Structural and guanosine triphosphate/diphosphate requirements for transit peptide recognition by the cytosolic domain of the chloroplast outer envelope receptor, Toc34.

Toc34 is a transmembrane protein located in the outer envelope membrane of chloroplasts and involved in transit peptide recognition. The cytosolic region of Toc34 reveals 34% alpha-helical and 26% beta-strand structure and is stabilized by intramolecular electrostatic interaction. Toc34 binds both chloroplast preproteins and isolated transit peptides in a guanosine triphosphate- (GTP-) dependent mechanism. In this study we demonstrate that the soluble, cytosolic domain of Toc34 (Toc34deltaTM) functions as receptor in vitro and is capable to compete with the import of the preprotein of the small subunit (preSSU) of ribulose-1,5-bisphosphate carboxylase-oxygenase into chloroplasts in a GTP-dependent manner. We have developed a biosensor assay to study the interaction of Toc34deltaTM with purified preproteins and transit peptides. The results are compared with the interactions of both a full-size preprotein and the transit peptide of preSSU with the translocon of the outer envelope of chloroplasts (Toc complex) in situ. Several mutants of the transit peptide of preSSU were evaluated to identify amino acid segments that are specifically recognized by Toc34. We present a model of how Toc34 may recognize the transit peptide and discuss how this interaction may facilitate interaction and translocation of preproteins via the Toc complex in vivo.

Lipid composition of outer leaflet of chloroplast outer envelope determines topology of OEP7.

OEP7, a 6.7-kDa outer envelope protein of spinach chloroplasts inserts into the outer envelope of the organelle independent of a classical cleavable targeting signal. The insertion of OEP7 was studied to describe the determinants for association with, integration into, and orientation of the protein in the outer envelope of chloroplasts. The insertion of OEP7 into the membrane is independent of outer membrane channel proteins and can be reconstituted with the use of protein-free liposomes. In situ, the binding of OEP7 to the membrane surface is not driven by electrostatic interaction because reduction of phosphatidylglycerol or phosphatidylinositol did not reduce the association with the liposomes. The positively charged amino acids flanking the transmembrane domain at the C terminus are essential to retain the native N(in)-C(out) orientation during insertion into chloroplasts. OEP7 inserts with reversed orientation into liposomes containing the average lipid composition of the outer envelopes. The native like N(in)-C(out) orientation is achieved by reduction of the phoshpatidylglycerol concentration mimicking the composition of the outer leaflet of the outer envelope of chloroplasts. We conclude that the unique lipid composition of the outer leaflet due to lipid asymmetry of the outer envelope is essential for the correct topology of OEP7.

Protein translocation into and across the chloroplastic envelope membranes.

Post-translational protein import into chloroplasts follows a common route characterised by the need for nucleoside-triphosphates at various steps and two distinct protein import machineries at the outer and inner envelope membrane, respectively. Several subunits of these complexes have been elucidated. In contrast, protein translocation into the chloroplastic outer envelope uses distinct and various but poorly characterised insertion pathways. A topological framework for single-membrane spanning proteins of the chloroplastic outer envelope is presented.

Neuroimaging in epilepsy.

Neuroimaging techniques have improved the understanding, diagnosis, and management of epilepsy. By providing excellent structural information, MRI is the technique of choice in evaluating patients with epilepsy. Functional imaging techniques, including MR spectroscopy, functional MRI, positron emission tomography, and single photon emission CT, permit noninvasive assessment of the epileptic substrate, its functional status, and neuroreceptors. The MRI-based techniques will potentially assume a greater role in the cost-effective workup of the patient. Currently, newer techniques such as magnetoencephalography, magnetic source imaging, and optical imaging are research tools.

Activity of high-dose cyclophosphamide in the treatment of childhood malignant gliomas.

Seventeen patients less than or equal to 20 years of age with newly diagnosed (n = 10) or recurrent (n = 7) malignant gliomas (anaplastic astrocytoma and glioblastoma multiforme) were treated with cyclophosphamide in association with hematopoietic cytokines (GM-CSF or G-CSF). Cyclophosphamide was given at a dose of 2 g/m2 daily for 2 days at 4-week intervals. Toxicity consisted of grade IV neutropenia and thrombocytopenia in 95% and 48% of cycles, respectively. There were no cyclophosphamide-related cardiac, pulmonary, or urothelial toxicities observed. Four of 10 patients with newly diagnosed disease demonstrated responses (three complete and one partial responses; one CR was only of 2 months duration). None of the seven patients with recurrent tumors demonstrated a response. We conclude that high-dose cyclophosphamide warrants further evaluation in children with newly diagnosed malignant glioma.

