Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphism analysis of polymerase chain reaction products.

Single-strand conformation polymorphism analysis of polymerase chain reaction products (PCR-SSCP analysis) was used for detection of mutations of the p53 gene in surgical specimens of human brain tumors. Six of 45 brain tumors showed mobility shifts in the analyses. These six tumors also showed loss of a normal allele. The samples were examined further by direct sequencing. Results showed that four of them had single-base substitutions and the other two had deletions of one and eight base pairs. Five of the six mutations detected were clustered in highly conserved regions of the p53 gene. The frequency of p53 gene mutations in primary brain tumors examined was 9.8%. We also found two new polymorphic markers in the p53 gene, one in intron 7 and the other in an Alu repeat in exon 11. Both markers could be detected by SSCP analysis. Using these two markers, we found two cases of loss of heterozygosity in other brain tumor specimens. Results suggested that aberrations of the p53 gene were not correlated with the malignancy of some types of brain tumors such as anaplastic astrocytoma and glioblastoma, contrary to previous observations on colorectal cancers.

Microsatellite instability and the PTEN1 gene mutation in a subset of early onset gliomas carrying germline mutation or promoter methylation of the hMLH1 gene.

High-frequent microsatellite instability (MSI-H) was detected in two of the 80 gliomas examined, whlie the other 78 gliomas showed microsatellite stable (MSS) phenotype. Both of the two MSI-H tumors were glioblastomas which developed in teenage patients. One of the patient was diagnosed as having Turcot's syndrome and had a germline mutation in the hMLH1 gene. Loss of expression due to promoter methylation was selectively observed in the wild type allele of the hMLH1 gene in the tumor of this patient. The other patient had neither a family history nor a past personal history of malignancy. Although no mutation in the mismatch repair genes was detected in the tumor of this patient, the level of expression of the hMLH1 gene was markedly decreased and the promoter sequence of the gene was highly methylated. In the tumor of this patient, the PTEN1 gene, one of the genes carrying microsatellite sequences in their coding regions, was altered by a slippage mutation within five adenine repeat sequences. These findings indicate that the genetic or epigenentic inactivation of the hMLH1 gene is involved in a subset of early-onset gliomas and the PTEN1 gene could be a downstream target for mutation as observed in glioblastoma without MSI.

Cloning and characterization of rat cellular nucleic acid binding protein (CNBP) cDNA.

We cloned and sequenced the cDNAs which code for rat cellular nucleic acid binding protein (CNBP). In-frame insertion/deletion differences were found among the clones at two sites in the open reading frame, suggesting alternative splicing of the message or the presence of multiple genes which code for this protein. The deduced amino acid sequence revealed that one rat CNBP sequence was completely identical to its human counterpart. This striking conservation, together with the fact that homologous genes have been found in various organisms including Schizosaccharomyces pombe, suggests that CNBP plays a basic biological role in eukaryotic cells. The recombinant GST-CNBP fusion protein produced in Escherichia coli bound to a G-rich single-stranded RNA and DNA in a sequence-specific manner.

A simpler, more robust method for the analysis of 8-oxoguanine in DNA.

The oxidized DNA base 8-oxoguanine has been commonly measured by enzymatic digestion of DNA to nucleosides followed by high-performance liquid chromatography (HPLC) separation of the adduct 8-oxodeoxyguanosine. There has recently been an enormous debate surrounding the validity of this approach, from which it has become clear that artifactual oxidation of the native base to 8-oxoguanine can occur at numerous stages in sample preparation. Hence, we have designed an alternative protocol to traditional enzymatic digestion of DNA which (i) limits the potential for artifactual oxidation, (ii) speeds up the assay markedly, (iii) increases the assay's sensitivity moderately, and (iv) addresses criticisms that have been raised concerning the efficiency of DNA digestion by nucleases. In short, we use the Escherichia coli repair enzyme formamidopyrimidine (Fapy) glycosylase to release the base 8-oxoguanine from full-length DNA, then separate 8-oxoguanine from high molecular weight molecules by ultrafiltration (10,000 Da exclusion) and analyze the base adduct by reverse-phase HPLC. Benefits of this approach include (i) rapid removal of the roughly million-fold molar excess of unaltered bases from the sample, (ii) reduction in the length of enzymatic incubations and the number of steps, (iii) elimination of high temperature incubation, (iv) a very clean chromatographic separation, and (v) rapid elution of the analyte and correspondingly greater throughput. Using this improved method, we have followed the induction of 8-oxoguanine in the DNA of peroxide-treated HeLa cells, an experiment that had proved cumbersome with traditional methods.

