Nestin expression in central nervous system germ cell tumors.

Central nervous system (CNS) germ cell tumors constitute a unique class of rare tumors that mainly affect children and adolescents. These tumors are believed to originate from displaced primordial germ cells. Recently, results of treatment of germ cell tumors have improved with use of radiotherapy and combination chemotherapy. However, some tumors have proven refractory to intensive treatment with surgery, radiation, and combination chemotherapy. Nestin is an intermediate filament protein expressed in undifferentiated cells during CNS development and in CNS tumors and is used as a marker of immature elements of tumors, including brain tumor stem cells. In this study, we examined for the first time nestin expression in 19 CNS germ cell tumors (nine pure germinomas, five germinomas with syncytiotrophoblastic giant cells, one yolk sac tumor, one choriocarcinoma, one embryonal carcinoma, and two mature teratomas). Nestin was expressed in 14 cases but was not expressed in three pure germinomas and two mature teratomas. Clinically, nestin-negative tumors did not exhibit dissemination, while all tumors that exhibited dissemination also strongly expressed nestin protein. These findings suggest that the detection of nestin expression could be useful in the management of CNS germ cell tumors, as an auxiliary predictor of dissemination and/or progression.

Surgical resection of tumors located in subcortex of language area.

OBJECT: Although functional mapping facilitates the planning of surgery in and around eloquent areas, the resection of tumors adjacent to language areas remains challenging. In this report, we took notice that the language areas (Broca's and Wernicke's) present at the perisylvian fissure. We posit that if there is non-essential language area on the inner surface of the Sylvian fissure, safe tumor resection may be possible even if the tumor is located under the language cortex. METHODS: The study population consisted of 5 patients with intrinsic brain tumors (frontal glioma, n = 3; temporal cavernous angioma, n = 1; primary malignant central nervous system lymphoma, n = 1) located in the perisylvian subcortex, in the language-dominant hemisphere. All patients underwent awake surgery and we performed intra-operative bipolar cortical functional language mapping. When the tumor was located under the language area, the Sylvian fissure was opened and the inner surface of the opercular cortex was exposed with the patient asleep, and additional functional mapping of that cortex was performed. This enabled us to remove the tumor from the non-functioning cortex. In our series, 4 of 5 patients had not language function on the inner surface of the operculum. Only one patient, a 52-year-old man with frontal glioblastoma (Case 3) had language function on the inner surface of the frontal operculum. CONCLUSION: We suggest that even perisylvian tumors located in the subcortex of the language area may be resectable via the nonfunctioning intrasylvian cortex by a transopercular approach without resultant language dysfunction.

Age-related changes in haematology and serum chemistry of Weiser-Maples guineapigs (Cavia porcellus).

Age-related changes in haematology and serum chemistry values were examined in male and female Weiser-Maples guineapigs (Cavia porcellus). Haematological changes that significantly (P<0.01) correlated with ageing were increased white blood cell and neutrophil counts in both sexes, decreased lymphocyte counts in both sexes, decreased reticulocyte and platelet counts in males, and decreased basophil counts in females. For serum chemistry, increases in total protein, triglycerides, blood urea nitrogen and creatinine were seen in both sexes, along with increases in total cholesterol in males and sodium in females. Decreased alkaline phosphatase in both sexes and decreased chloride in males were significantly (P<0.01) associated with age. These age-related changes are compared with the published literature.

Multimodal strategy for managing meningiomas in the elderly.

BACKGROUND: The incidence of brain tumors in elderly patients is increasing. It has become possible to treat meningiomas in the elderly by several modalities. We developed a successful multimodal strategy to treat these patients. METHODS: We registered 35 patients with meningiomas. Symptomatic meningiomas were treated surgically at the time of diagnosis (n=19). Of the 16 asymptomatic meningiomas, 5 were removed at the time of diagnosis. The other asymptomatic meningiomas (n=11) were treated conservatively and when the tumors increased in size, surgical treatment was considered. "Operated" patients with residual or recurrent tumors underwent radiosurgery with a gamma knife. FINDINGS: Surgical mortality and morbidity were 4% and 16%, respectively. Of the 25 "operated" patients, 21 (84.0%) had a good Karnofsky scale (> or =80%) at discharge. In all but two of the 11 patients with asymptomatic, conservatively treated meningiomas, the tumors did not increase during the follow-up period. Gamma knife radiosurgery, performed to treat 3 residual and 1 recurrent tumor, resulted in very good tumor control and none of the tumors increased after gamma knife surgery. CONCLUSIONS: Meningiomas in elderly patients require a multimodal approach. Our strategy, which includes surgery, radiosurgery, and conservative treatment, resulted in good tumor control and made it possible for patients to pursue their activities of daily life.