Malignant supratentorial glial-neuronal neoplasms: report of two cases and review of the literature.

OBJECTIVE: Malignant neoplasms exhibiting mixed populations of neuronal and glial cells occurring in the cerebral hemispheres of young adults and children are well recognized, but rare. A confusing array of diagnostic terms has arisen. We describe two patients with such tumors and review the literature concerning these interesting cases. PATIENTS: A 21-year-old man and a 5-year-old girl presented with large, cystic, intracerebral lesions on magnetic resonance images, which proved to be composite neoplasms exhibiting malignant neurons and astrocytes. RESULTS: The 21-year-old man had a frontal lobe mass with enhancing and nonenhancing regions, which corresponded to cerebral neuroblastoma and anaplastic astrocytoma, respectively. The presence of occasional microtubules and rare primitive presynaptic processes, accompanied by antisynaptophysin immunoreactivity, established the neuronal nature of the cells in the enhancing region. The nonenhancing region was composed of a moderately cellular neoplasm of fibrillar astrocytes that were mitotically active. The 5-year-old girl presented with a left parietal lobe neoplasm, which histologically was composed of lobular proliferations of neuroblasts and glia. The neuroblastic populations exhibited evidence of maturation with small anaplastic cells, spindle-shaped cells, and large dysmorphic ganglion cells. The glial tumor showed both well-differentiated fibrillary astrocytes with microcysts and anaplastic populations with central necrosis and pseudopalisading. CONCLUSIONS: Present classification systems devised to describe mixed neuronal and glial tumors do not adequately encompass the diversity of morphologies presented by these two cases. We conclude that the terms cerebral neuroblastoma-anaplastic astrocytoma for case 1 and cerebral ganglioneuroblastoma-glioblastoma for case 2 are preferred because they convey useful clinical information by reflecting concepts already encompassed by the World Health Organization's classification system of tumors of the central nervous system.

Neurosyphilis in patients with AIDS.

Syphilis has become much more prevalent because of the dramatic increase in immunocompromised patients. The increase in immunocompromised patients is mainly secondary to AIDS. This article is put forth to refamiliarize the reader with syphilis, specifically neurosyphilis. The neurologic symptomatology and neuroimaging characteristics are presented so that one can recognize the findings and consider the diagnosis of neurosyphilis when confronted with a patient with AIDS.

The limbic system. An overview of the anatomy and its development.

The limbic system has a pivotal role in attention, memory, and the emotions. The limbic lobe comprises four C-shaped arches stretching from the medial surface of the frontal lobe to the temporal pole. The anatomic relationships are elegantly demonstrated by MR imaging. This article provides an overview of the development and complex anatomy of the limbic system.

Hippocampus. Normal magnetic resonance imaging anatomy with volumetric studies.

MR imaging sections of the hippocampus in different orthogonal planes are illustrated. Matching coronal slices in T2-weighted and gradient echo sequences allow a comparative evaluation of the intricate structure of the hippocampus and its relationship with adjacent structures. Techniques in volumetric assessment of the hippocampus are discussed in light of recent advancement in MR imaging.

Hippocampal pathology.

Medial temporal sclerosis of the hippocampus and other lesions in the adjacent temporal lobe that can cause epilepsy are discussed in this article. The technical factors to consider to optimally image the hippocampus and criteria to diagnose medial temporal sclerosis are emphasized.

Anatomy and pathology of the septal region.

Comprising the septal area and the subcortical nuclei, the septal region is gray matter structures with widespread projection systems and different neurotransmitters. Although their function is poorly understood, lesions of the septal nuclei result in a syndrome of hyper-reactivity, amnesia, and hypersexuality. The pathologic processes affecting the septal region are discussed.

Limbic circuits and neuropsychiatric disorders. Functional anatomy and neuroimaging findings.

