High-level expression and molecular cloning of genes encoding Candida tropicalis peroxisomal proteins.

The development of peroxisomes in the cells of Candida tropicalis grown on oleic acid was accompanied by a markedly high expression of peroxisomal proteins. On the basis of this finding, the nuclear DNA library of this yeast was screened by differential hybridization, and 102 clones of oleic acid-inducible sequences were isolated. Seven coding regions were found to form clusters in three stretches of the genomic DNA. Five of the regions were identified as genes for peroxisomal polypeptides (PXPs). The coding sequence for PXP-2 hybrid selected an additional mRNA for PXP-4, the subunit of long-chain acyl coenzyme A oxidase, which was the most abundant PXP. PXP-2 and PXP-4 were close in apparent molecular weight and generated similar peptides when digested with a protease. The gene for PXP-4 was adjacent to that for PXP-2 on the genome and also hybridized to the mRNA coding for PXP-5. These and other similar results suggest that the genes for the peroxisomal proteins of this organism arose by duplication of a few ancestral genes.

Peroxisomal acyl-coenzyme A oxidase multigene family of the yeast Candida tropicalis; nucleotide sequence of a third gene and its protein product.

We have determined the complete nucleotide sequence of gene POX2, which encodes one of the major peroxisomal polypeptides (PXPs) of Candida tropicalis. POX2 is linked to gene POX4, which codes for a subunit (PXP-4) of long-chain acyl-CoA oxidase. Southern blot analysis revealed that POX2 had a significant homology to POX4, and also to gene POX5 which encodes a subunit (PXP-5) of the isozyme of acyl-CoA oxidase. PXP-2, the protein product of POX2, was co-purified with PXP-4 from the isolated peroxisomes. PXP-2 itself was a flavoprotein and likely to form an equimolar complex with PXP-4, although its enzymatic activity was uncertain. POX2 corresponds to a single open reading frame of 724 amino acids and has no introns. The N-terminal sequence and the calculated Mr of the deduced polypeptide were consistent with those of isolated PXP-2. The primary structure was highly homologous to those of PXP-4 and PXP-5 in respect of the amino acid sequence and the hydropathy profile. We conclude that POX2 is a third gene of the peroxisomal acyl-COA oxidase multigene family.

Major histocompatibility complex expression in brain of rats with graft-versus-host disease.

In a healthy state, the central nervous system (CNS) is believed to be an immunologically privileged site, which does not participate in the immune reactions of the rest of the body, and in which identifiable components of the immune system are rare or non-existent. In this study, an immunohistochemical examination of the CNS of F1 hybrid rats following induction of graft-versus-host disease (GVHD) was carried out to determine whether specific immune reactions in the normal CNS could occur during a systemic immune reaction. The results revealed extensive parenchymal and vascular expression of class I and II major histocompatibility complex (MHC) encoded cell surface molecules. The strongest expressors of class I and II molecules were endothelial cells and parenchymal cells, respectively, the latter being apparently activated microglia, in the cerebrum and cerebellum of rats with GVHD. In addition, occasional scattered lymphocytes were detected in the CNS of GVHD rats without blood-brain barrier disruption. Thus, evidence was obtained for the presence of immune responses such as MHC antigen expression and lymphocyte infiltration in the CNS during a strong systemic immune response such as GVHD, microglia and endothelial cells apparently playing an important role.

New aspects of sterol carrier protein 2 (nonspecific lipid-transfer protein) in fusion proteins and in peroxisomes.

