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The surgical procedures for treating chronic subdural hematoma (CSH) are carried out with a variety of different methods. The generally used operative method is a less invasive burr hole irrigation under local anesthesia. Recently, we have devised a new simple method using an endoscope to treat CSH. We have been using an endoscopically guided method in which a drainage tube is inserted in the hematoma cavity. We also analyzed the endoscopic findings in the hematoma cavities and reported a high recurrence rate in patients with clots in the hematoma cavity, which suggest the importance of clot removal to reduce recurrence. In this study, the advantage of endoscopic surgery for chronic subdural hematoma is demonstrated.

The outcome of multiple injures freqently depends on the priority of treatments, and the decision as to the procedures and timing of primary care is extremely important. We studied the patients with multiple trauma whounderwent emergency endovascular treatment for facial hemorrhage related to external carotid arterial injury. The subjects are 5 patients who underwent embolization of the external carotid artery by an endovascular approach among patients with multiple traumas who were brought to our hospital by ambulance. In these patients, the vital signs on arrival, interval between injury and intravascular surgery, type of brain injury, type and grade of concurrent injury and outcome were studied. Three patients showed hemorrhagic shock on arrival, and 1 patient showed hemorrhagic shock immediately after arrival. The mean interval between injury and endovascular surgery was 3.9 hours. All patients had skull base fracture, and abnormal intracranial lesions on initial CT including 4 focal injuries and 1 diffuse injury. Moderate to severe thoracic/abdominal injuries were noted in 3 patients. In the remaining 2 patients, there was no trauma in the thoracic or abdominal regions. Intraperitoneal hemorrhage with splenic injury was observed in 3 patients. In 3 of 4 patients died by hemorrhagic shock because of the delay of endvascular treatments. In trauma patients with persistent hemorrhage, emergency endovascular treatment should be considered as a primary survey for initial treatment without delay under intensive conservative treatment.

Buckwheat often causes severe allergic reactions, even when its ingestion level is extremely low. Therefore, buckwheat is listed in several countries as a common food allergen. In addition to common buckwheat and Tartarian buckwheat that are cultivated and consumed widely, wild buckwheat may be potentially allergenic. Food containing undeclared buckwheat poses a risk to patients with the buckwheat allergy. We describe in this report a PCR method to detect buckwheat DNA by using primers corresponding to the internal transcribed spacer region and the 5.8S rRNA gene. The method is buckwheat-specific and compatible with both cultivated and wild buckwheat of the Fagopyrum spp. Its sensitivity was sufficient to detect 1 ppm (w/w) of buckwheat DNA spiked in wheat DNA. This method should benefit food manufacturers, clinical doctors, and allergic patients by providing information on the presence of buckwheat contamination in food.

In addition to xeroderma pigmentosum (XP), mutations in the human XPG gene cause an early onset of Cockayne syndrome (CS) in some patients (XP-G/CS) with characteristics, such as growth retardation and a short life span. In the previous studies, we generated four Xpg mutant mice with two different C-terminal truncations, null, or a base substitution mutation to identify the protein region that causes the onset of CS, and found that the CS-causing mutations, null or a deletion of the last 360 amino acids, completely inhibited the NER activity of mouse XPG (Xpg), but the non-CS-causing mutations, XpgD811A (base substitution that eliminates the nuclease activity of Xpg) or XpgDeltaex15 (deletion of the exon 15 corresponding to the last 183 amino acids), resulted in the retention of residual NER activity. To understand why mutations that completely eliminate the NER activity of Xpg cause CS but those that abolish the nuclease activity without totally eliminating the NER activity of Xpg do not result in CS, we made a series of Xpg mutant mice with Xpa-null mutant allele and found that mice with the non-CS-causing deletion mutation (XpgDeltaex15) exhibited the CS phenotype when XPA was also absent but the base substitution mutation (XpgD811A) that eliminated the Xpg nuclease activity did not. These results indicate that Xpg has a second function, beside NER, and that the disruption of this second function (deletion of the last 183 amino acids) when combined with an NER defect causes CS. When we compared amino acid sequences corresponding to the exon 15 of Xpg, a significant homology was conserved among vertebrates, but not in Drosophila and Saccharomyces cerevisiae. These observations suggest that the second function of XPG may be conserved only in vertebrates and CS symptoms may occur in its absence.

* Fructan:fructan 6G-fructosyltransferase (6G-FFT) catalyses a transfructosylation from fructooligosaccharides to C6 of the glucose residue of sucrose or fructooligosacchrides. In asparagus (Asparagus officinalis), 6G-FFT is important for the synthesis of inulin neoseries fructan. Here, we report the isolation and functional analysis of the gene encoding asparagus 6G-FFT. * A cDNA clone was isolated from asparagus cDNA library. Recombinant protein was produced by expression system of Pichia pastoris. To measure enzymatic activity, recombinant protein was incubated with sucrose, 1-kestose, 1-kestose and sucrose, or neokestose. The reaction products were detected by high performance anion-exchange chromatography. * The deduced amino acid sequence of isolated cDNA was similar to that of fructosyltransferases and vacuolar type invertases from plants. Recombinant protein mainly produced inulin neoseries fructan, such as 1F, 6G-di-beta-D-fructofuranosylsucrose and neokestose. * Recombinant protein demonstrates 6G-FFT activity, and slight fructan:fructan 1-fructosyltransferase (1-FFT) activity. The ratio of 6G-FFT activity to 1-FFT activity was calculated to be 13. The characteristics of the recombinant protein closely resemble those of the 6G-FFT from asparagus roots, except for a difference in accompanying 1-FFT activity.

