Okayama University Medical School

A novel endonuclease of 55-kDa was found in rat liver mitochondria by a zymographic assay, in addition to the 29 kDa enzyme that is well-known as endonuclease G (Endo G). Subcellular localization of these enzymes in rat liver cells was examined by biochemical fractionation. Endo G was located in both nuclei and mitochondria as has been previously reported, while the 55-kDa enzyme was only detected in the mitochondrial fraction. The levels of the endonucleases in the mitochondria varied greatly among the rat organs, and the activity in the heart was about 30 times higher than that in the liver. The 55-kDa enzyme and Endo G were extracted from bovine heart mitochondria with 0.4 M NaCl. During purification the 55-kDa enzyme and Endo G were copurified because of their similar chromatographic behavior, so they were separated by gel filtration or electrophoresis in the presence of SDS and the proteins were then renatured. The nucleolytic properties of the 55-kDa enzyme resembled those of Endo G and other known mitochondrial nucleases. The enzyme degraded single-stranded DNA more rapidly than duplex DNA at a weak alkaline pH, requiring Mg²⁺ or Mn²⁺ but not Ca²⁺ or Zn²⁺. Nicks generated by the enzyme had 5′-P and 3′-OH ends. The 55-kDa enzyme, like Endo G, displayed an unusually strong preference to nick within a (dG)_n · (dC)_n tract.

The serum levels of the carbohydrate antigen sialyl Lewis X (SLEX) increase in liver diseases (Sunayama T, Okada Y, Tsuji T., J Hepatol 1994; 19: 451-458). However, it is not known whether the increased serum SLEX levels are associated with the increased levels of its carrier molecules and/or the increased density of SLEX per carrier molecule. By using of rabbit antibody against an SLEX-positive fraction from HepG2 culture supernatant, we developed an enzyme-linked immunosorbent assay to determine the serum levels of the carrier molecules of SLEX (CM_SLEX). The CM_SLEX-levels in patients with hepatocellular carcinoma were significantly higher than those of normal controls (P < 0.001) and benign chronic liver diseases, i.e., chronic active hepatitis, mild and severe form, and liver cirrhosis (P < 0.05). Patients with chronic persistent hepatitis and chronic active hepatitis, mild form, had higher CM_SLEX-levels than normal controls (P < 0.05). The serum CM_SLEX-levels did not differ significantly among benign liver diseases. We concluded that serum CM_SLEX-levels increase nonspecifically in liver diseases. This is a possible molecular mechanism for the increased serum SLEX levels in liver diseases.

A number of approaches have been put forward to monitor spinal cord ischemia during thoracic and thoracoabdominal aortic occlusion. However, none of these can ultimately prevent devastating complications which result from ischemic spinal cord injury. A direct measurement of the oxygen content of the spinal cord may accurately indicate the perfusion state, but in practice it is impractical. We surmised that intrathecal and/ or epidural oxygen concentration(I-pO₂ and E-pO₂, respectively) accurately reflect oxygen content in the spinal cord. So, we examined whether or not I-pO₂ and/or E-pO₂ correlated with the spinal cord pO₂ (S-pO₂) in dogs. In nine mongrel dogs, a model of graded spinal cord ischemia was developed by stepwise alternation of the level of aortic occlusion with an intraaortic balloon catheter. I-pO₂, E-pO₂ and S-pO₂ were measured with a mass spectrometer. Our results show that, both I-pO₂ and E-pO₂ significantly correlated with S-pO₂. I-pO₂ correlated with S-pO₂ better than E-pO₂ did. Therefore, I-pO₂ can be used as a new indicator for spinal cord ischemia, and I-pO₂ monitoring would be useful to prevent paraplegia associated with thoracic aortic surgery.

Using a transcranial Doppler blood flowmeter, the blood flow velocity (BFV) ratio of the middle cerebral artery (MCA) to the basilar artery (BA) was investigated in 12 patients with severe motor and intellectual disability syndrome. The BFV of the MCA was also investigated in 58 handicapped children, classified according to the severity of their motor and intellectual disability. The ratio of the MCA to the BA was lower by 2 SD from the mean of our previously reported standard value in 8 out of the 12 cases with severe motor and intellectual disability syndrome, suggesting a more profound decrease in the level of brain activity in the MCA area than that of the BA area. The BFV of the MCA mainly decreased in cases belonging to the category of the most severe motor disability (bed-ridden). Hence, it is suggested that motor disability is the main factor related to the decrease in the BFV of the MCA.