Sterol carrier protein 2 (SCP2) is a 13-kDa peroxisomal protein, identical to nonspecific lipid-transfer protein, and stimulates various steps of cholesterol metabolism in vitro. Although the name is reminiscent of acyl carrier protein, which is involved in fatty acid synthesis, SCP2 does not bind to lipids specifically or stoichiometrically. This protein is expressed either as a small precursor or as a large fusion (termed SCPx) that carries at its C-terminal the complete sequence of SCP2. SCPx exhibits 3-oxoacyl-CoA thiolase activity, as well as sterol-carrier and lipid-transfer activities. The N- and C-terminal parts of SCPx are similar to the nematode protein P-44 and the yeast protein PXP-18, respectively. P-44, which has no SCP2 sequence, thiolytically cleaved the side chain of bile acid intermediate at a rate comparable to that of SCPx. This, together with the properties of other fusions with SCP2-like sequence, suggests that the SCP2 part of SCPx does not play a direct role in thiolase reaction. PXP-18, located predominantly inside peroxisomes, is similar to SCP2 in primary structure and lipid-transfer activity, and protects peroxisomal acyl-CoA oxidase from thermal denaturation. PXP-18 dimerized at a high temperature, formed an equimolar complex with the oxidase subunit, and released the active enzyme from the complex when the temperature went down. This article attempts to gain insight into the role of SCP2, and to present a model in which PXP-18, a member of the SCP2 family, functions as a molecular chaperone in peroxisomes.

Metabolic significance and expression of Caenorhabditis elegans type II 3-oxoacyl-CoA thiolase.

The authors cloned the cDNA of the nematode Caenorhabditis elegans encoding a 44-kDa protein (P-44), which is similar to sterol carrier protein x (SCPx). Genomic DNA data and Northern blot analysis excluded the possibility of P-44 forming SCPx-like fusion protein. P-44 is required in the formation of bile acid in vitro from CoA esters of their enoyl-form intermediate in the presence of D-3-hydroxyacyl-CoA dehydratase/D-3-dehydrogenase bifunctional protein. Also, rat SCPx converts 24-hydroxy-form intermediate to bile acid under similar conditions. From this and other evidence, P-44 and SCPx were categorized as type II thiolase. The mRNA encoding P-44 was detected in every developmental stage of C. elegans: egg, larval stages, and adult. P-44, therefore, seems essential for the normal functioning of this organism.

Thiolase involved in bile acid formation.

The formation of cholic acid and chenodeoxycholic acid through cleavage of the side chains of CoA esters of 3alpha,7alpha,12alpha-trihydroxy-5beta-choles tan-26-oic acid and 3alpha,7alpha-dihydroxy-5beta-cholestan-26-oic acid is believed to occur in peroxisomes. Recently, we found a new peroxisomal enzyme, D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and suggested that this bifunctional protein is responsible for the conversion of 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-2 4-en-26-oyl-CoA and 3alpha,7alpha-dihydroxy-5beta-cholest-24-en-26-oyl-CoA to their 24-oxo-forms. In the present study, the products of this bifunctional protein reaction were analyzed by gas chromatography-mass spectrometry, and the formation of 24-oxo-27-nor-cholestanes was confirmed. Previously, we found a new thiolase in Caenorhabditis elegans, P-44, and suggested that P-44 and sterol carrier protein x, a peroxisomal protein, constitute a second group of 3-oxoacyl-CoA thiolases. The production of cholic acid and chenodeoxycholic acid from the precursors on incubation with the bifunctional protein and sterol carrier protein x or P-44 was confirmed by gas chromatography.

Glial fibrillary acidic protein (GFAP) expression and nucleolar organizer regions (NORs) in human gliomas.

The frequency of nucleolar organizer regions (NORs) in each glioma tissue and the relation between the expression of glial fibrillary acidic protein (GFAP) and the frequency of NORs was investigated. The number of Ag-NORs per cell for glioblastoma multiforme was significantly higher than that for anaplastic astrocytoma (P less than 0.05) and that for astrocytoma (P less than 0.01). The number of Ag-NORs per cell for GFAP-positive cells was significantly lower than that for GFAP-negative cells in each histopathological grade (P less than 0.01). Moreover, the linear relationship was demonstrated between the Ag-NORs numbers of GFAP-negative cells and bromodeoxyuridine (BUdR) labeling indices. From these results, it is concluded that many GFAP-positive glioma cells may have low growth potential in glioma tissue and GFAP-negative cells may have a close relation to cell proliferation. The combination of immunohistochemical and silver colloid staining is a useful method for investigating the biological characteristics of brain tumors.

De novo aneurysm formation after stereotactic radiosurgery of a residual arteriovenous malformation: case report.