Physical and functional interaction between BH3-only protein Hrk and mitochondrial pore-forming protein p32.

Bcl-2 homology domain (BH) 3-only proteins of the proapoptotic Bcl-2 subfamily play a key role as initiators of mitochondria-dependent apoptosis. To date, at least 10 mammalian BH3-only proteins have been identified, and it is now being realized that they have different roles and mechanisms of regulation in the transduction of apoptotic signals to mitochondria. Hrk/DP5 is one of the mammalian BH3-only proteins implicated in a variety of physiological and pathological apoptosis, yet the molecular mechanism involved in Hrk-mediated apoptosis remains poorly understood. In an attempt to identify cellular proteins participating in Hrk-mediated apoptosis, we have conducted yeast two-hybrid screening for Hrk-interacting proteins and isolated p32, a mitochondrial protein that has been shown to form a channel consisting of its homotrimer. In vitro binding, co-immunoprecipitation, as well as immunocytochemical analyses verified specific interaction and colocalization of Hrk and p32, both of which depended on the presence of the highly conserved C-terminal region of p32. Importantly, Hrk-induced apoptosis was suppressed by the expression of p32 mutants lacking the N-terminal mitochondrial signal sequence (p32(74-282)) and the conserved C-terminal region (p32 (1-221)), which are expected to inhibit binding of Hrk competitively to the endogenous p32 protein and to disrupt the channel function of p32, respectively. Furthermore, small interfering RNA-mediated knockdown of p32 conferred protection against Hrk-induced apoptosis. Altogether, these results suggest that p32 may be a key molecule that links Hrk to mitochondria and is critically involved in the regulation of Hrk-mediated apoptosis.

Thiol-oxidizing agent diamide and acidic pH enhance lipid peroxidation of rat heart mitochondria and cardiolipin-cytochrome c complex.

In this study, we investigated lipid peroxidation in rat heart mitochondria hydrolyzed by phospholipase A2 (PLA2) and lipid peroxidation in a mitochondrial-mimetic lipid peroxidation system, where phospholipids such as cardiolipin (CL) and cytochrome c (Cyt c) were first mixed together and then PLA2 and calcium chloride were added to the mixture (CL-Cyt c-PLA2 system). Production of hydroperoxy and hydroxy compounds of linoleic acid (LA) in the mixture was measured by high performance liquid chromatography. The ratio of the total amount of hydroperoxy and hydroxy compounds of LA to that of LA was calculated as an index for lipid peroxidation (1000 x mol/mol). The index for lipid peroxidation in the rat heart mitochondria hydrolyzed by PLA2 at the physiological pH of 7.4 was 22.8 +/- 2.2 (mean +/- SD, n = 4) and that at the acidic pH of 6.7 was 41.8 +/- 2.0. In the presence of the thiol (SH)-oxidizing agent diamide, the index was 47.0 +/- 2.6 (pH 7.4). In the CL-Cyt c-PLA2 system, lipid peroxidation seemed to be due to three mechanisms: (1) oxidation of the LA (nonreleased form) constituent of CL by Cyt c (oxidation of CL by Cyt c); (2) oxidation of free LA, released from CL, involving the oxidation of CL by Cyt c (free LA oxidation by the CL-Cyt c complex); and (3) oxidation of free LA, released from CL, by Cyt c and calcium ions (LA-Cyt c-Ca system). The lipid peroxidation of the CL-Cyt c-PLA2 system was also enhanced by the addition of diamide and by an acidic pH of 6.7. The fact that the SH-oxidizing agent enhanced the lipid peroxidation in the CL-Cyt c-PLA2 system suggested that SH groups in the hemoprotein played an inhibitory role in lipid peroxidation in the system.

