A novel Fe(II)/α-ketoglutarate-dependent dioxygenase from Burkholderia ambifaria has ß-hydroxylating activity of N-succinyl l-leucine.

An Fe(II)/α-ketoglutarate-dependent dioxygenase, SadA, was obtained from Burkholderia ambifaria AMMD and heterologously expressed in Escherichia coli. Purified recombinant SadA had catalytic activity towards several N-substituted l-amino acids, which was especially strong with N-succinyl l-leucine. With the NMR and LC-MS analysis, SadA converted N-succinyl l-leucine into N-succinyl l-threo-ß-hydroxyleucine with >99% diastereoselectivity. SadA is the first enzyme catalysing ß-hydroxylation of aliphatic amino acid-related substances and a potent biocatalyst for the preparation of optically active ß-hydroxy amino acids.

Quantum non-demolition readout of an electron spin in silicon.

While single-shot detection of silicon spin qubits is now a laboratory routine, the need for quantum error correction in a large-scale quantum computing device demands a quantum non-demolition (QND) implementation. Unlike conventional counterparts, the QND spin readout imposes minimal disturbance to the probed spin polarization and can therefore be repeated to extinguish measurement errors. Here, we show that an electron spin qubit in silicon can be measured in a highly non-demolition manner by probing another electron spin in a neighboring dot Ising-coupled to the qubit spin. The high non-demolition fidelity (99% on average) enables over 20 readout repetitions of a single spin state, yielding an overall average measurement fidelity of up to 95% within 1.2 ms. We further demonstrate that our repetitive QND readout protocol can realize heralded high-fidelity (>99.6%) ground-state preparation. Our QND-based measurement and preparation, mediated by a second qubit of the same kind, will allow for a wide class of quantum information protocols with electron spins in silicon without compromising the architectural homogeneity.

Activation of EVI1 transcripts with chromosomal translocation joining the TCRVbeta locus and the EVI1 gene in human acute undifferentiated leukemia cell line (Kasumi-3) with a complex translocation of der(3)t(3;7;8).

A cell line (Kasumi-3) established from acute myeloid leukemia (AML-M0) had unique phenotypes of undifferentiated leukemia cells with expression of both T cell and myeloid antigens. Kasumi-3 cells with t(3;7)(q26;q22) highly expressed a 6 kb transcript of EVI1, which is located on chromosome 3q26. Therefore, we further characterized the chromosomal breakpoint by pulsed-field gel electrophoresis near EVI1. We identified and isolated the chromosomal breakpoint at approximately 80 kb upstream from the 5' end of EVI1. Sequence analysis of the breakpoint revealed that the whole Vbeta region from T cell receptor beta (TCRbeta) at 7q35 was translocated to the upstream of EVI1. A 1.0 kb TCRbeta transcript was expressed in the Kasumi-3 cells, suggesting that TCRbeta rearrangement occurred as Dbeta-Jbeta joining events. Fluorescence in situ hybridization analysis revealed that the inverted chromosome 7q22-q35 segment between TCRbeta and the region proximal to the erythropoietin gene at 7q22 was translocated to the region distal to EVI1 in der(3). Since the telomeric region of chromosome 8 q was also translocated to the inverted chromosome 7q22-q35 segment in der(3), the chromosomal abnormalities of der(3) were defined as being der(3)t(3;7;8)(3pter-3q26::7q35-7q22::8q22 -8qter). It is suggested that a translocated enhancer element in the TCRbeta locus and/or loss of a negative regulatory element near EVI1 might function to enhance the EVI1 expression. Therefore, the enhanced EVI1 expression may contribute to the development of a subset of undifferentiated leukemia.

A repressor element in the 5'-untranslated region of human Pax5 exon 1A.