The results of the histological examinations of specimens of the tenosynovium of the flexor tendon, the epineurium and the transverse carpal ligament from two groups of Japanese patients with carpal tunnel syndrome (idiopathic and hemodialysis) were compared. Amyloid deposits, positively identified as β₂-microglobulin, appeared in all patients in the long-term hemodialysis group, but in no patients in the idiopathic group. Although the pathogenesis differed between the two groups, both resulted in nerve compression in the carpal tunnel. Therefore, surgical release is considered beneficial for both groups.

Even after successful operations, ugly postoperative skin scars are often distressing to patients and their parents. To judge the success of surgical methods and postoperative treatment, postoperative scars should be evaluated using a quantitative system. Height and width are easily measured, but scar redness is not. We have developed a simple and effective method for evaluating scar redness. According to the color definitions employed in computer graphics, each color can be expressed as RGB (red, green or blue) coordinates (r, g, b): 0 ≦ r, g, b ≦ 10. The degree of scar redness is defined by the following formula: redness score (RS) = (r₁ - r₀)² + (g₁ - g₀)² + (b₁ - b₀)². Here, (R₁, g₁, b₁) = coordinates of the scar color and (r₀, g₀, b₀) = coordinates of the surrounding skin color. RS was evaluated in 59 children (35 males, 24 females; ages 1 month to 12 years old) who had scar redness after congenital cardiac surgery. For each patient, scar color and surrounding skin color was identified on the color sample table. Scar redness was also evaluated by the conventional grading method: 1 = mild, 2 = moderate and 3 = severe. The RS of the colored scars ranged from 4 to 100 (38 ± 27). By the conventional grading method, 44 scars were grade 1, 15 grade 2 and none grade 3. RS was significantly higher among grade 2 than grade 1 patients, 52 ± 25 and 33 ± 27, respectively (P < 0.05). Given its subjectivity, the conventional grading method yields variable data; surrounding skin color, moreover, is not considered. Our new evaluation method using RS effectively and accurately defines scar and skin colors, and allows quantitative studies of these factors.

We report the results of phase I/II studies of preoperative multidisciplinary treatment of 14 patients with soft tissue sarcoma using hyperthermia from November 1990 to April 1995. The preoperative treatment was conducted with thermo-radio-chemotherapy in 11 cases of stage III, and with thermo-radiotherapy as well as thermo-chemotherapy in three cases of stages I and II. Hyperthermia was carried out twice a week with totals ranging from 4 to 14 times (average: 8.4 times); each session lasted 60min. Radiotherapy was administered four or five times per week, and the dose was 1.8 2Gy/fraction, with a total of 30-40Gy in a four week period. Chemotherapy was mainly in the form of MAID regimen (2-mercaptoethanesulphonic acid (mesna), adriamycin, ifosfamide and dacarbazine). The tumors were surgically resected in all patients after completing the preoperative treatment. The efficacy rate, as expressed by the percentage of either tumors in which reduction rate was 50% or more, or tumors for which post-treatment contrast enhanced CT image revealed low density volumes occupying 50% or more of the total mass, was 71 % (ten of the 14 tumors). The mean tumor necrosis rate in the resected specimens was 78%. The tumor necrosis rate was significantly high (P < 0.05) in patients whose Time ≥ 42°C was of long duration. Postoperative complications were observed in six patients; among these, two patients developed wound infection that required surgical treatment as a complication of surgery performed in the early stage following the preoperative treatment. After a mean postoperative follow-up of 27 months, distant metastasis occurred in four patients resulting in three fatalities. The three-year cumulative survival rate was 64.3%. No local recurrence was observed in any patient during the follow-up, thus confirming our hypothesis that preoperative multidisciplinary treatment has an excellent local efficacy. We think that it would be valuable to conduct, at many facilities, phase III studies on the treatment of soft tissue sarcoma by a combination of surgery and preoperative multidisciplinary treatment using hyperthermia, paying close attention to the interval between these two modalities.

High-resolution magnetic resonance venography (HR-MRV) of intracranial subependymal veins using a two-dimensional Fourier-transform time-of-flight technique was performed on normal volunteers and clinical cases of cerebral disease. For the pulse sequence, fast-field-echo sequence was used with the following parameters: TR/TE/ flip angle = 34ms/12ms/50deg., 256 x 256 matrix, 1 mm effective slice thickness, 150mm field of view, and one signal acquisition. Sequential vertical coronal sections were taken against the skull base. The anterior septal vein, the medial atrial vein, the anterior caudate vein and thalamostriate vein were detected in all subjects. In all clinical cases, HR-MRV was equal in diagnostic capability to conventional cerebral angiography.

