta T cells mediated by either IL-12 alone or IL-12 plus IL-2. We have, therefore, demonstrated that IL-12 can stimulate the cytotoxic activity of gammadelta T cells from glioblastoma patients, acting via the IL-2Rbeta component of the IL-2R and low-affinity IL-12R. IL-12 activation of patients' gammadelta T cells could possibly be of potential use in the treatment of glioblastoma patients.

Regulation of type V adenylyl cyclase by PMA-sensitive and -insensitive protein kinase C isoenzymes in intact cells.

Abstract Type V adenylyl cyclase (AC) was stably over-expressed in HEK293 cells (293AC-V). Forskolin-stimulated cAMP accumulation in 293AC-V was 5 times as great as that in control cells. PMA, a protein kinase C (PKC) activator, enhanced cAMP accumulation in 293AC-V cells dose-and time-dependently and this enhancement was abolished by staurosporine. Insulin also enhanced cAMP accumulation in 293AC-V cells. Co-transfection of PKC-zeta, but not PKC-alpha, potentiated the effects of insulin. These data suggest that type V AC activity is regulated in cells by PKC isoenzymes through different extracellular stimuli.

Isoform-dependent activation of adenylyl cyclase by proteolysis.

Recent findings have suggested that the cellular proteolytic system plays a major role in the regulation of various intra- and extra-cellular signaling. It was previously shown that proteolytic treatment of adenylyl cyclase leads to the activation of this enzyme. We demonstrate that this activation occurs in an adenylyl cyclase isoform-dependent manner. The type II isoform was strongly activated (approximately 500%), the type III isoform was modestly activated (approximately 30%),and the type V isoform was inhibited by trypsin. Activation of type II adenylyl cyclase occurred in trypsin dose- and time-dependent manners and was blocked by a trypsin inhibitor in a dose-dependent manner. Other proteases, such as thrombin and plasminogen, similarly activated the type II isoform, but not the others. Our data suggest that proteolytic activation is an isoform- and thus cell type-dependent mechanism of altering adenylyl cyclase catalytic activity.

A simple method for the propagation and purification of gamma delta T cells from the peripheral blood of glioblastoma patients using solid-phase anti-CD3 antibody and soluble IL-2.

Although gamma delta T cells make up no more than 10% of the peripheral blood mononuclear (PBM) cells, they appear to play an important role in host defense against tumor growth. In order to evaluate their functional activity against tumors and their response to various cytokines, large numbers of cells are required. Here, we describe a newly-devised method for the isolation and expansion of gamma delta T cells from the peripheral blood of cancer patients, in particular those with glioblastoma. Using this approach, a 1000-1500-fold increase in total cell numbers was achieved in two weeks, the proportion of gamma delta T cells in the expanded population being, on average, approximately 30% after 14 days of culture. The method therefore gives a yield of approximately 10-15 x 10(8) gamma delta T cells from only 5 ml of peripheral blood from glioblastoma patients and normal controls. The highly purified gamma delta T cells of glioblastoma patients were shown to bear both a high-affinity interleukin-2 receptor (IL-2R) and a low-affinity IL-12 receptor (IL-12R). They also displayed significant cytotoxicity against autologous tumor cells, but not against autologous fresh or IL-2-treated lymphocytes, and proliferated in response to IL-2, both effects being dependent on the dose of IL-2 used for activation. In addition, overnight incubation with 700 U/ml of IL-2 or 50 ng/ml of IL-12 resulted in significant cytotoxic activity of patients' gamma delta T cells against K562 target cells, the level of activity being almost the same as with similarly-treated gamma delta T cells from normal controls (P > 0.05). These results demonstrate that the patients' gamma delta T cells obtained using this method are intact in terms of cytotoxic function. Thus, this method not only makes it possible to produce large numbers of purified gamma delta T cells but also to produce populations containing both gamma delta T cells and NK cells, both active against tumor targets which might be suitable for clinical trials of adoptive-immunotherapy, especially in cancer patients for whom no effective therapy is available.

Role of natural killer cells in intraocular melanoma metastasis.