B-cell-specific activator protein (BSAP) encoded by the Pax5 gene plays a critical role during B-cell development. We have analyzed the 5'-flanking region plus the 5'-untranslated region (5'-UTR) of human Pax5 exon1A to clarify its regulatory mechanisms. Functional dissection of these regions by luciferase reporter assays indicated that a cluster of regulatory elements acts as a strong repressor between +320 and +453. Insertion of this segment between the heterologous simian virus 40 (SV40) promoter and the luciferase gene in both the sense and reverse orientation sharply reduced the luciferase activity, but insertion into the upstream of the SV40 promoter did not. This suggests that this segment must be located in the 5'-UTR to function effectively. A search through databases with the sequence of this segment did not reveal any known DNA binding factor site. Electrophoretic gel mobility shift assay (EMSA) experiments demonstrated that unknown factors bound to the fragment +408 to +429. Insertion of this fragment between the SV40 promoter and the reporter gene strongly suppressed the luciferase activity. Competitive EMSA indicated that the region between nucleotides +413 and +427 encompassed the binding site of the unknown factors and was hence regarded as a repressor element. Mutagenesis in this element significantly recovered reporter gene activity. These results suggest that the segment +320 to +453, especially the repressor element +413 to +427, in the 5'-UTR is involved in the regulation of Pax5 gene expression.

Long-term evaluation of reconstruction of the sellar floor with a silicone plate in transsphenoidal surgery.

OBJECT: The authors have used a silicone plate for reconstruction of the sellar floor during rhinoseptoplastic transsphenoidal surgery because it has greater elasticity and is easier to carve than nasal septal cartilage and sphenoid sinus bone. This study was designed to evaluate the usefulness of this technique based on the authors' experience during the past 7.6 years. METHODS: A silicone plate was used to reconstruct the sellar floor in 69 consecutive patients with sellar tumors that included 60 pituitary adenomas and nine Rathke's cleft cysts. The patients ranged in age from 16 to 82 years (mean 52 years). The postoperative position of the silicone plate could be clearly identified on sagittal or coronal magnetic resonance (MR) imaging as a very low intensity plate (void signal). No displacement or migration of the implanted silicone plate was observed on follow-up MR imaging in any patient. Infections of the lesion such as a pituitary abscess were not observed clinically or radiologically in any patient. Of the 16 patients with intraoperative cerebrospinal fluid (CSF) leakage, only one patient who had a ghost sella developed postoperative CSF rhinorrhea. In all seven patients who underwent repeated surgery for residual or recurrent tumor, the silicone plate that had been placed at the initial procedure was covered with a relatively thin fibrous capsule and the plate was well preserved. The silicone plate was easily removed at reoperation and was useful for detection of the sellar floor window made previously. CONCLUSIONS: These results indicate that a silicone plate can be useful for reconstruction of the sellar floor in rhinoseptoplastic transsphenoidal surgery.

Hyaluronic acid facilitates glioma cell invasion in vitro.

We investigated the effects of hyaluronic acid (HA) on the invasiveness of three human glioma cell lines (A172, T98G and U251) and their secretion of matrix metalloproteinases (MMPs), plasminogen activators (PAs), and hyaluronidase. The invasion of all three glioma cell lines was enhanced by the impregnation of Matrigel with HA in an in vitro invasion assay using 12 microns porosity polycarbonate filter transwells. The secretion of MMPs and PAs was not influenced by the presence of HA. Hyaluronidase activity was not detected in the culture media of any glioma cell line. HA also enhanced the motility of A172 and U251 glioma cells, but did not influence the motility of T98G glioma cells. The adhesion and spreading of all glioma cell lines were inhibited on HA-coated plates. HA, however, did not influence the proliferation of any of the glioma cell line. These results suggest that the presence of HA contributes to glioma cell invasion, which involves the stimulation of detachment and motility of glioma cells and the maintenance of proteinase secretion by glioma cells.

Captopril inhibits glioma cell invasion in vitro: involvement of matrix metalloproteinases.

We investigated the effects of captopril on the activity of matrix metalloproteinases (MMPs) secreted by T98G glioma cells and their invasiveness in vitro. On gelatin zymography, captopril inhibited gelatinolytic activities in the culture media of T98G cells. This inhibitory effect was reversed by the presence of excess zinc. In an in vitro invasion assay, invasion into the reconstituted basement membrane (Matrigel) by T98G glioma cells was inhibited by captopril in a dose-dependent manner. This inhibitory effect was also reversed by the addition of zinc to the culture media. However, at the effective concentration of captopril for the prevention of tumor cell invasion it did not inhibit the motility, adhesion to Matrigel or proliferation of T98G cells. These findings suggest that captopril inhibits the invasiveness of T98G glioma cells due to its MMP inhibitory activity, chelating zinc ions at the active center of MMPs.

