Recycle of Immobilized Endocellulases in Different Conditions for Cellulose Hydrolysis.

The immobilization of cellulases could be an economical alternative for cost reduction of enzyme application. The derivatives obtained in the immobilization derivatives were evaluated in recycles of paper filter hydrolysis. The immobilization process showed that the enzyme recycles were influenced by the shape (drop or sheet) and type of the mixture. The enzyme was recycled 28 times for sheets E' and 13 times for drops B'. The derivative E' showed the highest stability in the recycle obtaining 0.05 FPU/g, RA of 10%, and FPU Yield of 1.64 times, higher than FPU spent or Net FPU Yield of 5.3 times, saving more active enzymes. The derivative B showed stability in recycles reaching 0.15 FPU/g of derivative, yield of Recovered Activity (RA) of 25%, and FPU Yield of 1.57 times, higher than FPU spent on immobilization or Net PFU Yield of 2.81 times. The latex increased stability and resistance of the drops but did not improve the FPU/gram of derivative.

A model of plasma current through a hole of Rogowski probe including sheath effects.

In TST-2 Ohmic discharges, local current is measured using a Rogowski probe by changing the angle between the local magnetic field and the direction of the hole of the Rogowski probe. The angular dependence shows a peak when the direction of the hole is almost parallel to the local magnetic field. The obtained width of the peak was broader than that of the theoretical curve expected from the probe geometry. In order to explain this disagreement, we consider the effect of sheath in the vicinity of the Rogowski probe. A sheath model was constructed and electron orbits were numerically calculated. From the calculation, it was found that the electron orbit is affected by E × B drift due to the sheath electric field. Such orbit causes the broadening of the peak in the angular dependence and the dependence agrees with the experimental results. The dependence of the broadening on various plasma parameters was studied numerically and explained qualitatively by a simplified analytical model.

Local current density measurement using a Rogowski probe in Tokyo Spherical Tokamak-2.

A Rogowski probe consisting of a small multi-layer Rogowski coil, five magnetic pick-up coils, and a Langmuir probe was developed to measure the local current density and its direction. It can be moved along the major radius and can be turned around its axis. This probe was used to measure the current density profile near the last closed flux surface of Ohmic plasmas in Tokyo Spherical Tokamak-2. The current density profile was measured successfully with a signal to noise ratio of greater than 20.

Demonstration of improvement in the signal-to-noise ratio of Thomson scattering signal obtained by using a multi-pass optical cavity on the Tokyo Spherical Tokamak-2.

The multi-pass Thomson scattering (TS) scheme enables obtaining many photons by accumulating multiple TS signals. The signal-to-noise ratio (SNR) depends on the accumulation number. In this study, we performed multi-pass TS measurements for ohmically heated plasmas, and the relationship between SNR and the accumulation number was investigated. As a result, improvement of SNR in this experiment indicated similar tendency to that calculated for the background noise dominant situation.

Note: Multi-pass Thomson scattering measurement on the TST-2 spherical tokamak.

In multi-pass Thomson scattering (TS) scheme, a laser pulse makes multiple round trips through the plasma, and the effective laser energy is enhanced, and we can increase the signal-to-noise ratio as a result. We have developed a coaxial optical cavity in which a laser pulse is confined, and we performed TS measurements using the coaxial cavity in tokamak plasmas for the first time. In the optical cavity, the laser energy attenuation was approximately 30% in each round trip, and we achieved a photon number gain of about 3 compared with that obtained in the first round trip. In addition, the temperature measurement accuracy was improved by accumulating the first three round trip waveforms.

Dimerization of N-methyltransferases involved in caffeine biosynthesis.

Caffeine is synthesized from the precursor xanthosine through three methylation and one nucleoside removal steps. Methylation is catalyzed by N-methyltransferases, designated as CaXMT1, CaMXMT1 and CaDXMT1, which, respectively, convert xanthosine into 7-methylxanthosine, 7-methylxanthine into 3,7-dimethylxanthine, and 3,7-dimethylxanthine into 1,3,7-trimethylxanthine (caffeine). In the present study, we examined their cytological and biochemical properties using fusion proteins with fluorescent proteins. All three enzymes were found to localize in cytosol as visualized by green fluorescence protein fusions. The possibility of dimer formation among these enzyme proteins was examined in vivo by transient expression of bimolecular fluorescence complementation of yellow fluorescent protein (YFP) using onion epidermal cell layers. Results showed that each enzyme protein formed a homo-dimer in cytosol as seen by a clear reconstituted YFP fluorescence. In addition, each enzyme also formed a hetero-dimer with each of the other two enzymes in cytosol. The biological significance of dimerization among structurally resembling methyltransferases involved in caffeine biosynthesis is discussed.