The authors studied the role of natural killer (NK) cells in spontaneous metastasis of murine intraocular melanoma by transplanting murine B16 melanoma cells into the anterior chamber of the eye in syngeneic C57BL/6 mice and determining the number of metastatic lung tumor colonies after 40 days. Depletion of NK activity by anti-asialo GM1 serum dramatically enhanced metastasis and augmentation of NK activity by interferon inhibited it. The strong correlation between host NK activity and intraocular melanoma metastasis indicated that NK cells have an important role in spontaneous metastasis of intraocular melanoma.

Molecular forms of glutathione S-transferase and UDP-glucuronyltransferase as hepatic preneoplastic marker enzymes.

Changes in molecular forms of glutathione S-transferase (GST) and UDP-glucuronyltransferase (UDP-GT) as hepatic detoxicating enzymes were investigated during chemical hepatocarcinogenesis in the rat. The activity and the protein amount (formula; see text) itself of the GST-A form, which has fetal characteristics and is separable from other forms by CM-Sephadex column chromatography and by immunologic techniques, was much increased in gamma-glutamyl transpeptidase (gamma-GTP)-positive foci or hyperplastic nodules (HNs) induced by diethylnitrosamine and 2-fluorenylacetamide or 3'-methyl-4-dimethylaminoazobenzene. The activity of enzyme 1 (late fetal form) of UDP-GT assayed with o-aminophenol (o-GT) also increased with increased number of the foci or HNs, while the activity of enzyme 2 (neonatal form) assayed with phenolphthalein (p-GT) changed but little. The foci and HNs were stained more strongly than the nonnodular areas immunohistochemically using the antibody against purified GST-A or o-GT. The two activities were also increased in well-differentiated hepatomas, but they were decreased in moderately and poorly differentiated hepatomas, and activating enzymes such as cytochrome P-450 were markedly decreased from HN. GST-A and o-GT differ from fetal enzymes such as those of carbohydrate metabolism in that they are inducible in the short-term by drugs, including carcinogens, and they show the highest activities in HNs, and so they may be considered as hepatic preneoplastic (PN) antigens.

Prophylaxis of rotavirus gastroenteritis using immunoglobulin.

Oral inoculation of the human group A rotavirus MO strain (G serotype 3) into 5-day-old BALB/c mice causes gastroenteritis characterized by diarrhea. Using this small animal model, passive protection of suckling mice against human rotavirus infection was achieved with the use of immunoglobulin (IgY) from the yolks of eggs of rotavirus-immunized hens. When IgY against the rotavirus strain homotypic with the challenge virus (MO strain) was administered to mice, complete protection was achieved. After immunizing 8-month old pregnant Holstein cows with human rotavirus MO strain, colostrum containing neutralizing antibody to four different G serotypes of human rotavirus, designated Rota colostrum, was obtained. Rota colostrum completely protected suckling mice against rotavirus infection, and purified IgG obtained from Rota colostrum protected against infection with the homologous virus. After randomly grouping 20 infants from a baby care center, 10 infants received 20 ml of Rota colostrum for 2 weeks and 10 control infants received none. Rotavirus-associated diarrhea developed in 7 of the 10 infants in the control group. None of the three infants in the group daily receiving the Rota colostrum had such symptoms, and one of three infants in the group receiving treatment, every other day developed rotavirus-induced diarrhea. Oral administration of Rota colostrum seems to be an effective and safe means of preventing diarrhea caused by human rotavirus infection. Recently, the immunized cows were boosted by reinjection of 4 serotypes of human rotavirus into a superficial cervical lymph node two weeks after delivery, resulting in mass production of cow's milk containing a high-titered antibody to human rotavirus. Therefore, the hyperimmune cow's milk is a candidate for a "physiologically functional food" in Japan.

Crohn's disease associated with renal amyloidosis successfully treated with an elemental diet.