The effect of folic acid deficiency and MTHFR C677T polymorphism on chromosome damage in human lymphocytes in vitro.

We performed a comprehensive study on the genotoxic and cytotoxic effects of in vitro folic acid deficiency on primary human lymphocytes. Lymphocytes were cultured in medium containing 12-120 nM folic acid for 9 days in a novel cytokinesis-block micronucleus (CBMN) assay system (n = 20). Besides identifying optimal folic acid concentrations for in vitro genomic stability, we tested the hypothesis that lymphocytes from individuals homozygous for the C677T methylenetetrahydrofolate reductase (MTHFR) polymorphism (TTs, n = 10) are protected against chromosome damage relative to controls (CCs, n = 10) under conditions of folic acid deficiency. This hypothesis is based on the assumption that reduced MTHFR activity in TT lymphocytes causes a diversion of 5,10-methylene tetrahydrofolate toward thymidine synthesis, which minimizes uracil-induced double-stranded DNA breakage. Cells were scored for micronuclei, apoptosis, necrosis, nucleoplasmic bridges, and nuclear budding. The latter two endpoints are indicative of chromosome rearrangements and gene amplification, respectively, and to the best of our knowledge, this is the first report of their association with folic acid concentration. Folic acid concentration correlated significantly (P < 0.0001) and negatively (r, -0.63 to -0.74) with all markers of chromosome damage, which were minimized at 60-120 nM folic acid, much greater than concentrations assumed "normal," but not necessarily optimal in plasma. Two-way ANOVA revealed no effect of the MTHFR genotype on any of the endpoints. Results show that the C677T polymorphism does not affect the ability of a cell to resist chromosome damage induced by folic acid deficiency in this in vitro system.

The Healthy Neighborhoods Project: a local health department's role in catalyzing community development.

Studies show that community development approaches to health education may lead not only to improved social, economic, and health status but also to increased individual participation in health education and preventive health care activities. However, because of categorical funding restraints and philosophical issues, local health departments have rarely given control of defining project outcomes to the community. One such project was in a low-income urban neighborhood in the San Francisco Bay Area. In this Healthy Neighborhoods Project, the health department catalyzed community development and organization in a multiethnic public housing complex. As a result, an empowered community successfully advocated to improve public safety by installing street speed humps and increased street lighting. After project completion, residents initiated several additional health actions, including the removal of a neighborhood tobacco billboard. This article describes the project, which may serve as a model for other urban public health programs to explore their role in community empowerment.

[Enhancement of the effect of X-irradiation against cultured human glioblastoma cells by pretreatment with ACNU].

Human glioblastoma A-7 (GB A-7) cells can apparently recover from potentially lethal X-irradiation. The authors, using a colony-forming assay, studied the influence of pretreatment with 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) on the effectiveness of X-irradiation against GB A-7 cells grown in monolayers and as multicellular spheroids. Pre-exposure to ACNU inhibited the recovery of irradiated GB A-7 cells. In monolayer cells, the combination treatment was most effective when ACNU was applied 2 to 8 hours prior to irradiation, and the larger the X-ray dose, the more potent the effect. ACNU pretreatment was more effective against large spheroids (enhancement ratio 1.86) than against small ones (1.34). Large spheroids showed necrosis, whereas small ones did not. Isobolographic analysis disclosed that the effect of combining X-irradiation and ACNU is within an additive envelope at the surviving fraction of 10(-2), while supra-additive at the surviving fraction of 10(-3). These results suggest that the potency of X-irradiation is augmented by ACNU pretreatment through an interactive mechanism. Further, suppression of recovery from X-ray induced potentially lethal damage was influenced by the presence of necrosis.

Effects of X-irradiation alone and in combination with ACNU on human glioblastoma cells in vitro.

The combined effects of x-irradiation and 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloro-ethyl)-3-nitrosourea (ACNU) on multicellular glioblastoma A-7 spheroids were analyzed by means of cell survival and dose-response curves. The actual dose-response curve for small spheroids was almost identical to that estimated from the cell survival curve. It was strongly suggested that a small number of radiation-resistant cells, which were not detected in the cell survival curve, were present in large spheroids with central necrosis. The enhancing effect of ACNU was greater with large spheroids than with monolayer cells or small spheroids. A possible explanation for this is that ACNU is higher effective against the few radiation-resistant cells that may be present in larger spheroids.