Twelve cases of leptospirosis followed by the Infectious Diseases Clinic of the Cukurova University Medical School, Adana, Turkey, between January 1994 and November 1995 are reported. Included are their clinical manifestation, laboratory findings and serotype. Nine men and three women with an average age of 40.4 years were studied. Symptoms, clinical manifestations, laboratory findings and treatment of the disease are evaluated. All of the patients had fever and chills and the following symptoms: nausea and vomiting (91.6%), lower back pain and myalgia (58.3%), headache (50%), epistaxis (16.6%) and confusion (25%). The predominant clinical findings were jaundice (91.6%), hepatomegaly (41.6%), dyspnea (25%), conjunctival suffusion (33%), and nuchal rigidity (33%). Diagnosis was based on dark-field examination of the blood, cerebrospinal fluid and urine specimens. Also, microscopic agglutination tests (MAT) were carried out for serodiagnosis. MAT showed L. inter-rogans serovar icterohaemorrhagiae in 11 cases and L. interrogans serovar grippomosocova in one case. There was cross reaction with L. biflexa patoc in all cases. Agglutinations were tested in the same specimens twice and confirmed in the Microbiology Laboratory of the Etlik Veterinary Research Institute in Ankara. All cases were treated with penicillin and doxycycline. In the end; 83.3% of the patients were cured and 16.6% died due to hepatorenal failure.

Sections of the visual cortex of newborn (1-4 weeks after birth) and adult cats were stained with cationic iron colloid, aldehyde fuchsin or lectins (lectin Vicia villosa, soybean and Wisteria floribunda agglutinins). Many neurons in the adult cat visual cortex contained perineuronal sulfated proteoglycans detectable with cationic iron colloid and aldehyde fuchsin, or cell surface glycoproteins reactive to lectins. Double staining indicated that some of the lectin-labeled neurons were not stained with cationic iron colloid, and also that some of the cationic iron colloid-stained neurons were not labeled with lectins. The perineuronal sulfated proteoglycans and cell surface glycoproteins developed 3 weeks after birth. In the newborn cats 1-2 weeks after birth, no neurons were reactive to cationic iron colloid, aldehyde fuchsin or lectins. In the newborn cats 34 weeks after birth, it was clearly observed that the cytoplasm of the glial cells closely associated with the neurons containing the perineuronal sulfated proteoglycans showed an intense reaction to cationic iron colloid and aldehyde fuchsin, and that the Golgi complexes of the neurons with cell surface glycoproteins were intensely labeled with lectins. These findings suggest that the perineuronal sulfated proteoglycans are derived from the associated glial cells, and that the cell surface glycoproteins are produced by the associated nerve cells.

Characteristics of human hepatoma cell lines with the wild-type p53 were compared with those of human hepatoma cell lines with the mutant-type p53. The p21 protein located downstream of p53 was expressed in cell lines with the wild-type p53 but was not expressed in cell lines with the mutant-type p53. As to other tumor suppressor genes such as p16 and p27, there was no difference in their expression between both types of cell lines. In addition, no marked difference was observed in the activities of CDK2 and CDK4 between cell lines with the wild-type and the mutant-type p53. Phosphorylated Rb protein was detected in all cell lines except the HLE line, indicating that this cell line may have a deletion of and/or a mutation of the Rb gene. These results indicate that abnormalities of tumor suppressor genes other than p53, p16, p27, and Rb may be involved in hepatocarcinogenesis. The population doubling time of the wild-type p53 cells was significantly longer than that of the mutant p53 cells. Neither type of cell line showed a specific chromosome distribution which would indicate karyotype instability. The cell lines expressing the wild-type p53 produced tumors at lower frequency than those with the mutant p53 gene. Although there was no significant difference in effects of TGF-β1, EGF, cholera toxin, and db-cAMP on cell growth between the two types of cells, all three cell lines with the wild-type p53 were resistant to cytotoxicity of TNF-α, while two of the three with the mutant p53 were very sensitive to its cytotoxic effects.