We report a case of Crohn's disease associated with nephrotic syndrome due to renal amyloidosis in a 21-year-old man in whom remission of both Crohn's disease and the nephrotic syndrome has been maintained with an elemental diet. The patient developed toxic megacolon and nephrotic syndrome due to renal amyloidosis. Intensive intravenous prednisolone therapy with total parenteral nutrition was dramatically effective in treating the toxic megacolon and inducing remission in Crohn's disease and afterward, remission of the nephrotic syndrome. Remission of both conditions has been maintained for more than 2 years with the elemental diet. To our knowledge, this is the first confirmed case of Crohn's disease complicated with renal amyloidosis in which only slight proteinuria (below 0.3 g/day) was shown with an elemental diet used for a long period.

Computer simulation study on the conversion of spirolactone to enones over H-Y zeolite.

We report here the results of computer modeling studies and quantum chemical calculations on the spirolactone-to-enone conversion reaction over the zeolite catalysts, especially H-Y zeolite. We studied the adsorption mode of the molecules inside the supercage of H-Y and the mechanism of electron transfer between organic molecules and the framework of zeolite H-Y by density functional theory (DFT) calculations. Because the organic molecules considered in the present study are less symmetrical we docked the molecule inside the supercage of H-Y and energy minimization was then applied to these docked structures to yield representative low-energy binding sites for the molecules within the host structure. The interaction energy results show that the major interaction is between the methylene hydrogen of the molecule and the oxygen of the framework. The molecular electrostatic potential maps show that the ketonic oxygen of the reactant molecules abstract proton from Brönsted acid site. Thus the mechanism proposed by DFT calculation matches well with the experimental observations.

Tumor-specific cytocidal and immunopotentiating effects of relatively low molecular weight products derived from the basidiomycete, Agaricus blazei Murill.

Currently, some natural herbal extracts are believed to have a marked tumoricidal effect and low toxicity for normal tissues. We investigated the effect of relatively low molecular weight products extracted from the basidiomycete, Agaricus blazei Murill, on MethA tumor cell growth with the aim of producing synthetic derivatives based on these products. Inoculation of the low molecule fraction (LM) into the primary tumor of a two-tumor model resulted in the marked inhibition of the tumor, not only in the right flank, but also in the non-injected left flank. Chromatographic purification and physicochemical characterization showed the main tumoricidal activity to be located in a low molecule fraction-3 (LM-3), containing alpha-1,4-glucan-beta-1,6-glucan complex with an average molecular weight of 20 kDa. A11 LM fractions and crude ATF showed in vitro selective cytotoxicity for MethA tumor cells, having no effect on normal cells. Serum levels of immunosuppressive acidic protein (IAP) in mice receiving LM fractions, particularly LM-3, significantly increased, indicating the possible activation of granulocytes. We speculate that the inhibition of the distant tumor might be due to the increased migration of granulocytes, enhanced by the effect of extract injections at the primary tumor site.

Clinical effects of MND-19 (Inosiplex) on subacute sclerosing panencephalitis--a multi-institutional collaborative study--The Inosiplex-SSPE Research Committee.

A total of 151 cases of subacute sclerosing panencephalitis (SSPE), comprising 89 cases treated with MND-19 (Inosiplex) and 62 untreated cases, were retrospectively investigated as to background characteristics, survival rate and clinical course in order to compare the findings in the 2 groups of cases. The survival rate for the cases treated with MND-19 (MND-19-treated group) was significantly higher than that for the untreated cases (control group), which was also true on stratified analysis or on smoothing of the background factors by means of Cox's multiple regression model. Investigation of the clinical course revealed that progression through the disease stages was significantly slow in the MND-19-treated group, compared with in the control group. Global rating of the clinical course showed that a prolonged remission was obtained in more MND-19-treated cases than control cases. The measles virus antibody titer was in no way affected in the former group. Side effects of MND-19 were observed in 17 of the 89 treated cases (19.1%).

A case of adult moyamoya disease showing progressive angiopathy on cerebral angiography.