We report a case of a 19-year-old woman who underwent radiosurgical treatment of a residual arteriovenous malformation. Nine months after treatment, repeat angiography revealed a de novo paranidal aneurysm that was treated endovascularly. We postulate that changes in flow dynamics or vessel integrity after radiosurgery contributed to the formation of her de novo aneurysm.

An accurate adjustable applicator for magnetic resonance imaging-based stereotactic procedure using the Leksell G frame.

OBJECTIVE: An applicator system for the Leksell G frame was constructed to enable accurate placement of the frame for stereotactic magnetic resonance imaging (MRI) and successful stereotactic surgery. The applicator prevents inaccurate placement of the fiducial box on the patient's head and prevents contact of the frame holder with the patient's shoulder while in the MRI unit. It also helps to ensure optimal positioning of desired targets within the three-dimensional stereotactic space defined by the frame. METHODS: The applicator is made of transparent acrylic plates, which simulate the fiducial box that is attached to the frame for the preoperative stereotactic MRI study. An air cuff at the top supports the frame at any desired height and makes minute adjustments possible. Side cuffs help to keep the frame at the desired position from right to left. Indicators attached to the frame for the anterior fiducial plate prevent potential contact of the plate with the anterior posts and help avoid a poor fit caused by bending of the frame from excessive torque on the cranium fixation screws. Indicators for the MRI frame holder on the foot screws predict potential collision of the holder with the patient's shoulder before actually applying the holder on the frame. The applicator shows the range and limits of the Leksell stereotactic arc. RESULTS: This applicator system has been used effectively in more than 89 MRI-based functional stereotactic procedures. These include pallidotomy, thalamotomy, implantation of deep brain stimulators, and implantation of depth electrodes. It has functioned well and has facilitated excellent operative results in these cases. CONCLUSION: This simple frame applicator eliminates the need for reapplication of the stereotactic frame and additional imaging studies, thus providing successful and appropriate frame placement for stereotactic surgery.

Identification and localization of intracerebral vessels by microvascular doppler in stereotactic pallidotomy and thalamotomy: technical note.

OBJECTIVE: To identify intracerebral vessels in proximity to the target for thermocoagulation in functional neurosurgery, we use a microvascular doppler sensor held in a special supporting needle that fits in the straightening cannula for the thermocoagulation needle. TECHNIQUE: After insertion of the straightening cannula aimed at the stereotactic target, the microvascular doppler probe positioned at the tip of a supporting hollow needle is advanced through the cannula. The proximal micrometer gauge indicates the depth of the tip of the doppler probe. By setting the doppler device to the shortest focusing depth (0.1 mm), the maximum pulsatile vascular sound indicates the depth of the vessel. RESULTS AND CONCLUSION: A prominent vascular sound was identified in 3 of 13 cases. By adjusting the depth of the target, no major bleeding was experienced after thermocoagulation lesions were made. This technique secures and protects the fragile microvascular doppler and identifies any significant arterial vessels at the stereotactic target, thus avoiding vascular injury.

A dissecting aneurysm of the posteroinferior cerebellar artery: case report.

OBJECTIVE AND IMPORTANCE: We present a patient who experienced a subarachnoid hemorrhage secondary to a dissecting aneurysm of the right posteroinferior cerebellar artery (PICA). The use of an encircling clip in treating the aneurysm while preserving supply to brain stem perforators originating near the dissecting segment and the distal PICA territory was key in the operative management. CLINICAL PRESENTATION: A 48-year-old patient with a history of hypertension presented with subarachnoid hemorrhage confirmed by computed tomography of the brain. Successive cerebral angiography revealed a dynamic change in the configuration of the dissection, with expansion of the associated focal ectasia. OPERATIVE MANAGEMENT: At surgery, three brain stem perforators adjacent to the aneurysm were visualized. The dissecting segment was reconstructed with an encircling Sundt clip and muslin wrap, which preserved the flow through the PICA and brain stem perforators. CONCLUSION: A patient suffering from a dissecting PICA aneurysm and subarachnoid hemorrhage was successfully treated with direct surgical reconstruction of the parent artery, sparing the perforators to the medulla.

Nucleolar organizer regions in meningioma.