Maternal smoking has been linked to an increased risk for orofacial clefts. N'-nitrosonornicotine (NNN) is one of the tobacco-specific nitrosamines that has been shown to be linked to the deleterious effects of tobacco and could be linked to the formation of cleft palate birth defects. The effect of NNN on palatal fusion was examined using an in vitro organ culture model of palatal development. The organ cultures were exposed to NNN (0.01, 0.1, 1, 10 and 100 mM) and the effects on palatal development characterized at defined points. Palatal fusion was evaluated at embryonic day 13 (E13)+72 h by characterizing the remaining medial edge epithelium (MEE) and determining the extent of fusion compared to controls. The NNN-treated group (1 mM) had more MEE remaining in the palatal midline than the untreated group at E13+72 h (P<0.05). Changes in cell proliferation in the MEE resulting from NNN exposure were examined by BrdU incorporation in replicating DNA. Changes in the pattern of MEE cell death were examined by TUNEL. BrdU incorporation and TUNEL staining showed that the NNN (1 mM)-treated palates had more MEE cell proliferation and less apoptosis than the untreated-palates at E13+24 h (P<0.05). The mechanism altered by NNN was further evaluated by characterizations of extracellular signal-regulated kinase (ERK) 1/2, p38 and c-jun amino-terminal kinase (JNK). NNN at 1 mM induced ERK1/2 phosphorylation, but reduced p38 phosphorylation (P<0.05, P<0.01, respectively) in the MEE. The results suggest that NNN inhibited palatal fusion by effects on cell proliferation and MEE cell death.

A fructosyltransferase that transfers the terminal (2 --> 1)-beta-linked D-fructosyl group of fructo-oligosaccharides (1(F)(1-beta-D-fructofuranosyl)(n) sucrose, n >/= 1) to HO-6 of the glucosyl residue and HO-1 of the fructosyl residue of similar saccharides (1(F)(1-beta-D-fructofuranosyl)(m) sucrose, m >/= 0) has been purified from an extract of the bulbs of onion (Allium cepa). Successive column chromatography using DEAE-Sepharose CL-6B, Toyopearl HW65, Toyopearl HW55, DEAE-Sepharose CL-6B (2nd time), Sephadex G-100, Concanavalin A Sepharose, and Toyopearl HW-65 (2nd time) were applied for protein purification. The general properties of the enzyme, were as follows: molecular masses of 66 kDa (gel filtration chromatography), and of 52 kDa and 25 kDa (SDS-PAGE); optimum pH of c. 5.68, stable at 20-40 degrees C for 15 min; stable in a range of pH 5.30-6.31 at 30 degrees C for 30 min, inhibited by Hg(2+), Ag(+), p-chloromercuribenzoic acid (p-CMB) and sodium dodecyl sulfate (SDS), activated by sodium deoxycholate, Triton X-100 and Tween-80. The amino acid sequence of the N-terminus moiety of the 52-kDa polypeptide was ADNEFPWTNDMLAWQRCGFHFRTVRNYMNDPSGPMYYKGWYHLFYQHNKDFAYXG and the amino acid sequence from the N-terminus of the 25-kDa polypeptide was ADVGYXCSTSGGAATRGTLGPFGLL VLANQDLTENTATYFYVSKGTDGALRTHFCQDET. The enzyme tentatively classified as fructan: fructan 6(G)-fructosyltransferase (6G-FFT). The enzyme is proposed to play an important role in the synthesis of inulin and inulinneo-series fructo-oligosaccharides in onion bulbs.

Transforming growth factor (TGF)-beta3 is an important contributor to the regulation of medial edge epithelium (MEE) disappearance during palatal fusion. SMAD2 phosphorylation in the MEE has been shown to be directly regulated by TGF-beta3. No phospho-SMAD2 was identified in the MEE in Tgf-beta3-null mutant mice (Tgf-beta3-/-), which was correlated with the persistence of the MEE and failure of palatal fusion. In the present study, the cleft palate phenotype in Tgf-beta3-/- mice was rescued by overexpression of a Smad2 transgene in Keratin 14-synthesizing MEE cells following mating Tgf-beta3 heterozygous mice with Keratin 14 promoter directed Smad2 transgenic mice (K14-Smad2). Success of the rescue could be attributed to the elevated phospho-SMAD2 level in the MEE, demonstrated by two indirect evidences. The rescued palatal fusion in Tgf-beta3-/-/K14-Smad2 mice, however, never proceeded to the junction of primary and secondary palates and the most posterior border of the soft palate, despite phospho-SMAD2 expression in these regions at the same level as in the middle portion of the secondary palate. The K14-Smad2 transgene was unable to restore all the functional outcomes of TGF-beta3. This may indicate an anterior-posterior patterning in the palatal shelves with respect to TGF-beta3 signaling and the mechanism of secondary palatal fusion.

Immunoelectron microscopy and a quantitative analysis of immunogold labeling of a glutamine synthetase (GS; EC 6.3.1.2) revealed that, in mesophyll cells of mature leaves of Azolla filiculoides, almost all GS was present in chloroplasts. By contrast, in hair cells, abundant labeling of GS was observed both in chloroplasts and in the cytoplasm. In hair cells of cyanobiont-free plants, the labeling of GS of both chloroplasts and cytoplasm was very weak compared to that of cyanobiont-containing plants. The findings suggest that hair cells play an important role in the assimilation of ammonia released by the cyanobiont.

The activity of soluble invertase, and the variation in glucose, fructose and sucrose contents in onion bulbs (Allium cepa) during long-term storage at 10 degrees C and 20 degrees C were investigated. Invertase activity increased progressively after 8 weeks to 0.084 and 0.092 nkat/g fresh weight (FW), then sharply to 0.29 and 0.35 nkat/g FW at 20 degrees C and 10 degrees C, respectively, and remained high during 5 weeks. Then, activity decreased abruptly to 0.039 and 0.041 nkat/g, and remained low during the last 8 weeks and close to that observed initially. Glucose increased to 17.73 and 14.62 mg/g FW after 4 weeks at 20 degrees C and 10 degrees C, respectively, then decreased sharply between week 5 and week 7 to 4.13 and 4.91 mg/g FW, respectively, and remained rather stable ranging from 9 and 10 mg/g FW at both temperatures. Fructose showed a similar pattern and was 14.8 and 21.68 mg/g FW at 20 degrees C and 10 degrees C, respectively. Between week 10 and week 24, fructose ranged from 5 and 6 mg/g FW, and from 6 and 7 mg/g FW at 20 degrees C and 10 degrees C, respectively. Sucrose increased to 19.63 and 14.43 mg/g FW at 20 degrees C and 10 degrees C, respectively, decreased during 3 weeks, and then increased randomly from 5.69 to 9.42 mg/g FW at 20 degrees C, but remained in a steady state at 10 degrees C ranging 5.03 +/- 0.78 mg/g FW. During the last 6 weeks, the sucrose content was higher at 20 degrees C than at 10 degrees C. The fructose-glucose ratio varied during the first 8 weeks but remained at a steady level during the last 16 weeks. The (glucose+fructose)/sucrose ratio increased randomly at 10 degrees C, whereas at 20 degrees C the ratio increased during 10 weeks then decreased progressively during the final 14 weeks.