This study was designed to investigate the induction of apoptosis during the reperfusion phase following warm liver ischemia in vivo. We evaluated apoptotic bodies (ABs) in sections stained with hematoxylin and eosin (H. E.) and positive hepatocytes in sections stained by the in situ nick end labeling method (TUNEL method) during the reperfusion phase up to 48 h after a 70% liver ischemia for 30 or 60min in duration (30 or 60 min group). The peak number of ABs in H. E.-stained sections was observed at 1 to 3 h in the 30 min group and 3 to 6 h in the 60 min group. The number of ABs gradually fell as the length of the perfusion period increased, and few ABs were observed at 24 and 48 h after reperfusion. A peak number of TUNEL-positive hepatocytes was recognized at 3 h after reperfusion in both groups, after which the numbers decreased gradually. DNA extracted from both groups was electrophoresed on a 1.5% agarose gel. In both groups, a ladder-like pattern over smear pattern was recognized at 3 h after reperfusion. These results show that hepatocyte apoptosis was induced during the early phase of reperfusion after rat liver ischemia morphologically and biochemically, which suggests that hepatocyte apoptosis may be associated with ischemia and reperfusion injury.

To clarify the nature of nitrogen metabolism between branched chain amino acid (BCAA) and glutamine (GIn) in liver failure, we measured arterial plasma concentrations of GIn and ¹⁵N uptake to amino-N and amide-N of GIn in normal and D-galactosamine-induced fulminant hepatic failure (FHF) rats after ¹⁵N-leucine (Leu) injection. Fifteen, 30 and 60 min after Leu injection, the arterial plasma concentrations of GIn were significantly higher in FHF rats than in controls. The concentrations of amino-¹⁵N GIn were also significantly higher in FHF rats than in controls at 5, 15, 30 and 60 min after injection. The concentrations of amide-¹⁵N GIn did not significantly differ between FHF and controls at 5, 15 and 30 min. However, at 60 min, the concentration was significantly higher in the FHF rats. The higher uptake of ¹⁵N to amino-N of GIn in FHF rats suggests the presence of an enhanced ability to synthesize GIn from Leu in FHF rats. The higher uptake of ¹⁵N to amide-N of GIn in FHF rats at 60 min after injection suggests that excessive administration of BCAA to patients with severely impaired urea-cycle capacity suffering with hepatic failure may lead to greater levels of hyperammonemia.

Two rare cases of carpal tunnel syndrome caused by calcification in the carpal tunnel are reported. One case involved a tumorous calcification consisting of basic calcium phosphate, and the other involved a diffuse calcification consisting of a mixture of calcium pyrophosphate dihydrate and basic calcium phosphate. These cases suggest that the shape of carpal tunnel calcifications is influenced by the nature of calcifying substance itself, i.e., whether it is heterogenous or homogenous.

Lumbar X-ray findings and clinical manifestations were investigated in 10 patients who underwent posterior fusion with or without Harrington instrumentation for idiopathic scoliosis between 1965 and 1975. The subjects were 4 men and 6 women, who ranged from 10 to 17 years of age at the time of surgery (mean, 12 years and 9 months). The postoperative follow-up period ranged from 20 to 30 years (mean, 24 years and 7 months). All patients were followed-up at our institution. Three patients received posterior fusion without instrumentation, and Harrington instrumentation was used in 7 from 1967 onwards. The distal end of the fusion was L2 in 4, L3 in 4, and L4 in 2 patients. Pain, evaluated by Moskowitz's criteria, was stage 1 in 5 and stage II in 5 patients (none of them had stage III or IV). In X-ray evaluation, graded according to Lawrence's classification, grade III changes were noted in 2 patients; one with thoracolumbar fusion with Harrington instrumentation to the L4 vertebra and the other patient was assessed at 30 years post-surgery. According to White-Panjabi's criteria, instability was noted in 1 patient with Harrington fixation including the L4 vertebra. Clinical manifestations and X-ray abnormalities were less severe than anticipated at 20 years plus post-surgery, although a tendency for deterioration was observed in patients with fusion including the L4 or patients followed up for more than 30 years post-surgery.

To understand the development of the trabecular meshwork of the eye, floating cellular aggregates (multicellular spheroids) were formed from human trabecular cells in a non-adherent environment of culture and incubated for up to one month. Dissociated trabecular cells formed multicellular spheroids within one day in the non-adherent environment, and apoptosis continued to occur in the spheroids which had been initially filled with cells. The final structure after one month appeared as a meshwork of cells with large extracellular spaces. Epidermal and basic fibroblast growth factor (EGF and bFGF) protected trabecular cells in the spheroids from apoptosis and, as a result, kept the spheroids filled with cells even after one month. In the absence of excess EGF or bFGF, the multicellular spheroids grown in vitro from human trabecular cells mimicked the mesh-like structure of normal trabecular tissue. In constrast, under an excess of these growth factors, spheroids of high cellularity, resembling the abnormal trabecular tissues of patients with congenital glaucoma, were formed.