Seventy-eight cases of meningioma and related tumors were examined independently using a simple and reproducible argyrophilic method for the demonstration of nucleolar organizer regions (AgNORs) and staining with bromodeoxyuridine monoclonal antibody. The mean number of AgNORs per cell and the bromodeoxyuridine labeling index were shown to be linearly related (r = 0.84, P less than 0.001). The mean AgNOR number was 2.99 for meningeal sarcoma, 2.29 for anaplastic meningioma, 2.08 for hemangiopericytic meningioma. 1.72 for recurrent meningioma without atypical histological findings, and 1.52 for nonrecurrent meningioma. We noted that the mean number of AgNORs reflected the cellular kinetics of a tumor and was related to histological grade and clinical behavior.

Radiosurgery-induced microvascular alterations precede necrosis of the brain neuropil.

OBJECTIVE: Radiosurgery is used as a therapeutic modality for a wide range of cerebral disorders. It is important to understand the underlying causes of deleterious side effects that may accompany gamma-irradiation of brain tissue. In this study, structural alterations in rat cerebral vessels subjected to gamma knife irradiation in vivo were examined, for elucidation of their potential role in necrosis formation. METHODS: A maximal center dose of 75 Gy was delivered to the rat parietal cortex with a 4-mm collimator, and changes occurring before necrosis formation were assessed 3.5 months after irradiation. Transmission electron microscopy, using horseradish peroxidase as a tracer, and scanning electron microscopy with vascular casting were performed. RESULTS: The capillary network in the irradiated area exhibited thickening and vacuolation of the basement membrane. The capillary density in the irradiated area was lower and the average capillary diameter was larger, compared with the nonirradiated side. These results indicate that substantial changes in the neuropil do not occur 2 weeks before the time of definite necrosis formation, whereas changes in the basement membrane are prominent. CONCLUSION: The necrotic response to intermediate doses of focused-beam irradiation appears after a considerable latency period and then progresses rapidly. This contrasts with previously reported responses to fractionated whole-brain irradiation, in which damage occurs slowly and gradually. Alterations in the microvascular basement membrane precede overt cellular changes in neuronal and vascular cells and provide an early index of cerebrovascular dysfunction in regions destined to undergo necrosis.

A burr hole button to secure the electrode cable in depth electrode placement. Technical note.

A simple magnetic resonance imaging-compatible buttonlike device was devised to fix a depth electrode cable securely in the burr hole used for its insertion during surgery for depth electrode placement. The button is tightly fixed in the burr hole and it holds the cable without allowing protrusion or tension on the wound.

Cerebral microvascular architecture following experimental cold injury.

The sequential changes in microvascular architecture following local cold injury in rat brains were studied post mortem by scanning electron microscopy and the vascular casting method. The findings were compared with the results of immunohistochemical studies of injured endothelial cells using the bromodeoxyuridine (BUdR) and anti-BUdR monoclonal antibody technique. Repair of the microvascular architecture had begun by the 3rd day after injury, with hematogenous cells and reactive astrocytes present in the edematous brain participating in the regenerative process. The normal microvascular architecture was reconstructed starting from the edge of the lesion nearest to the brain surface. On the other hand, in the most severely injured part of the brain surface, newly formed microvascular architecture appeared, resembling that of the developing fetal and newborn rat cortex. Seven days after injury, the entire microvascular architecture in the region of the lesion had been reconstructed.

Optimizing accuracy in magnetic resonance imaging-guided stereotaxis: a technique with validation based on the anterior commissure-posterior commissure line.