We reviewed the records of 1,335 minor head injury patients with initial Glasgow Coma Scale (GCS) scores of 15 treated by our neurosurgery service between January 1998 and December 2000. Skull X-ray was performed in 945 patients (71%), and Computed tomography (CT) was performed in 590 patients (44%). Skull fracture was shown radiographically in 24 patients (2.5%), and abnormalities on the initial CT were seen in 29 patients (4.9%). The most frequent intracranial lesion on CT was acute epidural hematoma with skull fracture. Significantly more intracranial lesions were found in those with a fracture than in those without by chi2 analysis. Post-traumatic vomiting was significantly associated with radiographical abnormalities, but headache and nausea did not increase the risk of skull fracture and intracranial lesions on the CT. Patients required neurosurgical intervention in 4 cases, and all of those were acute epidural hematoma with skull fracture. In this study, the first thing we should do for asymptomatic minor head injury patients with a GCS score of 15 is to investigate the presence of a skull fracture by skull X-ray. Head trauma patients with a skull fracture and post-traumatic vomiting should undergo CT to facilitate detection of intracranial lesions, even when there are no abnormal neurological signs.

In addition to xeroderma pigmentosum (XP), mutations in the human XPG gene cause early onset of Cockayne syndrome (CS) in some patients (XPG/CS). The CS-causing mutations in such patients all produce truncated XPG proteins. To test the hypothesis that the CS phenotype, with characteristics such as growth retardation and a short life span in XPG/CS patients, results from C-terminal truncations, we constructed mutants with C-terminal truncations in mouse XPG (Xpg) (from residue D811 to the stop codon [XpgD811stop] and deletion of exon 15 [Xpg Delta ex15]). In the XpgD811stop and Xpg Delta ex15 mutations, the last 360 and 183 amino acids of the protein were deleted, respectively. To generate Xpg mutant mice, we devised the shortcut knock-in method by replacing genomic DNA with a mutated cDNA fragment (cDNA-mediated knock in). The control mice, in which one-half of Xpg genomic DNA fragment was replaced with a normal Xpg cDNA fragment, had a normal growth rate, a normal life span, normal sensitivity to UV light, and normal DNA repair ability, indicating that the Xpg gene partially replaced with the normal cDNA fragment retained normal functions. The XpgD811stop homozygous mice exhibited growth retardation and a short life span, but the Xpg Delta ex15 homozygous mice did not, indicating that deletion of the last 360 amino acids results in the CS phenotype but deletion of the last 183 amino acids does not. The XpgD811stop homozygous mice, however, exhibited a slightly milder CS phenotype than did the Xpg null mutant mice, indicating that the XpgD811stop protein still retains some Xpg function that affects the severity of the CS phenotype.

Hypertensive intracerebral hemorrhage (HIH) occurring simultaneously in different locations is rare. We encountered 11 such cases between January 1990 and November 2002. The diagnosis of all 11 cases was established by computed tomography, and the location of the hematomas was: cerebellum and basal ganglia in 5 cases, pons and basal ganglia in 4, and subcortex and basal ganglia in 2. Our patients were analyzed with respect to clinical characteristics, pathogenesis of multiple hematomas, and indication of operation. These patients represented 1% of all 1,069 patients we encountered with HIH. As past history, there were no characteristic disorders except hypertension. There were no characteristic initial symptoms suggesting that hemorrhage had occurred simultaneously. Both supra- and infra-tentorial hematomas were observed in 80% of the patients, and the size of the multiple hematomas was proportional in principle. Cerebellar hematomas were often mild, and pontine hematomas were often severe. The outcome in those patients whose neurological grading was 1 to 3 was good with conservative therapy or surgical treatment. The severity, treatment methods, and outcomes in these patients were similar to those in patients with single HIH, which suggests only a slight influence of multiple lesions on outcome. As for the possible mechanism of simultaneous multiple hemorrhages, we speculated that bleeding occurred simultaneously in the different regions, or that the initial bleeding was followed after a short time by secondary bleeding due to high intracranial pressure and circulatory disturbance. In patients with cerebellar hematoma, initial symptoms suggested the development of secondary hemorrhage after primary hemorrhage. The surgical treatment for multiple hematomas should be determined by the location and maximum axis of the hematoma. We proposed that cerebellar hematomas should be removed if the supra-tentorial hematoma is small.

Aspiration pneumonia is a common cause of death in older people, and the pathophysiology is a chronic respiratory failure with a mild airway inflammation. In this study, we established a mild inflammatory pneumonia model using Porphyromonas gingivalis (Pg) pathogen-infected mice. It elucidated the effects of Pg-infected pneumonia on proinflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin-6 (IL-6), and IL-1beta production in both lung tissue and serum. We also elucidated production of soluble (s) TNF receptor (R) s, because TNF-alpha is considered to be a dominant inflammatory mediator. Lung TNF-alpha levels significantly increased at 2 h after infection, and rapidly returned to basal level at 24 h. Consistent with increase of TNF-alpha, remarkable increase of sTNFR2 but not sTNFR1 was detected in lung tissue from 2 to 72 h. Interestingly, sTNFR2/sTNFR1 ratio was significantly enhanced at 2 h in serum. In addition, lung IL-1beta and IL-6 levels also significantly increased from 2 to 24 h. Importantly, we found that IL-6 levels in serum reflected its local level. These results may suggest that systemically produced sTNFR2 and IL-6 could be a key role to modulate proinflammatory activities of TNF-alpha in Pg-induced lung inflammation simulated aspiration pneumonia.