Involvement of protein kinase C-epsilon in signal transduction of thrombopoietin in enhancement of interleukin-3-dependent proliferation of primitive hematopoietic progenitors.

We studied the effect of thrombopoietin (TPO) on interleukin-3 (IL-3)-dependent bone marrow cell colony formation of mice to clarify the role of protein kinase C (PKC) in the signal transduction of TPO for the proliferation of primitive hematopoietic progenitors. TPO alone hardly yielded colonies. However, TPO in combination with IL-3 increased colony numbers synergistically from 2- to 4-fold, compared with those supported by IL-3 alone. Serial observation of colony development showed that TPO may hasten the appearance of colonies by shortening the dormant period (G(0)) of primitive progenitors. Immunocytochemical studies on PKC isoforms in progenitor cells stimulated with TPO have revealed that the expression pattern of PKC-epsilon is changed, but not that of PKC-alpha, -beta, -gamma, -delta, or -zeta. Selective PKC inhibitors, such as calphostin C and GF 109203X, and PKC-epsilon-specific translocation inhibitor peptide abrogated the enhancing effect of TPO on IL-3-dependent colony formation and the changes in the intracellular expression pattern of PKC-epsilon. These data taken together suggest that TPO has a direct effect on primitive progenitors and enhances IL-3-dependent colony formation, at least partly through the activation of PKC-epsilon.

Diagnosis of cyanide intoxication by measurement of cytochrome c oxidase activity.

Cytochrome c oxidase (CCO), a mitochondrial enzyme, is inactivated by cyanide or carbon monoxide (CO) intoxication. We measured CCO activity, in the major organs of the rat at various times after death caused by cyanide intoxication. Tissue samples were homogenized, and the CCO activity in the mitochondrial fraction was measured using ferrous cytochrome c as the substrate. The CCO activity inhibition was highest in the brain, although the cyanide concentration was lowest level. As a result of this and the clinical symptoms displayed, we consider the brain to be the primary organ of cyanide intoxication. As cyanide is highly toxic to humans, in small amounts and many patients and victims have already had some medical care, it is difficult to detect cyanide in criminal investigations. The CCO activities in various organs remained significantly low for 2 days after the cyanide intoxication, suggesting that the diagnosis may be possible by measuring not only the cyanide concentration but also the CCO activity.

Development of rigor mortis is not affected by muscle volume.

There is a hypothesis suggesting that rigor mortis progresses more rapidly in small muscles than in large muscles. We measured rigor mortis as tension determined isometrically in rat musculus erector spinae that had been cut into muscle bundles of various volumes. The muscle volume did not influence either the progress or the resolution of rigor mortis, which contradicts the hypothesis. Differences in pre-rigor load on the muscles influenced the onset and resolution of rigor mortis in a few pairs of samples, but did not influence the time taken for rigor mortis to reach its full extent after death. Moreover, the progress of rigor mortis in this muscle was biphasic; this may reflect the early rigor of red muscle fibres and the late rigor of white muscle fibres.

Rho-associated kinase phosphorylates MARCKS in human neuronal cells.

Myristoylated alanine-rich C kinase substrate (MARCKS) is a filamentous actin bundling protein and has multiple sites for phosphorylation, by which the biochemical function is negatively regulated. However, the role of such phosphorylation in physiological functions, particularly in neuronal functions, is not well understood. Using a phosphorylation-site specific antibody, we detected the phosphorylation of MARCKS at Ser159 by various protein kinases. Rho-kinase, protein kinase A, and protein kinase C, could introduce (32)P into human recombinant MARCKS in vitro and the phosphorylation site was confirmed to be the Ser159 residue. In human neuronal teratoma (NT-2) cells, lysophosphatidic acid (LPA) induced MARCKS phosphorylation dose- and time-dependently. This phosphorylation was sensitive to Rho-kinase inhibitor HA1077. However, the phosphorylation induced by PDBu was lesser sensitive. In a skinned NTera-2 cell system, Ca(2+)-independent and GTP gamma S/ATP-stimulated phosphorylation at Ser159 was also sensitive to pre-treatment C3 toxin and HA1077. These findings suggest that the Ser159 residue of MARCKS is a target of LPA-stimulated Rho-kinase in neuronal cells.