OBJECT: Some of the earliest successful frame-based stereotactic interventions directed toward the thalamus and basal ganglia depended on identifying the anterior commissure (AC) and posterior commissure (PC) in a sagittal ventriculogram and defining the intercommissural line that connects them in the midsagittal plane. The AC-PC line became the essential landmark for the localization of neuroanatomical targets in the basal ganglia and diencephalon and for relating them to stereotactic atlases. Stereotactic/functional neurosurgery has come to rely increasingly on magnetic resonance (MR) imaging guidance, and methods for accurately determining the AC-PC line on MR imaging are being developed. The goal of the present article is to present the authors' technique. METHODS: The technique described uses MR sequences that minimize geometric distortion and registration error, thereby maximizing accuracy in AC-PC line determinations from axially displayed MR data. The technique is based on the authors' experience with the Leksell G-frame but can be generalized to other MR imaging-based stereotactic systems. This methodology has been used in a series of 62 stereotactic procedures in 47 adults (55 pallidotomies and seven thalamotomies) with preliminary results that compare favorably with results reported when using microelectrode recordings. The measurements of the AC-PC line reported here also compare favorably with those based on ventriculography and computerized tomography scanning. CONCLUSIONS: The methodology reported here is critical in maintaining the accuracy and utility of MR imaging as its role in modern stereotaxy expands. Accurate parameters such as these aid in ensuring the safety, efficacy, and reproducibility of MR-guided stereotactic procedures.

Correlation of in vitro bromodeoxyuridine labeling index and DNA aneuploidy with survival or recurrence in brain-tumor patients.

There are no previous reports correlating the in vitro bromodeoxyuridine (BUdR) labeling index (LI) with the clinical outcome in patients with brain tumors. The reliability of the LI as a predictor of patient survival or recurrence was examined in this study of 66 human brain tumors (19 gliomas, 18 meningiomas, and 29 others). Anti-BUdR staining was performed on surgically extirpated tumor tissue that had been treated with BUdR as previously described. Correlation of the BUdR LI with patient survival or tumor recurrence rate was carried out by the method of Kaplan and Meier. Deoxyribonucleic acid (DNA) aneuploidy was estimated in 52 cases. The results of this study indicate that BUdR LI values correlated well with the clinical course of patients with brain tumor. In comparison with patients with higher LI's, there was both a significantly higher survival rate for tumors other than meningiomas and a higher recurrence-free rate for meningiomas in patients with LI's of less than 4% and 1%, respectively. Although there was a tendency for patients without tumor aneuploidy to show better survival data than the others, no statistical difference was observed. These results suggest that the in vitro BUdR labeling method is reliable for prediction of a patient's prognosis, whereas prognosis on the basis of DNA aneuploidy alone is uncertain.

Analysis of loss of heterozygosity on chromosome 10 in patients with malignant astrocytic tumors: correlation with patient age and survival.

OBJECT: The most frequent genetic abnormality in human malignant gliomas is loss of heterozygosity (LOH) on chromosome 10. Candidate genes on chromosome 10 that are associated with the prognosis of patients with anaplastic astrocytoma (AA) and glioblastoma (GBM) were evaluated. METHODS: The authors used 12 fluorescent microsatellite markers on both arms of chromosome 10 to study LOH in 108 primary astrocytic tumors. The LOH on chromosome 10 was observed in 11 (32%) of 34 AAs and 34 (56%) of 61 GBMs. No LOH was detected in 13 low-grade gliomas. Loss of heterozygosity was not detected in any AA in the seven patients younger than 35 years, but it was discovered in 41% of the patients older than 35 years. The prognostic significance of LOH at each locus was evaluated in 89 patients older than 15 years; 33 (37%) had supratentorial AAs and 56 (63%) had supratentorial GBMs. The Cox proportional hazards model, adjusted for patient age at surgery, the preoperative Karnofsky Performance Scale score, and the extent of surgical resection revealed that LOH on marker D10S209 near the FGFR2 and DMBT1 genes was significantly associated with shorter survival in patients with AA. The LOH on markers D10S215 and D10S541, which contain the PTEN/MMAC1 gene between them, was significantly associated with shorter survival in patients with GBM. CONCLUSIONS: In the present study it is found that LOH on chromosome 10 is an age-dependent event for patients with AAs and that LOH on marker D10S209 near the FGFR2 and DMBT1 loci is a significantly unfavorable prognostic factor. It is also reported that LOH on the PTEN/MMAC1 gene is a significantly unfavorable prognostic factor in patients with GBM.

Anticonvulsant effects of gamma surgery in a model of chronic spontaneous limbic epilepsy in rats.