Previous studies suggested that the onset of phenytoin-induced gingival overgrowth depended on serum phenytoin concentration. Cytochrome P450 2C (CYP2C) plays an important role in phenytoin metabolism. Recently, single nucleotide polymorphisms in the coding region of CYP 2C influencing phenytoin metabolism were identified. The purpose of the present study was to see if CYP 2C polymorphisms might relate to the onset and severity of phenytoin-induced gingival overgrowth. Twenty-eight epileptic patients taking phenytoin aged 15 to 75 (mean age: 42.2 years old, 20 males and 8 females) and 56 unrelated healthy subjects aged 30 to 48 (mean age: 36.8 years old, 48 males and 8 females) were examined for CYP 2C polymorphisms. All epileptic subjects were examined for the degree of gingival overgrowth, daily phenytoin dose and serum phenytoin concentration. The results indicated about 7% of the subjects including epileptic and healthy subjects examined were positive for CYP 2C9*3. However, the degree of gingival overgrowth did not directly correlate with CYP 2C polymorphisms. Nevertheless, the subjects with severer gingival overgrowth exhibited significantly higher serum phenytoin concentration, indicating that phenytoin metabolism is an important determinant for the severity of the disease. Additionally, CYP 2C9*3 carriers exhibited significantly higher serum drug concentration to drug dose. Therefore, we concluded although the gene analysis is not directly related to diagnose the disease itself, it can be utilized in estimating serum phenytoin concentration from drug dose, which in turn serves to predict the future development and clinical course of the disease.

Soil microorganisms play an important role in maintaining soil pH at levels suitable for other soil organisms. To clarify the biological neutralization mechanism in soil, we isolated soil microorganisms showing a high ability to neutralize acids and studied their characteristics. From our taxonomic study, three isolated strains were identified as filamentous fungi, namely Mucor sp., Aspergillus fumigatus, and Aureobasidium pullulans. These strains could secrete basic materials, such as ammonia, for neutralization, grow in the medium at pH 4.0 and increase the pH of the medium to approximately 8.0. These microbial cells could neutralize not only nitric acid but also sulfuric and hydrochloric acids. The strains could also grow by utilizing nitric acid as a sole nitrogen source. In the soil containing these organisms, the pH was maintained in the neutral range by the buffering action of basic materials that they secrete. These results suggest that these fungal cells are useful for protecting the soil from acidification by acid rain.

Antioxidative activity and protective effect against ethanol-induced gastric mucosal damage of potato protein hydrolysate (potato peptides, Po-P) were studied in vitro and in vivo. The Po-P obtained by proteolysis with Amano P and pancreatin inhibited linoleic acid oxidation either by 83%, at its coexistent 0.005% in a ferric thiocyanate assay system or by 32% at its coexistent 0.0002% in a beta-carotene decolorization assay system. Meanwhile Po-P were orally administered to male Wistar rats at doses of 12.5-100 mg/kg of body weight (BW) 30 min prior to ethanol injection. Consequently the ethanol-induced gastric damage was significantly reduced in a dose-dependent manner in the Po-P administered rat. The highest effect was observed in the group dosed with 100 mg Po-P/kg BW; the inhibition ratio was 69.6%. The extent of antioxidation or protection against ethanol-induced gastritis was quite similar to those of the respective peptides from casein, corn protein and ovalbumin, suggesting that the potato protein hydrolysate could serve as a useful food ingredient in practical eating habits.

Five novel oligosaccharides (tetra-, penta- and hexa-saccharides) were synthesized by glucosyltransfer from beta-D-glucose 1-phosphate to isokestose (O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside) or nystose (O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside) using Thermoanaerobacter brockii kojibiose phosphorylase. The oligosaccharides were identified as 2(2-alpha-D-glucopyranosyl)(m)isokestose; [O-alpha-D-glucopyranosyl-(1-->2)](m)-O-[beta-D-fructofuranosyl-(2-->1)](2)-alpha-D-glucopyranoside: m=1, 2, and 3, and 2(2-alpha-D-glucopyranosyl)(n)nystose; [O-alpha-D-glucopyranosyl-(1-->2)](n)-O-[beta-D-fructofuranosyl-(2-->1)](3)-alpha-D-glucopyranoside: n=1 and 2 using gas liquid chromatography analysis of the methyl derivatives, and MALDI-TOF-MS and NMR measurements of the newly formed oligosaccharides. 1H, 13C NMR signals of each saccharide were assigned using 2D-NMR techniques, including COSY, HSQC, HSQC-TOCSY, HMBC, CH(2)-selected E-HSQC, and CH(2)-selected E-HSQC-TOCSY.

The developmental competence of in vitro cultured embryos vitrified-warmed at an early cleavage stage (2- or 4, 8-cell stage) was examined by both direct transfer into recipient animals and after in vitro manipulation for chimeric mice production using embryonic stem (ES) cells. Vitrified-warmed embryos transferred at the morulae and blastocyst stages showed fetus development comparable to control embryos, although blastocyst development of vitrified-warmed embryos was significantly slower than that of controls. When vitrified-warmed early cleavage stage embryos were used for chimeric mouse production using ES cells, 1 to 10% of the injected or aggregated embryos developed into chimeric neonates and germ-line chimeric mice were obtained from all ES cell lines. This study indicates that embryos developed in vitro from vitrified-warmed embryos have equivalent competence with unvitrified embryos irrespective of stage of vitrification and that these vitrified-warmed embryos maintain adequate viability even after in vitro manipulation such as aggregation and microinjection with ES cells.

The favorable effect of burr hole irrigation and closed drainage system has been reported in the treatment of chronic subdural hematoma (CSH). After analyzing the relationship of the direction of the drainage tube and recurrence, we have suggested that the residual air after surgery night be important as one of the factors causing recurrence. The present study included 128 chronic subdural hematomas in 102 patients who had been treated in our institute between January, 1996 and October, 2000. We attempted to place the tip of the external drain at the occipital side within the hematoma cavity between January, 1996 and December, 1998 (occipital drainage group), while we tried to insert the tip of the external drain at the frontal side using an endoscope between January, 1999 and October, 2000 (frontal drainage group). We focused on the residual air on CT, the direction of the drainage, recurrence of the hematoma and the size of the hematoma. Some statistical analyses were conducted using these factors. The recurrence rate was slightly lower in the frontal drainage group than that in occipital drainage group. There was no difference in the residual rate of air immediately after the surgery on CT and the recurrence rate between the 2 groups. However, the interval preceding the recurrence was statistically significantly longer in the frontal drainage group. A higher rate of recurrence was noted for cases in which the volume of hematoma was more than 70 ml. The residual rate of air 1 week after surgery was significantly higher in the recurrent group than in non-recurrent group. In this study, it is demonstrated that a long-standing residual of air after surgery is one of the causes of the recurrence of CSH. We must contrive to reduce the residual air after surgery for the treatment of CSH.