Production of amyloid beta protein precursor as a proteinase inhibitor by human astrocytic tumors.

The production of amyloid beta protein precursor (APP), which is a potent inhibitor of matrix metalloproteinases and serine proteinases, in human astrocytic tumors (n = 17) and normal brain tissues (n = 3) was investigated. We found proteinase inhibitory activity at around 120 kD by trypsin reverse zymography in the culture media of explant cultures of anaplastic astrocytomas and glioblastomas, but not in those of astrocytomas and normal brain tissues. Immunohistochemistry using a monoclonal antibody against human APP demonstrated that APP was detectable mainly in tumor and endothelial cells. Semiquantative analysis of western blotting revealed that immunoreactivity for APP in the culture media of tumor explant cultures appeared to be increased associated with the malignancy of astrocytic tumors. These findings suggest that APP production may be related to the malignant progression of human astrocytic tumors.

MR imaging and CT of pituitary abscess: case report and review.

Pituitary abscess is a rare disorder. However, preoperative diagnosis is important to prevent a cranial approach leading to severe meningitis. A case of a 55 year-old woman with pituitary abcess is reported. The patient was admitted with a several-week history of frontal headache and no signs of inflammation. Computed tomographic (CT) scan showed a slightly low-density suprasellar expanding mass lesion with an enhanced thin wall in the pituitary region. Magnetic resonance imaging showed a homogenous high-intensity signal relative to brain parenchyma on T1-weighted images with an enhanced thin wall and a homogenous low-intensity signal on T2-weighted images. This was histologically shown to be a pituitary abscess. Our case and review of the available literature suggest that pituitary abscess generally shows a homogenous low-density on CT scan and a homogenous low- to iso- and high-intensity signals or homogenous high- and low-intensity signals on T1- and T2-weighted images, respectively, with a cystic appearance and enhanced smooth wall.

Experimental and clinical study of detection of glioma at surgery using fluorescent imaging by a surgical microscope after fluorescein administration.

Total resection is the optimal treatment for malignant gliomas. However, an unexpected residual tumor mass is sometimes found on magnetic resonance imaging performed after an operation because of a macroscopically unclear margin of the tumor at surgery. This study was designed to evaluate the effectiveness of fluorescent imaging by a surgical microscope after fluorescein administration for the detection of gliomas at surgery. For this study, we produced two filters for the excitation and emission of fluorescein that can be easily fitted to and removed from a surgical microscope manually during the operation. For the experimental study, Wistar rat brains bearing C6 glioma were removed at appropriate intervals after intravenous administration of 10-20 mg kg-1 body weight of sodium fluorescein, and their surface and coronal section through the tumor were observed using a surgical microscope with the filters. In clinical cases, 1000 mg of sodium fluorescein was intravenously administered to five patients with glioma before tumor resection. In the experimental study, the C6 glioma itself and the edematous brain adjacent to the tumor (within 2-3 mm of the gross surface of the tumor) were well stained a brilliant yellowish green for a few hours. The normal brain was not stained. In clinical cases, the tumors were stained a brilliant yellowish green under fluorescent observation at surgery. The patients had no side effects. At all times the fluorescent observation could be quickly changed to ordinary observation by removing the filters from the surgical microscope. The tumor was also stained a faint yellow under ordinary nonfluorescent observation. Although this contributed to detection of the tumor, the fluorescent staining demarcated the tumor more clearly than nonfluorescent staining. These results suggest that this imaging technique by a surgical microscope with special filters at surgery may be practical and useful for detection of gliomas and warrants further clinical evaluations.

Analysis of the proliferative potential of tumor cells after stereotactic radiosurgery for recurrent astrocytic tumors.