Aberrant expression of beta- and gamma-catenin is an independent prognostic marker in oral squamous cell carcinoma.

Alteration in expression of E-cadherin and catenins is associated with loss of differentiation, acquisition of an invasive phenotype and poor clinical outcome in many types of cancer. To identify molecular prognostic markers, membrane expression levels of E-cadherin, and alpha-, beta- and gamma-catenin in biopsy samples (n=135) of oral squamous cell carcinoma (OSCC) were evaluated immunohistochemically in relation to preoperative tumour-related features, clinical course and prognostic value, and were found to be significantly correlated with an endophytic growth pattern and pathologically proved lymph-node metastasis. Alteration of expression of E-cadherin, and alpha-, beta- and gamma-catenin was also significantly correlated with poor disease-specific 5-year survival (P=0.0096, 0.0434, 0.0005 and 0.0005, respectively). Multivariate Cox proportional hazard regression analysis showed that alteration of beta- and gamma-catenin expression was a significantly independent prognostic parameter for survival (P=0.0112 and 0.0088, respectively), as was the case with endophytic growth pattern and advanced N-category. These results indicate that patients with OSCC and absent or reduced membrane expression of beta- and gamma-catenin should be considered a high-risk group for regional lymph-node metastasis and poor prognosis.

[Persistent primitive olfactory artery: report of 5 cases].

Five cases of a persistent primitive olfactory artery are reported. The anomalous artery coursed anteromedially along the ipsilateral olfactory nerve and made a hairpin turn, supplying the circulation of the anterior cerebral artery. It was proposed that a primitive embryonic olfactory artery had remained as this artery. A cerebral aneurysm was located at the hairpin curve of the artery. Hemodynamic stress is considered to have an important role in aneurysm formation. Any aneurysm occurring at an artery should be considered for surgical treatment.

Inhibition of matrix metalloproteinases prevents allergen-induced airway inflammation in a murine model of asthma.

Although matrix metalloproteinases (MMPs) have been reported to play crucial roles in the migration of inflammatory cells through basement membrane components in vitro, the role of MMPs in the in vivo accumulation of the cells to the site of inflammation in bronchial asthma is still obscure. In this study, we investigated the role of MMPs in the pathogenesis of bronchial asthma, using a murine model of allergic asthma. In this model, we observed the increase of the release of MMP-2 and MMP-9 in bronchoalveolar lavage fluids after Ag inhalation in the mice sensitized with OVA, which was accompanied by the infiltration of lymphocytes and eosinophils. Administration of tissue inhibitor of metalloproteinase-2 to airways inhibited the Ag-induced infiltration of lymphocytes and eosinophils to airway wall and lumen, reduced Ag-induced airway hyperresponsiveness, and increased the numbers of eosinophils and lymphocytes in peripheral blood. The inhibition of cellular infiltration to airway lumen was observed also with tissue inhibitor of metalloproteinase-1 and a synthetic matrix metalloproteinase inhibitor. These data suggest that MMPs, especially MMP-2 and MMP-9, are crucial for the infiltration of inflammatory cells and the induction of airway hyperresponsiveness, which are pathophysiologic features of bronchial asthma, and further raise the possibility of the inhibition of MMPs as a therapeutic strategy of bronchial asthma.

Cell cycle-associated accumulation of tissue inhibitor of metalloproteinases-1 (TIMP-1) in the nuclei of human gingival fibroblasts.

We first confirmed an earlier immunohistochemical study showing that immunoreactive TIMP-1-like protein accumulated in the nuclei of human gingival fibroblasts (Gin-1 cells), reaching a maximum in the S phase of the cell cycle (Li, H., Nishio, K., Yamashita, K., Hayakawa, T. and Hoshino, T. (1995). Nagoya J. Med. Sci. 58, 133-142). Then we isolated this protein from a nuclear extract of Gin-1 cells and demonstrated it to be identical to human recombinant TIMP-1 by western blotting, by a sandwich enzyme immunoassay for TIMP-1 and by an assay for matrix metalloproteinase inhibition. The amount of TIMP-1 in the cytosolic fraction of quiescent Gin-1 cells after stimulation by fetal calf serum increased continuously for 48 hours, whereas that in the nuclear extract showed a maximum at 24 hours (S phase) and significantly decreased thereafter. Gin-1 cells expressed mRNAs for both TIMP-2 and TIMP-3 together with mRNA for TIMP-1. However, neither TIMP-2 nor TIMP-3 proteins seemed to accumulate in the nuclei of Gin-1 cells. These facts strongly suggest that TIMP-1 accumulates specifically in the nuclei of Gin-1 cells in a cell cycle-dependent manner.