On the genesis of chronic subdural hematoma (CSH), it is one of the possible explanations that a head injury triggers uncertain cause off for the formation of the hematoma membrane, where the bleeding occurs to form and enlarge the hematoma. Although some reports on the staging classification based on computed tomography (CT) findings are available the natural course or history of the hematoma formation is not yet known. As previously reported, we have been using an endoscopically guided method in which a drainage tube is placed in the most frontal area of the cavity so as to evacuate the residual air efficiently. In this study, we analyzed the endoscopic findings in hematoma cavities and compared them with the clinical data, focusing on the post-trauma interval and recurrence of the hematoma. Between January 1999 and October 2000, we had an opportunity to observe the inner aspect of 60 hematomas in 48 patients. A trabecular structure was found in 39 hematoma cavities (65%), whereas apparent clot formation was observed in 18 cavities (30%). Septum formation leading to a multi-loculated hematoma was observed in 10 cavities (17%). Twenty-six patients with 32 hematomas with the obvious history of head injury were classified into 4 groups according to the post-traumatic interval to the surgery, which are stage I; less than 30 days, stage II; 31 to 60 days, stage III; 61 to 90 days and stage IV; more than 91 days. Clot formation and a trabecular structure were frequently seen in stages II and III. Clot formation was statistically and significantly seen in the recurrent group. The results suggest that CSHs increase in size in stages II and III, when clot formation and a trabecular structure are frequently observed. Endoscopic observation of hematomas may assist to know hematomas, and clot formation may be a warning of hematoma recurrence.

Two novel oligosaccharides, tetra-and penta-saccharides were synthesized by fructosyl transfer from 1-kestose to 4G-beta-D-galactopyranosylsucrose with a purified 1F-fructosyltransferase of asparagus roots and identified as 1F-beta-D-fructofuranosyl-4G-beta-D-galactopyranosylsucrose, O-beta-D-fructofuranosyl-(2-->1)-beta-D-fructofuranosyl-O-[beta-D-galactopyranosyl-(1-->4)]-alpha-D-glucopyranoside and 1F(1-beta-D-fructofuranosyl)2-4G-beta-D-galactopyranosylsucrose, [O-beta-D-fructofuranosyl-(2-->1)]2-beta-D-fructofuranosyl-O-[beta-D-galactopyranosyl-(1-->4)]-alpha-D-glucopyranoside, respectively. Both oligosaccharides were scarcely hydrolyzed by carbohydrase from rat small intestine. Human intestinal bacterial growth by 1F-beta-D-fructofuranosyl-4G-beta-D-galactopyranosylsucrose was compared with that by the tetrasaccharides, stachyose and nystose. Bifidobacteria utilized 1F-beta-D-fructofuranosyl-4G-beta-D-galactopyranosylsucrose to the same extent as stachyose or nystose. On the other hand, the unfavorable bacteria, Clostridium perfringens, Escherichia coli and Enterococcusfaecalis, that produce mutagenic substances did not use the synthetic oligosaccharide.

Cells isolated from Xpg (the mouse counterpart of XPG)-disrupted mice underwent premature senescence and showed early onset of immortalization, suggesting that Xpg might be involved in genetic stability. Recent studies showed that human XPG, in addition to its function in the nucleotide excision repair (NER), was involved in the repair of oxidative base damages such as thymine glycol (Tg) and 8-oxo-guanine (8-oxoG), and this may explain the genetic instability observed in Xpg-deficient cells. To clarify this point, we determined spontaneous mutation frequencies and the type of spontaneous base substitution mutations in cells obtained from normal and Xpg-deficient mice using the supF shuttle vector (pNY200) for mutation assay. The spontaneous mutation frequency of the supF gene in pNY200 propagated in the Xpg-deficient cells was about three times higher than that in normal cells, indicating the importance of Xpg in reducing the frequency of spontaneous mutations. The frequency of spontaneous base substitution mutations at A:T sites, particularly that of the A:T to C:G transversion, increased markedly in the Xpg-deficient cells.

Laboratory mice carrying the nonfunctional xeroderma pigmentosum group G gene (the mouse counterpart of the human XPG gene) alleles have been generated by using gene-targeting and embryonic stem cell technology. Homozygote animals of this autosomal recessive disease exhibited signs and symptoms, such as postnatal growth retardation, reduced levels of activity, progressive ataxia and premature death, similar to the clinical manifestations of Cockayne syndrome (CS). Histological analysis of the cerebellum revealed multiple pyknotic cells in the Purkinje cell layer of the xpg homozygotes, which had atrophic cell bodies and shrunken nuclei. Further examination by an immunohistochemistry for calbindin-D 28k (CaBP) showed that a large number of immunoreactive Purkinje cells were atrophic and their dendritic trees were smaller and shorter than in wild-type littermates. These results indicated a marked degeneration of Purkinje cells in the xpg mutant cerebellum. Study by in situ detection of DNA fragmentation in the cerebellar cortex demonstrated that some deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin in situ nick labeling (TUNEL)-positive cells appeared in the granule layer of the mutant mice, but few cell deaths were confirmed in the Purkinje layer. These results suggested Purkinje cell degeneration in the mutant cerebellum was underway, in which much Purkinje cell death had not appeared, and the appearance of some abnormal cerebellar symptoms in the xpg-deficient mice was not only due to a marked Purkinje cell degeneration, but also to damage of other cells., (Copyright 2001 Wiley-Liss, Inc.)