We analyzed the effectiveness of stereotactic radiosurgery (SRS) for recurrent astrocytic tumors histologically. Five patients were followed by pathological examination after radiosurgery treatment of recurrent astrocytic tumors. Histological diagnoses at the time of the last operation before SRS were Daumas-Duport grade II in two patients and grade IV (glioblastoma) in three patients. No histological diagnoses at the time of SRS were identified in any patients. Contrast enhanced lesions enlarged gradually on magnetic resonance (MR) images after SRS, and local control by SRS was judged as progressive disease radiologically in all patients. Four of five patients received re-operation after SRS, and the other patient died without re-operation and underwent post-mortem examination. After SRS, Ki-67 labeling indices (LIs) of recurrent astrocytomas initially diagnosed as grade II were 2.6% and 1.1%. These LIs were relatively lower than those of the control group of patients with recurrent grade II astrocytomas that were not treated by SRS. Ki-67 LIs of three glioblastomas after SRS were 23.5%, 18.6%, and 17.8%. These LIs were significantly lower than those before SRS (2.3%, 4.5%, and 0.9%). In the autopsy case, there was a significant difference between the LI of tumor cells in the radiosurgically treated region (0.9%) and that in the untreated region (29.2%). These results suggest that the proliferative potential of malignant astrocytic tumors in the radiosurgically treated area is reduced after SRS, and that radiological enlargement of enhanced lesions on MR images is due to propagation of the residual tumor cells that were not covered by radiosurgical target volume or to radiation necrosis. SRS may be a useful therapeutic tool in multidisciplinary treatment of malignant gliomas.

Nutrient removal and starch production through cultivation of Wolffia arrhiza.

Wolffia arrhiza, a small weed found mostly in tropical and subtropical water environments, exhibits a high growth rate and consequently absorbs large amounts of nitrogen and phosphorus. Its vegetative frond contains 40% protein on a dry weight basis and its turion, which is the dormant form, has a similar starch content. The applicability of this weed to nutrient removal from secondary-treated waste water combined with starch resource production was evaluated. The nitrogen and phosphorus removal capabilities of the vegetative frond and the optimal conditions for inducing of the formation of turions from harvested biomass of vegetative fronds for the production of starch were investigated using artificial nutrient solutions. The vegetative frond showed high contents of nitrogen (6-7% of the total dry weight) and phosphorus (1-2% of the total dry weight). The nutrient removal rates of the vegetative frond were estimated to be 126 mg-N/m(2)/d and 38 mg-P/m(2)/d under a continuous flow condition. For turion formation from the vegetative fronds, a low nutrient concentration and a high plant density were most effective. Under the optimum conditions, the starch production rate was estimated to be 6 g-starch/m(2) (nutrient removal tank)/d.

Procedural antecedents of behavioral contrast: a re-examination of errorless learning.

Behavioral contrast reliably occurred in pigeons following errorless discrimination training, contrary to Terrace's (1963) observations. In the main experiment, a 60-sec green keylight, associated with a variable-interval 30-sec schedule of reinforcement alternated with a 60-sec period of extinction when the key was dark. Such aspects of the discrimination training procedure as: (1) the amount of prior nondifferential exposure to the positive stimulus before the discrimination was instituted, and (2) the rapidity with which the negative stimulus was introduced (whether progressively or abruptly) directly influenced the amount of behavioral contrast produced. This occurred independently of the number of errors made by a pigeon during acquisition of the discrimination. In a series of control experiments, substitution of a red keylight for the dark key during extinction resulted in greater behavioral contrast, while an increase to 3 min in the duration of the green keylight associated with reinforcement attenuated the behavioral contrast effect.

Ruptured dissecting aneurysms of both anterior cerebral arteries.

A case of bilateral dissecting aneurysms of the anterior cerebral arteries presenting with subarachnoid haemorrhage is reported. Dissection involved the proximal A2 portions of both anterior cerebral arteries. Dissection of the left anterior cerebral artery was responsible for the subarachnoid haemorrhage. Both aneurysms were treated by coating with cotton and fibrin glue.

Magnetic resonance angiography of experimental cerebral vasospasm.