[Proteus mirabilis brain abscess in a neonate].

A case of neonatal brain abscess was reported. This female infant was born by spontaneous vertex delivery at 38 weeks gestation, weighing 3.1 kg. There were no antenatal and perinatal complications. The Apgar's score was 8 points and her head was 32.8 cm in circumference. Fifty-six hours after birth, she had a clonic general convulsion for about 3 minutes. On the next morning, convulsion recurred, and she was transferred to our hospital. When admitted to the Pediatrics, she was exhausted and irritable. But after hospitalization, she improved without convulsions. CT scan, performed 7 days after birth, revealed large low density areas in both frontal lobes. Electroencephalography showed spike discharge at all leads. The head circumference gradually increased and subsequent CT scans revealed enlargement of the bifrontal low density areas. The first operation was done 28 days after birth. A large amount of yellowish grey pus was aspirated from both sides and the cavity was irrigated. Tobramycin was administered into the abscess cavity. Culture of the pus grew proteus mirabilis. The postoperative conditions were good. But two weeks later her head started to enlarge with the bulging fontanelle. CT scan revealed marked dilation of the ventricles and enlargement of the left frontal abscess. At 2 months of age, second operation was done. Left frontal abscess was punctured and serous pus was aspirated. Proteus mirabilis was cultured again from the pus. Tobramycin was administered into the cavity via the drainage tube every day for a week. As the content of the abscess cavity became clear, V-P shunt and cyst-peritoneal shunt were performed. After shunting procedure, the increase of head circumference stopped and the fontanelle became flat and soft.(ABSTRACT TRUNCATED AT 250 WORDS)

[Follow-up of primary pontine hemorrhage of the young adult by CT scan].

A case of primary pontine hemorrhage of the young was reported. He was 22 years of age, who did not have the history of hypertension. He suddenly noticed disturbance of skilled act of his right hand and speech disturbance. He was admitted to our clinic two days after the onset. Neurological examination revealed right facial palsy, disturbance of delicate movement of his right hand and slight dysarthria. Lumbar puncture, after six days from the onset, yielded watery clear cerebrospinal fluid containing 6/3 cells/mm3, 27 mg/dl of protein and 65.1 mg/dl of sugar. An opening pressure was 75 mmH2O. The vertebral angiography revealed no evidence of mass lesion and vascular anomalies. CT scan demonstrated a pontine hematoma, the size of this hematoma sequentially enlarged in follow-up CT scan. His clinical symptoms were gradually aggravated, i.e. cerebellar sign, urinary disturbance and involuntary movement. Conservative therapy was performed and his symptoms gradually improved. The size of hematoma was reduced in follow-up CT scan. The genesis, diagnosis and treatment of primary pontine hemorrhage of the young were discussed.

[Agenesis of the corpus callosum associated with interhemispheric cyst. A case report].

A 7-year-old boy was admitted because of convulsive seizures. He was diagnosed as agenesis of corpus callosum associated with interhemispheric cyst by CT scan and angiography. Metrizamide CT cisternography and cystography disclosed no communication between interhemispheric cyst and the third ventricle. By means of metrizamide CT cisternography and cystography, the authors considered this interhemispheric cyst gradually increased by some kinds of ball-valve mechanism. Cysto-peritoneal shunt was carried out. Postoperative course was uneventful and follow-up CT revealed disappearance of interhemispheric cyst and typical findings of the acallosal brain. Improvement of clinical symptoms and EEG findings occurred after operation. Agenesis of the corpus callosum associated with interhemispheric cyst is rare. The authors can find only one case of this brain anomaly in the literature in which there is no communication between interhemispheric cyst and the third ventricle. Metrizamide CT cisternography and cystography was very useful in diagnosis and selection of treatment.

[A case of glioblastoma in which early diagnosis was difficult by MRI].