The possibility exists that residual air after surgery is one cause of recurrence of chronic subdural hematoma. We have devised a new simple method which decreases postoperative residual air, using external drainage and an endoscope. First, we make endoscopic observations of the inner aspect of the hematoma cavity. Then, we insert external drainage apparatus into the most frontal area of the hematoma cavity, we regard this location as the most appropriate place to ensure most effective drainage. The present study included 37 chronic subdural hematomas in 32 patients who had been treated between January and December, 1999. Their ages ranged from 48 to 86 years old, with an average of 72 years. Insertion of external drainage in the most frontal area of the hematoma cavity was successfully achieved in 27 (73%, Group I) out of 37 cases and resulted in no recurrence. In the remaining 10 hematomas (27%, Group II), external drainage was not able to be inserted in the most frontal area, and four hematomas (40%) had recurrence (p < 0.01 vs Group I). Insertion in the most frontal area of the hematoma cavity decreases residual air after surgery, and may be effective for the prevention of recurrence of chronic subdural hematoma.

Blood glucose and insulin responses and gastric emptying were examined in rats intubated with sucrose or soluble starch that contained adenosine, inosine and cytosine. The increase in serum glucose and insulin levels in the rats following loading with sucrose (2.5 g/kg of body weight) or soluble starch (1.875 g/kg of body weight) was significantly reduced by the administration of adenosine, inosine and cytosine (0.0625-0.125 g/kg of body weight). The gastric emptying rates were only marginally affected by the nucleoside administration. The activities of sucrase, maltase, isomaltase and glucoamylase in a crude preparation from the small intestinal mucosa of rats were mildly inhibited by the nucleosides. The decrease in blood glucose and insulin levels may have been in response to a decrease in glucose absorption caused by the inhibiting effect of the nucleosides on the mucosal enzymes that digest sucrose, maltose, and malto- and isomalto-oligosaccharides.

The xeroderma pigmentosum group G (XP-G) gene (XPG) encodes a structure-specific DNA endonuclease that functions in nucleotide excision repair (NER). XP-G patients show various symptoms, ranging from mild cutaneous abnormalities to severe dermatological impairments. In some cases, patients exhibit growth failure and life-shortening and neurological dysfunctions, which are characteristics of Cockayne syndrome (CS). The known XPG protein function as the 3' nuclease in NER, however, cannot explain the development of CS in certain XP-G patients. To gain an insight into the functions of the XPG protein, we have generated and examined mice lacking xpg (the mouse counterpart of the human XPG gene) alleles. The xpg-deficient mice exhibited postnatal growth failure and underwent premature death. Since XPA-deficient mice, which are totally defective in NER, do not show such symptoms, our data indicate that XPG performs an additional function(s) besides its role in NER. Our in vitro studies showed that primary embryonic fibroblasts isolated from the xpg-deficient mice underwent premature senescence and exhibited the early onset of immortalization and accumulation of p53.

Complementation analysis of a polyhydroxyalkanoate (PHA)-negative mutant of Aeromonas caviae proved that ORF3 in the pha locus (a 402-bp gene located downstream of the PHA synthase gene) participates in PHA biosynthesis on alkanoic acids, and the ORF3 gene is here referred to as phaJ(Ac). Escherichia coli BL21(DE3) carrying phaJ(Ac). under the control of the T7 promoter overexpressed enoyl coenzyme A (enoyl-CoA) hydratase, which was purified by one-step anion-exchange chromatography. The N-terminal amino acid sequence of the purified hydratase corresponded to the amino acid sequence deduced from the nucleotide sequence of phaJ(Ac) except for the initial Met residue. The enoyl-CoA hydratase encoded by phaJ(Ac) exhibited (R)-specific hydration activity toward trans-2-enoyl-CoA with four to six carbon atoms. These results have demonstrated that (R)-specific hydration of 2-enoyl-CoA catalyzed by the translated product of phaJ(Ac) is a channeling pathway for supplying (R)-3-hydroxyacyl-CoA monomer units from fatty acid beta-oxidation to poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis in A. caviae.

The Ku p70 and Ku p80/XRCC5 genes are involved in DNA double-strand break repair and V(D)J recombination, and their gene products are the components of the DNA-dependent protein kinase. We have determined the chromosomal locations of the mouse Ku p70 and Ku p80/XRCC5 genes by both in situ hybridization and molecular linkage analysis: the Ku p70 gene was localized to mouse chromosome 15 and rat chromosome 7, and the Ku p80/XRCC5 gene was localized to mouse chromosome 1 and rat chromosome 9. Both genes were mapped to a region of conserved linkage homology among three species, i.e., the mouse, rat, and human. Molecular linkage analysis using interspecific backcross mice revealed that the murine Ku p70 locus was localized 0.7 cM terminal to D15Mit1 and that the murine Ku p80/XRCC5 locus was 0.7 cM proximal to D1Mit46. To determine the size and tissue transcription specificity of the mouse Ku p70 and Ku p80/XRCC5 mRNA, Northern blot analysis was carried out with six mouse tissues. Each tissue expressed one species of the Ku p70 gene transcript with 2.4 kb and one species of the Ku p80/XRCC5 gene transcript with 2.6 kb. In the latter case, however, the brain showed two sizes of transcript, 2.6 and 2.9 kb.

A cDNA and a gene encoding endo-inulinase from Penicillium purpurogenum were isolated, and were cloned for the first time. Two oligonucleotide probes, which were synthesized based on the partial amino acid sequences of the purified endo-inulinase, were used to screen a cDNA library. A 1.7-kb DNA fragment encoding endo-inulinase was isolated and analyzed. A single open reading frame, consisting of 1548-bp, was found to encode a polypeptide that comprised a 25-amino acid signal peptide and 490-amino acid mature protein. All the partial amino acid sequences of the purified enzyme were discovered in the deduced ones. The deduced amino acid sequences of endo-inulinase had similar sequences to those of fructan hydrolases. A 3.5-kb chromosomal DNA fragment encoding endo-inulinase was also isolated and analyzed. The same ORF with cDNA clone as identified. There were no introns in the endo-inulinase gene.