Plasma 5-hydroxytryptamine (serotonin), tryptophan, neopterin and cortisol levels were measured in patients with depressive cancer cachexia and in healthy controls during the same time period. Patients with advanced cancers had significantly raised neopterin, a marker of endogenous gamma-interferon (IFN-γ) production, and cortisol values, but decreased serotonin and tryptophan levels. Much work has been done to elucidate the possible role of serotonin in depressive states. IFN-γ induces a high level of indoleamine dioxygenase (IDO), a tryptophan degrading enzyme, and high cortisol levels induce high tryptophan oxygenase activity, which in turn increases metabolism along the tryptophannicotinic acid pathway. These results suggest that persistent immune activation and intense adrenal activity occur in patients with cancer cachexia, resulting in disorders involving tryptophan metabolism followed by depression in cancer cahexia.

Mental retardation is detected in 20-30% of children with epilepsy at hospitals specializing in treatment of childhood epilepsy. However, the incidence of mental deterioration in childhood epilepsy is not high. In this study, mental deterioration was found in 52 (1.8%) of the 2,880 children with epilepsy at Okayama University Hospital. The patients showing mental deterioration mostly suffered from specific epileptic syndromes, such as West syndrome, Lennox-Gastaut syndrome, severe myoclonic epilepsy in infancy and epilepsy with continuous spike-waves during slow wave sleep. These types of epilepsy show generalized electroencephalographic (EEG) abnormalities. It is presumed that mental deterioration is caused by the total effects of prolonged diffuse EEG abnormalities and the age of the patients. Antiepileptic drugs exert a relatively minor effect on mental deterioration.

A simian cell line, Si-IIA, harboring Epstein-Barr-virus (EBV) -related herpesvirus (Si-IIA-EBV), produces malignant lymphoma in rabbits when administered by intravenous inoculation. In this study, we analyzed the Si-IIA-EBV genome and compared it with human EBV and herpesvirus macaca fascicularis 1 (HVMF 1 ), which is associated with B-cell lymphoma developing in SIV-infected immunosuppressed monkeys. DNA from Si-IIA-EBV was amplified by the polymerase chain reaction using three different primer pairs complementary to human EBV (B95-8) DNA; two of the primer pairs covered part of the long internal repeat 1 region (IR 1) and the third covered part of the BRRF 1 region. Direct sequencing of the three PCR products revealed that Si-IIA-EBV DNA had about 82% nucleotide homology to the human EBV DNA in all three regions and 92.4% homology to HVMF1 in the IR1 region. The blotting pattern by Southern blot analysis was different between Si-IIA-EBV and human EBV.

We purified a 44-kDa nuclear protein from salt-extract of permeable mouse ascites sarcoma cells in an effort to isolate factors involved in the repair of acid-depurinated DNA. It was copurified with a major AP endonuclease (APEX nuclease) by sequential column chromatography then further purified by sodium dodecyl sulphate-poly-acrylamide gel electrophoresis as a possible DNA repair support factor. Its partial amino acid sequences were determined, and a cDNA clone for the protein was isolated from a mouse T-cell cDNA library using long degenerate oligonucleotide probes deduced from the amino acid sequence. The complete nucleotide sequence of the cDNA (1.7 kilobases) was determined. Northern hybridization using this cDNA detected two transcripts: 1.8kb being the major one and 2.6 kb being the minor one. The complete amino acid sequence for the protein predicted from the nucleotide sequence of the cDNA indicates that the 44-kDa protein consists of 394 amino acids with a calculated molecular weight of 43,698. In tests performed thus far, the recombinant 44-kDa protein expressed in Escherichia coli has not expressed any repair-support activity. It remains to be analyzed whether the protein attains this activity after appropriate posttranslational modifications. Most parts of the 44-kDa protein cDNA and the deduced amino acid sequence were found to be identical to those of the protein p38 -2G4, recently reported as a cell cycle-specifically modulated nuclear protein of 38kDa. The p38-2G4 may be a truncated form of the present 44-kDa protein.