The authors report a case of glioblastoma in which MR images with Gd-DTPA enhancement changed rapidly during the early stage. A 61 year-old male presented with sudden right facial spasm and dysarthria. However, both a plain and an enhanced CT failed to demonstrate any abnormal lesions. On the other hand, T2 weighted MR image revealed a well circumscribed high intensity lesion in the left frontal lobe without mass effect. This lesion could not be differentiated from cerebral infarction, since no contrast enhanced lesion was able to be observed in T1 weighted MR image with Gd-DTPA. His symptoms gradually became aggravated and at 3 months from the onset, MR image with Gd-DTPA disclosed a small enhanced lesion in the left frontal lobe near the cortical surface. After 6 months from the onset, he suffered from right hemiparesis and motor aphasia. The MR image with Gd-DTPA at this time showed a large enhanced lesion in the left frontal lobe with mass effect. He was admitted to our hospital, and subtotal removal of the tumor and intraoperative radiation was carried out. The patient did well postoperatively without additional neurological deficit, and then he received additional radiation therapy. It should be noted that Gd-DTPA enhanced MR image might fail to reveal the lesion of glioblastoma in its early stage, while T1 weighted image discloses only the gyral swelling.

[Multiple meningiomas with Werner's syndrome--a case report].

A case of multiple meningiomas with Werner's syndrome is reported. He was 45-year-old man, who complained change of character and slight right motor weakness for 4 months. His symptoms were characteristic small stature, premature senility, scleroderma-like changes and other manifestations. Glucose tolerance test showed a diabetic pattern. CT scan and cerebral angiography revealed multiple meningiomas. After chemical embolization of the feeding artery, we removed these meningiomas successfully. We also reviewed the literature on Werner's syndrome with meningioma.

[A case of intracranial osteochondroma originating with psychomotor epilepsy].

A 28-year-old woman was admitted to our clinic due to psychomotor epilepsy. Craniogram, CT scan demonstrated a calcified lesion in the frontal, parasagittal region. MR proton images showed a mixed hyper-and hypodense lesion. The tumor originated from the frontal bone and was totally removed using airdrill safely. Histological findings revealed osteochondroma originating in the frontal bone is rare, and diagnostic procedures and surgical management of this tumor are discussed.

[Multiple brain tumors with von Recklinghausen's disease].

Thirty-three cases of von Recklinghausen's disease, nine of which had multiple brain tumors, were experienced in our clinic. In this report, these nine cases were reported and discussed, especially representative two cases in detail. Nine cases had various combinations of multiple brain tumors: bilateral acoustic neurinomas in two cases, multiple neurinomas in two cases, multiple meningiomas in one case and multiple neurinomas and meningiomas in two cases. In two cases, multiple brain tumors were totally extirpated at one stage, but both died within ten months. In three cases, partial extirpation of multiple brain tumors were performed at one stage, one died thirteen years after the operation and two are alive but dependent on their families. In recent two cases, two stage operations were performed, by which tumors were successfully removed totally. These postoperative courses were good. The other two cases were treated conservatively. One is alive and working, but has severe auditory disturbance. The other died after six years from onset. Autopsy was performed in this case, which revealed multiple neurinomas in the left 3rd and bilateral 5-12th cranial nerves and numerous meningiomas. In the review of the cases with more than three brain tumors in von Recklinghausen's disease were discussed.

[Cerebral cysticercosis: a case report with special reference to CT and MRI findings].

A case of cerebral cysticercosis was reported with special reference to CT and MRI findings. A 51-year-old man, who had traveled to Taiwan in 1984, was admitted to the hospital for further examination concerning right hemiconvulsion, on January 23, 1988. On admission, a slightly euphoric state and Foster Kennedy syndrome (left: optic atrophy, right: choked disc) were observed. CT scan revealed multiple cystic lesions with ring-like enhancement around basal cisterns. MRI showed intensity of cyst contents parallel to CSF (on T1 and T2 weighted images) and high intensity rim around the cysts (especially on T2 weighted image). An operation was performed and the cysts were almost totally removed. The cyst was confirmed pathologically as cysticercosis, but no scolex was found in it. Postoperative immunological study on the serum and CSF was negative. In the diagnosis of atypical cystic lesions around basal cisterns, cerebral cysticercosis, rare in Japan, should also be considered, and it was emphasized that MRI was very useful for the diagnosis of such cysts.

[Results in oligodendroglioma: postoperative radiotherapy combined with chemotherapy].

The retrospective analysis of 39 cases of supratentorial oligodendroglioma included the years 1965 - 84. The 5-year and 10-year survival rates were 73.1% and 32.2%, respectively. Although oligodendrogliomas were benign tumor, the 10-year survival rate was not good. Recurrence of tumor appeared in 9 cases and some cases were noticed about 10 years after the treatment. So, long term follow-up is important for oligodendrogliomas. Our treatment for oligodendrogliomas was surgical removal and postoperative radiochemotherapy (radiation + ACNU + FT-207 + PSK) for several years. The previous cases who had only subtotal removal of tumor showed short survival time. On the other hand, the cases treated with postoperative radiotherapy or radiochemotherapy showed relatively good results.