US31 is a UV-sensitive mutant cell line (rodent complementation group 8) derived from a mouse T cell line L5178Y. We analyzed removal kinetics for UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts in US31 cells using monoclonal antibodies against these photoproducts. While nearly all (6-4) photoproducts were repaired within 6 h after UV-irradiation, more than 70% of cyclobutane pyrimidine dimers remained unrepaired even 24 h after UV-irradiation. These kinetics resembled those of Cockayne syndrome (CS) cells. Since US31 cells had a low efficiency of cell fusion and transfection, which hampered both complementation tests and gene cloning, we constructed fibroblastic complementation group 8 cell line 6L1030 by fusion of US31 cells with X-irradiated normal mouse fibroblastic LTA cells. Complementation tests by cell fusion and transfection using 6L1030 cells revealed that rodent complementation group 8 corresponded to CS complementation group A.

We have molecularly cloned and sequenced the mouse counterpart of the human repair gene XPG/ERCC5 cDNA. The mouse xpg cDNA had a single long open reading frame predicted to encode 1170 amino acid residues (predicted M(r) of 130,753). Northern blot analysis has been carried out to determine the size and tissue transcription specificity of the mouse xpg mRNA. The xpg gene expressed one species of transcript with 4.3 kb at similar levels in five mouse tissues examined. We have determined the chromosomal location of the xpg gene by both in situ hybridization and molecular linkage analysis. The xpg gene was localized at 2.3 cM proximal to the microsatellite locus D1Mit18 on the R-positive B band of mouse chromosome 1. By in situ hybridization with the mouse xpg probe, the rat homolog of the mouse xpg was localized on q22.3 band of rat chromosome 9, which has been known to have a conserved linkage homology to mouse chromosome 1. In the case of human, the XPG/ERCC5 gene has been reported to be assigned to human chromosome 13q32.3-q33.1, where any conserved linkage homology to mouse chromosome 1 has not been found so far. Thus, these results show new regions of conserved linkage homology among mouse chromosome 1, rat chromosome 9, and human chromosome 13q.

We have isolated a human excision repair gene ERCC5 which complements the defect of the mouse UV-sensitive mutant XL216 (rodent complementation group 5). Here we report cDNA cloning of human and mouse ERCC5 genes using an exon containing an ERCC5 fragment as a probe. The ERCC5 cDNA encodes a predicted 133-kDa nuclear protein that shares some homology with the product of the yeast DNA repair gene RAD2. Transfection with mouse ERCC5 cDNA restored normal levels of UV resistance to XL216 cells. Microinjection of ERCC5 cDNA specifically restored the defect of xeroderma pigmentosum group G cells (XP-G) as measured by unscheduled DNA synthesis, and XP-G cells stably transformed with ERCC5 cDNA showed nearly normal UV resistance.

The genomic DNA fragment encoding the excision repair gene, ERCC5, was mapped by direct R-banding fluorescence in situ hybridization. The signals were localized to human chromosome 13q32.3-q33.1. This result was in agreement with previous reports, and the gene was assigned to a narrower region.

A gene for ethionine resistance isolated from the yeast Saccharomyces cerevisiae DKD-5D-H conferred on the yeast cells resistance to seleno-L-methionine and capability to produce S-adenosyl-L-methionine in the cells. An enzymatic study of the L-methionine synthetic pathway of L-methionine proto- and auxotrophs and in dried yeast cells with or without the gene suggested that the cloned gene for ethionine resistance is responsible for the activity of S-adenosyl-L-methionine synthase. To produce S-adenosyl-L-methionine by yeast cells transformed with the ethionine resistance gene, some culturing conditions were determined.

The mouse lymphoma L5178Y cell line and its radiosensitive variants M10 and LX830 were examined for DNA synthesis after X-irradiation. The dose-response curves show that the rates of DNA synthesis immediately after exposure are reduced in a dose-dependent fashion and that the extents of reduction in these 3 cell lines are similar to one another. But a difference was observed in the recovery of DNA synthesis with time of incubation. The recovered levels in M10 and LX830 cells were much higher than those in L5178Y cells at high doses of X-rays. These results are discussed in relation to radioresistant DNA synthesis in ataxia telangiectasia cells.

Polymorphonuclear leukocytes from the gingival crevicular fluid (CF-PMNs) of patients with generalized severe periodontitis were examined using electron microscopy and immunocytochemical techniques. CF-PMNs were found to contain numerous phagocytic vacuoles. This suggests that CF-PMNs actively phagocytized various substances from the environment. Immunocytochemical staining with FITC-conjugated IgG, IgM, and IgA reagents and TRITC-conjugated C3 reagent was applied to CF-PMNs as well as peripheral blood PMNs incubated with cell-free crevicular fluid. The cytoplasm of PMNs exhibited numerous granular foci of immunofluorescence. This finding suggests that these proteins were acquired from the environment by PMNs. The coincidental appearances of immunoglobulins and C3 in a single cell were considered to be immune complexes phagocytized by CF-PMNs in generalized severe periodontitis.

Three X-ray-sensitive mutants (LX821, LX827 and LX830) have been isolated from mouse-lymphoma L5178Y cells. These mutants are much more sensitive to the lethal effects of ionizing radiation than the parental L5178Y cells but are as resistant to ultraviolet radiation as L5178Y cells. We have previously isolated a mutant M10 that is sensitive to methyl methanesulfonate (MMS) and cross sensitive to ionizing radiation and 4-nitroquinoline 1-oxide (4NQO). Unlike M10 cells, newly isolated mutants were not sensitive to MMS and were less sensitive to 4NQO. These results indicate that new mutants may be deficient in the repair of DNA damage specific to ionizing radiation. LX821 and LX827 cells were concomitantly resistant to 5-bromodeoxyuridine, whereas LX830 cells were not.

The X-ray-sensitive mutant M10 and the UV-sensitive mutant Q31 of mouse lymphoma L5178Y cells are both sensitive to killing by 4-nitroquinoline-1-oxide (4NQO). Since cell hybridization experiments showed that the 4NQO sensitivities in M10 and Q31 cells behaved as codominant traits (Shiomi et al., 1982c), it is not possible to determine by complementation test whether the M10 and the Q31 mutations responsible for 4NQO sensitivities are allelic. We have obviated this difficulty by selecting double mutants that are sensitive to both X-rays and UV. From X-ray-sensitive M10 cells, two UV-sensitive mutants (XU 1 and XU 2) were isolated by a cell-suspension spotting method. XU 1 and XU 2 were found to belong to the same complementation group as Q31 (group I). Double mutants XU 1 and XU 2 were 30-37-fold more sensitive to 4NQO than parental L5178Y cells, whereas the single mutants M10 and Q31 were only 6-8-fold more sensitive to 4NQO than L5178Y cells in terms of D10 values (dose required to reduce survival to 10%). These results show that the M10-Q31-double mutations enhance 4NQO sensitivity synergistically, indicating that the M10 and the Q31 mutations relevant to 4NQO sensitivities are non-allelic. The implications of this finding are discussed.