In moyamoya disease, progression of carotid occlusive lesion in an adult patient is very rare. We report a case of adult moyamoya disease with acute angiographical stage progression and hemodynamic deterioration. A 56-year-old female complaining of left motor weakness visited our department. On MRI, infarct lesion was found in the right white matter. On cerebral angiography, occlusive lesions in the bilateral internal carotid arterial siphons and moyamoya vessels were found. The right side was stage V and the left side was stage III. On IMP-SPECT, decreased cerebral hemodynamic reserve of the right cerebral hemisphere was found. In this patient, right STA-MCA anastomosis was performed. After operation, she became possible to walk and discharged to home. Five months after operation, good collateral formation and improvement of hemodynamic reserve in the right hemisphere were found. However, on left carotid arteriography, the anterior and middle cerebral arteries were only slightly imaged, and the disease state progressed to stage IV. In addition, decreased blood flow and hemodynamic reserve were appeared in the left hemisphere.

A case of bilioduodenal fistula treated with a self-expandable metallic stent.

We report the case of a 72 year-old female patient who suffered from biliary fistulae. The biliobiliary and bilioduodenal fistulae appeared after an operation for biliary bleeding. Conventional therapy for biliary fistula would be the disconnection of the fistula by either conservative or operative treatment. In the present case, however, it was preferable to enlarge the fistula to drain bile juice into the duodenum, rather than to close the fistula because it would have been difficult to achieve a tight adhesion with this operation. The enlargement by a plastic tube stent failed to drain the bile juice into the duodenum, because the sludge made the tube stenotic. Therefore, a self-expandable metallic stent was applied in this case. An expandable stent was used because a large final caliber is necessary to prevent stenosis of the fistula by sludge and mucosal hyperplasia. After insertion of a self-expandable metallic stent by the percutaneous transhepatic biliary drainage route, the patient has not suffered from cholestasis and cholangitis for the last 30 months. It can therefore be concluded that enlargement of the fistula by a self-expandable metallic stent is a convenient therapy for such biliointestinal fistulae.

[Spontaneous intracranial hypotension complicating subdural hematoma: unilateral oculomotor nerve palsy caused by epidural blood patch].

We report a case of a 41-year-old man with a 1-month history of postural headache due to spontaneous intracranial hypotension (SIH). His MRI revealed bilateral chronic subdural hematoma (CSH) and diffuse dural enhancement after gadolinium infusion. Indium-111 radionuclide cisternography revealed a CSF leak from the cervico-thoracic junction and rapid accumulation of radioisotope in the bladder. Postural headache failed to resolve with prolonged bed rest. The patient became restless and suffered recent memory disturbance. We therefore decided to treat the CSF leak with an epidural blood patch. After the procedure, the patient's headache resolved completely. However one day later, left oculomotor nerve palsy developed. MRI revealed enlargement of the left CSH with mass effect and midline shift. After hematoma drainage, the patient became alert and oculomotor palsy recovered gradually. To treat cases of CSH with SIH, the best method is to repair the CSF leakage and treat subdural hematoma at the same time. If the patient shows depressed consciousness, we recommend initial drainage of the subdural hematoma, because, following the repair of CSF leakage, mass effect such as uncal herniation may occur.

[Efficacy of antibiotic prophylaxis in clean neurosurgical operations: a comparison of seven-day versus one-day administration].

We compared seven-day and one-day administration of antibiotics as prophylaxis in clean neurosurgical operations. A total of 175 patients were examined. Seven-day administration (protocol A) was studied retrospectively from May 1997 to May 1998 in 87 patients of 46 craniotomies and 41 burr hole operations. One-day administration (protocol B) was studied prospectively from June 1998 to July 1999 in 88 patients of 39 craniotomies and 49 burr hole operations. Protocol A received 1.5 g of sulbactam/ampicillin = 1:2 (SBT/ABPC) preoperatively, and further doses were continued at 12-hour intervals for seven days after the operation. Protocol B received 1.5 g of SBT/ABPC preoperatively and intraoperative additional antibiotics were administered at 5-hour intervals, and only one single dose was administered after the operation. In both of the protocols, 60 mg of tobramycin was mixed with 500 ml of saline for irrigation of operative fields. No postoperative infection occurred in either of the two protocols. We conclude that one-day administration of prophylactic antibiotics is enough to prevent postoperative infections in clean neurosurgical operations.