[Effect of X-rays combined with ACNU and O6-ethylguanine on rat subcutaneous gliomas].

Rat gliomas of subcutaneously transplanted RGc-6 cells were irradiated with X-ray either alone, or combined with ACNU, and the cell-survival was assayed in vitro. Cell-survival curve composed of two components by X-irradiation alone indicated the presence of a hypoxic cell fraction. We have previously shown that combined treatment with ACNU apparently made the effect of X-ray on spheroids in vitro of the same cell line of RGc-6 more powerful. Although treatment of rat gliomas with ACNU administered at 2 hrs prior to X-irradiation was most effective, it resulted in only the additive effect of the independent action of the two agents. Further treatment by O6-ethylguanine prior to ACNU administration and X-irradiation apparently increased the strength of the effect of ACNU combined with X-ray to the dose-modifying factor for X-ray of 1.8. The result indicated that the combination of O6-ethylguanine prior to ACNU administration and X-irradiation may clinically enhance the effect of X-ray against apparent ACNU-resistant glioma cells such as RGc-6 cells.

[Chronic subdural hematoma associated with malignancy: report of three cases].

Three cases of chronic subdural hematoma (CSH) associated with malignancy are reported. Case 1; A one-year-old girl was referred for vomiting and convulsions. Left CSH was removed, and her symptoms disappeared. Cytological examination of chronic subdural hematoma revealed abnormal white blood cells. A clinical diagnosis of acute monocytic leukemia was made after the laboratory examination. Remission was achieved by chemotherapy, but she died one year after the operation. Case 2; A 72-year-old woman was referred for right hemiparesis and urinary incontinence. Left CSH was irrigated, and her clinical symptoms immediately disappeared. Cytological examination of chronic subdural hematoma revealed abnormal white blood cells. A clinical diagnosis of chronic lymphocytic leukemia was made after the laboratory examination. No treatment was given since there were no clinical symptoms of chronic lymphocytic leukemia. Case 3; A 70-year-old woman who had been affected with early gastric cancer and mammary cancer for the previous two years was admitted to our clinic because of headache, right hemiparesis and consciousness disturbance. Left CSH was irrigated, and her clinical symptoms improved. However, there was a tendency to bleed because disseminated intravascular coagulation had occurred, and CT showed bilateral subdural hematoma. A second irrigation was performed, but her symptoms did not improve. Left acute subdural hematoma, which was removed by craniotomy, occurred three days after the second operation. Pathological examination of the outer membrane of the subdural hematoma revealed invasion of adenocarcinoma. She died three days after the third operation. It is recommended that both the cytological and the histological examinations be performed when possible, since they are simple to perform and very useful in some cases.

[Clinical analysis of vitreous hemorrhage combined with ruptured aneurysm].

Eight cases of vitreous hemorrhage with ruptured intracranial aneurysm (Terson's syndrome) were experienced in our clinic from April, 1978 to April, 1983. They consisted of five males and three famales between 45 and 69 years old. All cases had unconscious attacks and three of them experienced repeated episodes of subarachnoid hemorrhage within 24 hours. CT scan, performed 24 hours within the attack, revealed remarkable high density areas at prepontine and suprasellar cisterns, interhemispheric and sylvian fissures. Ruptured aneurysms, confirmed by cerebral angiography and operative findings, were three anterior communicating artery aneurysms, three internal cerebral artery aneurysms, one middle cerebral artery aneurysm and one anterior cerebral artery aneurysm. We performed radical operation for the ruptured aneurysm, six cases at acute atage, one at 11 days and one at 18 days after the attack. Postoperative courses were all good. Five cases had bilateral vitreous hemorrhages and three had unilateral ones. At first they were treated conservatively. Visual acuity of six cases, 13 eyes improved gradually, but three cases, four eyes did not improve after four or five months after the attack. So we let ophthalmologist to perform vitrectomy, which showed effective results. Vitreous hemorrhage following a ruptured intracranial aneurysm is not a so rare complication than has been assumed and also its prognosis is not so poor. In most cases vitreous hemorrhage following a ruptured intracranial aneurysm should be treated conservatively, but in selected cases effective results would be given by vitrectomy.