A multiple mutagen-sensitive mutant (XUM1) of mouse T-cell lymphoma line, L5178Y, is hypersensitive to ionizing radiation, ultraviolet (UV) light, and cross-linking agents (such as mitomycin C). The frequency of transfection for XUM1 cells after exposure to calcium phosphate-coprecipitated pSV2neo DNA was more than 10(4)-fold less effective than that for Ltk-aprt- (LTA) cells. Other transfection methods (DEAE-dextran and polybrene-DMSO) were not effective for L5178Y and XUM1 cells. The transfection-proficient trait of LTA cells was demonstrated to be genetically dominant by examining the the transfection frequency in hybrid clones constructed between XUM1 and LTA cells. To circumvent the problem with XUM1, the LTA genes necessary for transformation processes were introduced into XUM1 cells by constructing hybrids between XUM1 and LTA cells irradiated with X-rays which causes directional chromosome elimination for hybrid cells. Four of 194 hybrid clones tested were transfection-proficient and hypersensitive to UV (XL102, XL107, XL215, and XL216). All four clones were not hypersensitive to X-rays or mitomycin C. The frequencies of transfection for XL102 and XL216 were nearly the same level as that for LTA cells. The efficiency of transfection for XL107 and XL215 was 10 to 100-fold lower than that for LTA cells.

The X-ray analysis of 8,5'-(N-carbamoyloxy)-2-dimethylamino- methylene-9-(2',3'-0-isopropylidene-beta-D-ribofuranosyl)guanine (2a) has disclosed that some bond angles are significantly larger or smaller than the corresponding angles of normal ribonucleosides. Abnormally longer or shorter bond lengths have also been found. The base-sugar torsion angle of 2a has proved to reside between the C1'-C2' and C1'-C3' lines, and quite near the latter line. Some new 8,5'-carbamoyloxy bridged adenosines were also synthesized as 2',3'-protected forms.

Three mutagen-sensitive mutants, MS-1, M10 and Q31, have been isolated from mouse L5178Y cells. MS-1 cells are sensitive to methyl methanesulfonate (MMS), M10 cells are cross-sensitive to X-rays, MMS and 4-nitroquinoline 1-oxide (4NQO), and Q31 cells are cross-sensitive to UV and 4NQO. Lines resistant to 6-thioguanine (TGr) and 5-bromo-2'-deoxyuridine (BUr) were isolated from L5178Y and these three mutagen -sensitive mutants. All the TGr lines were sensitive to 5-bromo-2'-deoxyuridine and HAT medium and all the BUr lines were sensitive to 6-thioguanine and HAT medium. The hybrids homozygous for the mutagen-sensitive markers showed nearly the same sensitivity to UV, 4NQO, X-rays and MMS as their parental TGr and BUr lines. The hybrids constructed by fusing L5178Y BUr and TGr lines from each of MS-1, M10 and Q31 displayed the normal UV, X-ray and MMS resistancy of L5178Y cells. Thus the UV-, X-ray- and MMS-sensitive markers in MS-1, M10 and Q31 were recessive in somatic cell hybrids. The 4NQO-sensitive phenotype, however, behaved codominantly in somatic cell hybrids.

The mutant mouse lymphoma cell M10, which is sensitive to methyl methanesulfonate and ionizing radiation, was compared with the parental L5178Y cells for mutation induction after gamma-irradiation. The rate of induced mutations to 6-thioguanine resistance in L5178Y cells was 2-3 X 10(-7) per R, as determined after exposures ranging from 25 to 500 R. The induced mutation frequency per unit dose per locus in M10 cells was about 4 times higher than that in L5178Y cells at the lower doses of exposure (25-75 R), but it declined sharply at the higher doses of gamma-rays (100-150 R). The rate of induced mutation per unit cell killing in M10 cells was nearly the same as that in L5178Y cells when they were compared at the levels of lower cell killing.

26 mutants with increased sensitivity to the lethal effects of mitomycin C (MMC) were isolated from mouse lymphoma L5178Y cells by a replica-plating technique. Most of them were about 5-10 times more sensitive in terms of D37 values to MMC than were parental cells. 5 of the MMC-sensitive mutants isolated from independently mutagenized cell populations were further analyzed. They were highly sensitive to the killing by decarbamoyl (DC) MMC, a monofunctional derivative of MMC, but were not sensitive to ultraviolet radiation, X-rays, 4-nitroquinoline-1-oxide or methyl methanesulfonate. These 5 mutants were classified into at least 2 genetic complementation groups. The implication of these mutations in cross-link and mono-adduct repair of DNA damage induced by MMC and DCMMC is discussed.

Two-methyl-methanesulfonate-sensitive strains have been isolated, one of which, M10, was cross-sensitive to X-rays as reported before. Sensitivities of parental L5178Y, M10, and newly isolated MS-1 cells to various mutagens were examined. Mutagens tested were UV, X-rays, 4-nitroquinoline 1-oxide (4NQO), caffeine and alkylating agents; methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and mitomycin C (MMC). In terms of D37 values, M10 cells were 2.5-7 times more sensitive to EMS, MMC and 4NQO as well as to MMS and X-rays than were parental L5178Y cells, while the new mutant MS-1 was about 3 times more sensitive to MMS, EMS, MMC and caffeine than were parental cells. The characteristics in sensitivities of M10 cells to X-rays, alkylating agents and 4NQO resemble resemble some ataxia telangiectasia cells; and MS-1 cells to alkylating agents and caffeine are novel among mammalian cell mutants so far reported. Sensitivity of M10 cells to mutagens has so far been stable for one year, and that of MS-1 cells was stable for 6 months in continuous culture.