The purpose of this study was to observe spastic cerebral arteries by magnetic resonance angiography (MRA) and to establish acetazolamide reactivity of these vessels. After control studies using MRA and conventional angiography, subarachnoid haemorrhage (SAH) was induced on day 0 in 7 Japanese monkeys. MRA and conventional angiography were then repeated on day 7 to observe the development of cerebral vasospasm. Reactivity of cerebral vessels to acetazolamide was also studied in both control animals (angiography before SAH) and on day 7 after SAH. Cerebral vasospasm was detected by both conventional angiography and MRA on day 7. The arteries on the side of the clot were more spastic than those on the control side. MRA was superior to conventional angiography in demonstrating dilatation of both control arteries (before SAH induction) and vasospastic arteries (on day 7 after SAH) after administration of acetazolamide.

Production of tissue inhibitor of metalloproteinases-1 (TIMP-1) by human astrocytic tumors.

Production of tissue inhibitor of metalloproteinases-1 (TIMP-1), a specific inhibitor of matrix metalloproteinases, was investigated in human astrocytic tumors (n = 15) and normal brain tissues (n = 3). Enzyme immunoassay indicated that TIMP-1 levels in the culture media of tumor explants were significantly higher in glioblastomas as compared with anaplastic astrocytomas, astrocytomas, and normal brain tissues. Immunohistochemistry using a monoclonal antibody against TIMP-1 demonstrated that TIMP-1 was detectable mainly in tumor cells, especially in glioblastoma. These results suggest that increased expression of TIMP-1 is associated with the malignant progression of astrocytic tumors.

Self-recognition ability in mentally retarded adolescents.

Self-recognition abilities of 30 mentally retarded adolescents were measured using an optical system and a psychophysical scaling procedure that results in a recognition threshold. Compared with college freshmen, the experimental group had significantly higher thresholds (p = .00003). Males were better than females at recognizing full-face self-images (p = .0238). Results were discussed in terms of the retarded adolescents' greater dependency on specific visual cues. The method is discussed as an advance over previous self-recognition methods because of accuracy and the capability of measuring a range of self-recognition abilities.

[Successful treatment with administration of systemic high dose methotrexate and intraarterial carboplatin for osteosarcoma of the skull: a case report].

An 11-year-old boy was admitted with a painless lump in the right frontal area as his chief complaint. On admission, physical and neurological examinations were normal except for the localized lump constituting the frontal lesion. Skull roentgenogram showed an osteoblastic and osteolytic mass with radiating bony spicules "sunray appearance" around the coronal suture. CT scans demonstrated it more clearly. The initial surgery was performed in July 1993. Pathological specimen showed osteosarcoma of the skull. The patient was transferred to our hospital for chemotherapy. The regimen consisted of intravenous high dose methotrexate 9,000 mg given for 6 hours with leucovorin rescue in a single course. Following 4 courses of high dose methotrexate, he underwent residual tumor resection. Two courses of intraarterial carboplatin 150 mg and methotrexate 200 mg were also given. Histologically, the lesion showed complete remission of the tumor, and no viable tumor cells were seen. Two additional courses of high dose methotrexate were given. The patient is doing well without evidence of recurrence. This case indicates that the chemotherapy with systemic high dose methotrexate and intraarterial carboplatin and methotrexate is effective for skull osteosarcoma.

[Antitumor effect of MX2, a new morpholino anthracycline against C6 glioma cells and its combination effect with photodynamic therapy in vitro].

MX2, a new lipophilic morpholino anthracycline, has been reported to have superior chemotherapeutic effects to adriamycin against murine and human tumor cells. In this study the chemotherapeutic effect of MX2 against C6 glioma cells was examined as well as the photocytotoxicity of MX2 and the combination effect of MX2 and photodynamic therapy (PDT) in vitro. Colony formation is inhibited even with only 2 hour treatment with MX2 in a dose-dependent manner. In this colony forming efficiency assay the drug concentration required for 50% inhibition of colony formation for C6 glioma cells was 24.0 +/- 4.5 ng/ml. Mild photocytotoxicity of MX2 against C6 glioma cells was observed at a high concentration (100 ng/ml) of MX2 following exposure to white light but not red light. In combination, MX2 and the photosensitizer haematoporphyrin derivative (HpD) exhibited an additive cytotoxic effect against C6 glioma cells when the cells were treated with MX2 either immediately after red light illumination following incubation with HpD or at an interval of 24 hours before incubation with HpD. We conclude that MX2 may be clinically useful against malignant glioma alone, and in combination with other therapies such as PDT.