OBJECT: The management of intractable epilepsy remains a challenge, despite advances in its surgical and nonsurgical treatment. The identification of low-risk, low-cost therapeutic strategies that lead to improved outcome is therefore an important ongoing goal of basic and clinical research. Single-dose focal ionizing beam radiation delivered at necrosis-inducing and subnecrotic levels was investigated for its effects on seizure activity by using an established model of chronic recurrent spontaneous limbic seizures in rats. METHODS: A single 90-minute period of repetitive electrical stimulation (inducing stimulus) of the hippocampus in rats elicited a single episode of status epilepticus, followed by a 2- to 4-week seizure-free period. Spontaneous recurrent seizures developed subsequently and persisted for the duration of monitoring (2-10 months). Simultaneous computerized electroencephalography and video recording were used to monitor the animals. After the establishment of spontaneous recurrent seizures, bilateral radiation centered in the ventral hippocampal formation was administered with the Leksell gamma knife, aided by a stereotactic device custom made for small animals. A center dose of 10, 20, or 40 Gy was administered using a 4-mm collimator. Control animals were subjected to the same seizure-inducing stimulus but underwent a sham treatment instead of gamma irradiation. In a second experiment, the authors examined the effects of gamma irradiation on the proclivity of hippocampal neurons to display epileptiform discharges. Naive animals were irradiated with a single 40-Gy dose, as already described. Slices of the hippocampus were prepared from animals killed between 1 and 178 days postirradiation. Sensitivity to penicillin-induced epileptiform spiking was examined in vitro in slices prepared from control and irradiated rat brains. CONCLUSIONS: In the first experiment, single doses of 20 or 40 Gy (but not 10 Gy) reduced substantially, and in some cases eliminated, behaviorally and electrographically recognized seizures. Significant reductions in both the frequency and duration of spontaneous seizures were observed during a follow-up period of up to 10 months postradiation. Histological examination of the targeted region did not reveal signs of necrosis. These findings indicate that single-dose focal ionizing beam irradiation at subnecrotic dosages reduces or eliminates repetitive spontaneous seizures in a rat model of temporal lobe epilepsy. In the second experiment, synaptically driven neuronal firing was shown to be intact in hippocampal neurons subjected to 40-Gy doses. However, the susceptibility to penicillin-induced epileptiform activity was reduced in the brain slices of animals receiving 40-Gy doses, compared with those from control rats that were not irradiated. The results provide rational support for the utility of subnecrotic gamma irradiation as a therapeutic strategy for treating epilepsy. These findings also provide evidence that a functional increase in the seizure threshold of hippocampal neurons contributes to the anticonvulsant influence of subnecrotic gamma irradiation.

1995 AUR Memorial Award. Gamma knife irradiation-induced changes in the normal rat brain studied with 1H magnetic resonance spectroscopy and imaging.

RATIONALE AND OBJECTIVES: The pathogenesis of brain injury following radiosurgery is poorly understood. To better elucidate the relationship between blood-brain barrier disruption and metabolic derangements, we used magnetic resonance (MR) imaging and 1H MR spectroscopy to detect early changes from focused single-fraction, high-dose irradiation injury in rat brains. METHODS: Using the Leksell gamma knife, we irradiated the frontoparietal cortex of 11 male Wistar rats with a single dose of 120 Gy. Four weeks later, we sequentially performed water-suppressed 1H MR spectroscopy and gadopentetate dimeglumine-enhanced T1-weighted MR imaging. Metabolic maps were created of n-acetylaspartate (NAA), creatine and choline (Cr/Cho), and lactate from the MR spectroscopy data set. Detection of irradiation injury among the tested modalities was assessed by receiver operating characteristic analysis and by quantitative signal intensity changes. Pathologic confirmation of irradiation damage was obtained in all rats. RESULTS: Gadopentetate dimeglumine-enhanced T1-weighted MR imaging was the only imaging modality that detected statistically significant signal intensity changes (p < .05). No reproducible changes in the metabolites of interest could be detected by 1H MR spectroscopy. CONCLUSION: In our animal model, blood-brain barrier disruption was a reproducible, integral finding of single-fraction, high-dose irradiation injury. No reproducible metabolic derangements of ischemia or necrosis were detected by 1H MR spectroscopy, possibly because of dose-latency effects or sensitivity issues.