Interaction between tissue inhibitor of metalloproteinases-2 and progelatinase A: immunoreactivity analyses.

By immunoreactivity analysis using monoclonal antibodies, we showed that the C-terminal domain [R415-631; R is residue] of progelatinase A [pro-matrix metalloproteinase-2 (proMMP-2); EC 3.4.24.24] affected the immunoreactivity of a one-step sandwich enzyme immunoassay (sandwich EIA) for tissue inhibitor of metalloproteinases-2 (TIMP-2) in exactly the same way as does proMMP-2 [Fujimoto, Zhang, Iwata, Shinya, Okada and Hayakawa (1993) Clin. Chim. Acta 220, 31-45], confirming that the C-terminal domain ("tail" portion of TIMP-2 participates in the binding with the C-terminal domain of proMMP-2. We also demonstrated that not only the C-terminal domain but also the N-terminal domain (R1-417) of proMMP-2 bound to TIMP-2 in a 1:1 molar ratio. The binding of each individual domain to TIMP-2, however, was weak enough that either domain could be fully replaced by proMMP-2 through the same binding sites as does proMMP-2, and also that the high-order structure of proMMP-2 allows a more stable binding to TIMP-2. We further confirmed that TIMP-2 complexed with the N-terminal domain of pro-MMP-2 had fully inhibitory activity against the collagenolytic activity of MMP-1. We also demonstrated that either the interstitial collagenase-TIMP-2 complex or the gelatinase B(MMP-9)-TIMP-2 complex was able to form a ternary complex with proMMP-2 in a 1:1 molar ratio, clearly indicating that there are two distinct binding sites, one specific for proMMP-2 complex, but the binding seemed to be less stable than the binding with TIMP-2 alone. Even in the presence of a 10-fold molar excess of the N-terminal domain, ternary complex formation was not observed between the N-terminal domain and the MMP-9--TIMP-2 complex. These clear differences might be ascribed to some significant conformational change(s) evoked in the TIMP-2 molecule, or hindrance of a part of the N-terminal domain binding site of TIMP-2 by complex formation with MMP-9.

[Studies on distortion of the optic nerve by the internal carotid artery].

The optic nerve (ON) is sometimes distorted by an atherosclerotic internal carotid artery (IC) which we observe during operations. However the distortion seldom causes visual dysfunction. In this paper, 102 nonoperative cases without severe visual disturbance or marked effect on the visual system were studied, using coronal section of MRI and IC angiography, to ascertion ON distortion by IC. The grading of the distortion was determined by coronal section of MRI. Grade (Gr) 0: No distortion of ON, Gr 1: Distortion of ON without chiasmal dislocation, Gr 2: Distortion of ON with chiasmal dislocation. The rate of Gr 0, Gr 1, and Gr 2 were 62.7, 21.6 and 15.7%, respectively. The age of Gr 0, Gr 1 and Gr 2 were 55.4 +/- 19.6, 63.5 +/- 14.7 and 68.9 +/- 5.0 years old, respectively (Gr 2 was different from Gr 0). The occupying rate was calculated by the following formula. The areas were measured by IC angiography of the optic canal: The area of IC in optic canal/The area of optic anal x 100%. The rate of Gr 0, Gr 1 and Gr 2 were 24.2 +/- 19.8, 76.3 +/- 13.2 and 62.0 +/- 28.2%, respectively (Gr 1 and 2 differed from Gr 0). With a rate of more than 70%, the patient had ON distortion by IC.(ABSTRACT TRUNCATED AT 250 WORDS)

Mice lacking alpha 1 (IX) collagen develop noninflammatory degenerative joint disease.

Type IX collagen is a nonfibrillar collagen composed of three gene products, alpha 1(IX), alpha 2(IX), and alpha 3(IX). Type IX molecules are localized on the surface of type II-containing fibrils and consist of two arms, a long arm that is crosslinked to type II collagen and a short arm that projects into the perifibrillar space. In hyaline cartilage, the alpha 1(IX) collagen transcript encodes a polypeptide with a large N-terminal globular domain (NC4), whereas in many other tissues an alternative transcript encodes an alpha 1(IX) chain with a truncated NC4 domain. It has been proposed that type IX molecules are involved in the interaction of fibrils with each other or with other components of the extracellular matrix. To test this hypothesis, we have generated a mouse strain lacking both isoforms of the alpha 1(IX) chain. Homozygous mutant mice are viable and show no detectable abnormalities at birth but develop a severe degenerative joint disease resembling human osteoarthritis.