RIZ, the retinoblastoma protein interacting zinc finger gene, is mutated in genetically unstable cancers of the pancreas, stomach, and colorectum.

The retinoblastoma protein interacting zinc finger (RIZ) gene is a candidate tumor suppressor gene on 1p36, a region frequently rearranged in a wide variety of human tumors. As the RIZ gene harbors several microsatellites within its coding region, it is a candidate for an inactivating mutation in microsatellite instability (MSI) mediated carcinogenesis. In this study, we examined mutations of two poly adenine tracts, A(8) and A(9), within the coding region of the RIZ gene, in MSI-high (MSI-H) primary cancers occurring in the pancreas, stomach, and colorectum. Frameshift mutations were found in one (10%) of 10 pancreatic, four (36%) of 11 gastric, and two (25%) of eight colorectal cancers. These results indicate that mutations of the RIZ gene play an important role in the pathogenesis of some MSI-H cancers.

[A case of giant skull myofibroma occupying left anterior cranial fossa].

We report a rare case of giant skull myofibroma occupying left anterior cranial fossa. A 53-year-old woman presented with left exophthalmos for 2 years. Neurological examination showed left exophthalmos, disturbance of bilateral visual acuity, and bitemporal hemianopsia. A CT scan revealed an ossifing mass at left anterior cranial fossa. On magnetic resonance images, the tumor showed iso-intensity on T 1-weighted image, heterogeneous high intensity on T 2-weighted image, and was heterogeneously well-enhanced after administration of Gd-DTPA. The tumor was fed mainly by middle meningeal artery. The patient underwent surgery and the tumor was removed totally. Histological diagnosis of the tumor was myofibroma. The patient has been followed every other month by MRI without any adjuvant therapy. There has been no tumor recurrence for 19 months. There is no other myofibroma in her body, therefore the patient was diagnosed as solitary myofibroma of the skull. Our case is the first report of solitary myofibroma of the skull because we could not find any reports on solitary myofibroma of the skull in the past literature.

Structure, expression and mutational analysis of the hBRAG gene on 10q in the frequently deleted region in human endometrial cancer.

We previously reported that chromosome arm 10q is one of the target regions of allelic loss in human endometrial cancer. To identify the gene in this region responsible for endometrial cancer, we further characterized this region and localized the hBRAG gene. The function of hBRAG has not yet been fully studied, and there is the possibility that this gene works as a tumor suppressor. This gene consist of 7 exons and 6 introns encoding 503 amino acids; all the introns start with GT and end with AG in agreement with the GT-AG rule. Expression of this gene was studied by Northern hybridization and suppressed expression was observed in one (SK-UT-1B) of the six endometrial cancer cell lines. Mutation analysis in 38 primary EC tissues and six EC cell lines disclosed no genetic alterations. The genomic structure of hBRAG elucidated in this study should contribute to the future analysis of the hBRAG gene.

Onset of rigor mortis is earlier in red muscle than in white muscle.

Rigor mortis is thought to be related to falling ATP levels in muscles postmortem. We measured rigor mortis as tension determined isometrically in three rat leg muscles in liquid paraffin kept at 37 degrees C or 25 degrees C--two red muscles, red gastrocnemius (RG) and soleus (SO) and one white muscle, white gastrocnemius (WG). Onset, half and full rigor mortis occurred earlier in RG and SO than in WG both at 37 degrees C and at 25 degrees C even though RG and WG were portions of the same muscle. This suggests that rigor mortis directly reflects the postmortem intramuscular ATP level, which decreases more rapidly in red muscle than in white muscle after death. Rigor mortis was more retarded at 25 degrees C than at 37 degrees C in each type of muscle.

cis-9,10-Methylenehexadecanoic acid inhibits contractility and actomyosin ATPase activity of guinea pig myocardium.

Superfusion with a cyclopropane fatty acid, cis-9, 10-methylenehexadecanoic acid (10-300 microM), reduced the contractility of papillary muscle isolated from guinea pigs in a dose-dependent manner. cis-9,10-Methylenehexadecanoic acid also inhibited the Mg(2+)-ATPase activity of guinea pig papillary myocardium by about 40% at 400 microM. Since cis-9, 10-methylenehexadecanoic acid 4 microM inhibited the K(+)-EDTA-ATPase activity inherent in myosin's catalytic activity by about 25%, the fatty acid was thought to interact with the catalytic center of the myosin molecule.

[Radiation induced glioblastoma: a case report].

We report a surgical case of a 54-year-old woman with a radiation induced glioblastoma. At the age of 34, the patient was diagnosed to have a non-functioning pituitary adenoma. It was partially removed followed by 50 Gy focal irradiation with a 5 x 5 cm lateral opposed field. Twenty years later, she suffered from rapidly increasing symptoms such as aphasia and right hemiparesis. MRI showed a large mass lesion in the left temporal lobe as well as small mass lesions in the brain stem and the right medial temporal lobe. These lesions situated within the irradiated field. Magnetic resonance spectroscopy revealed relatively high lactate signal and decreased N-acetyl aspartate, choline, creatine and phosphocreatine signals. Increased lactate signal meant anaerobic metabolism that suggested the existence of a rapidly growing malignant tumor. Thus, we planned surgical removal of the left temporal lesion with the diagnosis of a radiation induced malignant glioma. The histological examination revealed a glioblastoma with radiation necrosis. MIB-1 staining index was 65%. Postoperatively, her symptoms improved, but she died from pneumonia 1 month after the surgery. An autopsy was obtained. The lesion of the left temporal lobe was found to have continuity to the lesion in the midbrain, the pons and the right temporal lobe as well. High MIB-1 staining index suggested that a radiation induced glioblastoma had high proliferative potential comparing with a de novo and a secondary glioblastoma.

Effect of cytochrome c on the linoleic acid-degrading activity of porcine leukocyte 12-lipoxygenase.

Hemoproteins are known to have quasilipoxygenase activity that converts linoleic acid (LA) to its hydroperoxides. However, it is not still clear whether, like lipoxygenases, hemoproteins can produce LA hydroperoxides when the LA is part of a mixture containing many different saturated and unsaturated fatty acids. In this study, we found that such hemoprotein as cytochrome c (Cyt c) did not produce LA hydroperoxides from the phospholipase A(2) (PL-A(2)) hydrolysis products of egg yolk phosphatidylcholine (PC). We also found that traces of hydroperoxides and a high concentration of the target unsaturated fatty acid (LA) needs to be present in a fatty acid mixture before the quasi-lipoxygenase activity of Cyt c becomes apparent. We also attempted to elucidate how Cyt c interact with porcine leukocyte 12-lipoxygenase (12-LOX). Hemoproteins are known to possess pseudo-lipohydroperoxidase activity, and can remove the hydroperoxides of unsaturated fatty acids from a reaction mixture. However, we found that Cyt c catalyzed the reaction by which hydroperoxides degrade LA, and thus enhanced the LA-degrading activity of 12-LOX. This hemoprotein-induced promotion of the ability of 12-LOX to degrade LA was observed even when the reaction mixture contained many different saturated and unsaturated fatty acids.

Postmortem changes in cytochrome c oxidase activity in various organs of the rat and in human heart.

Cytochrome c oxidase (COX), a mitochondrial enzyme, is inactivated by cyanide or carbon monoxide (CO) intoxication. To test whether cytochrome c has potential as an indicator of these toxins in cadavers, we measured COX activity in the main organs of the rat, and in the human heart, at various times after death. Each tissue sample or organ was homogenized and the COX activity in the mitochondrial fraction was measured using ferrous cytochrome c as the substrate. COX activity was significantly higher in rat brain, heart and kidney than in lung and liver from 0 to 4 days after death. The loss of COX activity was significantly slower in the brain and heart than in the lung, liver and kidney. Most importantly, COX activity correlated with the time-since-death for each of the rat organs we tested (r2=0.70-0.95), but for the human heart (r2=0.47). It may be possible that COX activity is likely to be a useful indicator of the time-since-death, and is worth pursuing as an indicator of the tissue cyanide and CO content.