A one-step sandwich enzyme immunoassay for tissue inhibitor of metalloproteinases-2 using monoclonal antibodies.

A one-step sandwich enzyme immunoassay system was developed with a pair of monoclonal antibodies against two individual oligopeptides prepared from the amino acid sequence of the human tissue inhibitor of metalloproteinases-2 (TIMP-2). The assay system consisting of two simultaneous immunoreactions used a solid phase monoclonal antibody and a horse-radish peroxidase-labeled monoclonal antibody. The system detected a free form of TIMP-2 and that complexed with active forms of matrix metalloproteinases (MMPs) giving a different sensitivity for each MMP but not TIMP-2 complexed with the precursor of 72 kDa gelatinase/type IV collagenase (MMP-2). The sensitivity of the system was 1.6 microgram/l (16 pg/assay) and linearity was obtained between 6.3 and 50 micrograms/l (63-500 pg/assay). TIMP-2 levels in the sera of 20 patients with rheumatoid arthritis (68 +/- 25 micrograms/l, mean +/- S.D.) and 13 patients with hepatocellular carcinoma (76 +/- 46 micrograms/l) were significantly higher (P < 0.05) than those of 18 normal subjects (5.6 +/- 7.4 micrograms/l). In contrast, the levels in the sera of 10 patients with gastric cancer (45 +/- 18 micrograms/l) and 7 patients with cancer of the uterus (36 +/- 13 micrograms/l) were significantly lower (P < 0.05 or P < 0.01) than those of normal subjects. Immunoreactivity analyses suggested that the precursor of MMP-2 in normal sera exists in a complexed form with TIMP-2 by interacting with the C-terminal domain of TIMP-2.

[Clinical course of acute intracerebral hematoma which shows a fluid level presentation by X-ray CT].

Three cases of intracranial hematomas which showed a fluid level presentation and/or mixed density in the acute stage by X-ray CT were reported. Case 1 is a 7-year-old boy who had the epidural and intracerebral hematomas three hours after the traffic accident. During the course, intracerebral hematomas which showed a fluid-level presentation had grown both 6 and 17 hours after the episode. Case 2 is a 39-year-old female who had received an aortic valve replacement 20 years ago. She was diagnosed as cerebral infarction due to the occlusion of the right middle cerebral artery. Intracerebral hematomas with a fluid level presentation developed in the infarcted area 20 days after the embolectomy. Case 3 is a 67-year-old female who had a mild subarachnoid hemorrhage by the traffic accident. Intracerebral hematoma with mixed density developed 68 hours after the episode and it enlarged again 89 hours after the accident. All three cases showed the abnormalities in the coagulofibrinolytic activities during the hemorrhage and showed a growth of the hematomas. It should be noticed that the case which shows a mixed density and/or a fluid level presentation in the X-ray CT requires more intensive care in both neurosurgical and general management.

[Traumatic occlusion of the bilateral vertebral arteries associated with fracture of the cervical spine--a case report].

A case of traumatic occlusion of the bilateral vertebral arteries associated with fracture of the cervical spine is reported. A 34-year-old man, having no previously noted medical problems, fell to the bottom of a bathtub with a depth of 80 cm, and hit the vertex fronto-parietal region of his head. He was transferred to our hospital 6 hours after his fall with a crush fracture of the cervical spine at the C6 level. On admission he was alert, but having pain in the vertex region, dysarthria, blurred vision and hemiparesis. Roentgenograms confirmed a crush fracture of the C6 vertebral body. Computed tomograms of the brain revealed a high density of basilar artery. Cervical traction with a Halo brace was then carried out. Twelve hours after the trauma, left oculomotor and right facial palsy appeared followed by bilateral oculomotor palsy and respiratory difficulty. At the 14th hour, he displayed bilateral Babinski's signs and tetraparesis. Tetraparesis became complete with right-side Horner's syndrome at 16 hours. Cerebral arteriograms performed 20 hours after the trauma showed a complete occlusion of the right vertebral artery and an incomplete occlusion of the left vertebral artery at the C6-7 intervertebral disk space. Conscious level deteriorated to a 200 level on the Japan coma scale 28 hours after the trauma and to a 300 level after 32 hours. Computed tomograms revealed a marked low density on the cerebellum and brain stem 38 hours after the accident. He expired on the 22nd day after the trauma.(ABSTRACT TRUNCATED AT 250 WORDS)