The limbic system seems to be involved in the pathophysiology of neuropsychiatric disorders, including dementia, schizophrenia, affective disorders, and amnestic disorders. These findings are subtle and largely went undetected until the advent of modern neuroimaging. This article discusses some of the neuroimaging findings in these disorders.

False positive images in the follow-up of patients with brain tumors.

In recent years, major advances in the diagnosis and treatment of patients with brain tumors have been seen. Today, evaluation of the central nervous system almost always includes magnetic resonance imaging (MRI). The appearance of a new lesion on the MRI scan of a patient previously treated for a central nervous system (CNS) tumor raises concern for recurrent disease with the need for selection of new, potentially toxic therapy. However, the sensitivity of MRI may allow demonstration of new lesions which are not due to tumor. We now report three patients with medulloblastoma who demonstrated new enhancing lesions on MRI following treatment of their tumors with surgery (3 patients), chemotherapy (2 patients), and radiotherapy (2 patients). Two patients underwent resection of the lesion revealing gliosis. One patient had serial imaging that showed disappearance of the lesions. This suggests that not all new enhancing lesions in previously treated brain tumor patients represent tumor. Histologic proof of a suspicious lesion should be demonstrated prior to initiation of new therapy.

Gliomatosis cerebri presenting with optic nerve involvement: MRI.

Gliomatosis cerebri is a rare form of astrocytoma characterized by extreme infiltration of the brain structure in conjunction with a relative paucity of clinical findings. We describe the MRI findings in a patient with gliomatosis cerebri widely infiltrating the brain who presented with symptoms related to involvement of the optic nerves and chiasm. Contrast-enhanced MRI showed enlargement of the optic nerves and chiasm with pathological enhancement; T2-weighted images showed extensive infiltration of the brain by tumor. Histopathologic examination of the biopsy specimen showed anaplastic astrocytoma with gemistocytic predominance and a diagnosis of gliomatosis cerebri was reached.

Successful treatment of childhood pilocytic astrocytomas metastatic to the leptomeninges with high-dose cyclophosphamide.

Leptomeningeal dissemination of childhood pilocytic astrocytoma (PA) is a rare event with little information available regarding therapy. We report here four children with disseminated PA whom we treated with high doses of cyclophosphamide with clinical benefit. The patients were aged 2.5 to 8 years. Three patients presented with PA localized in the posterior fossa, initially treated with surgical resection (n = 3) and radiotherapy (n = 1). Leptomeningeal dissemination occurred at 32, 44, and 8 months from diagnosis, respectively. The fourth patient presented with an optic pathway tumor with leptomeningeal dissemination at diagnosis. At commencement of cyclophosphamide therapy, disease was present in the subarachnoid space (intracranial, n = 2; spinal, n = 4), cerebral ventricles (n = 2), and primary site (n = 3). Histology was identical at diagnosis and recurrence in the two biopsied cases and cerebrospinal fluid was negative in all cases. Treatment was with cyclophosphamide 4-5 g/m2/cycle given every 4 weeks for a total of two cycles (n = 1) and four cycles (n = 3). One patient achieved disease stabilization (duration 27 months at the time of publication) and three patients experienced significant reductions in tumor burden. Subsequent intrathecal therapy was administered to two patients. Two patients developed disease progression at 10 and 9 months from cessation of chemotherapy. The one re-treated patient responded to further, lower dose, cyclophosphamide. This is the first report of the use of high dose cyclophosphamide for disseminated PA. The recurrence of disease in two cases with a further response to lower dose cyclophosphamide has implications for the optimal duration of therapy for these low grade, aggressive tumors.

Single-voxel proton brain spectroscopy exam (PROBE/SV) in patients with primary brain tumors.

OBJECTIVE: The Single-Voxel Proton Brain Exam (PROBE/SV) is an automated MR spectroscopic technique. The purpose of this study was to investigate the use of PROBE/SV as a diagnostic tool in patients with primary brain tumors and to compare our findings with the known information obtained from conventional nonautomated MR spectroscopic techniques. SUBJECTS AND METHODS: We used PROBE/SV to image 10 normal adults and 46 patients with primary brain tumors: 29 glioblastoma multiformes (GBMs), five anaplastic astrocytomas, and 12 low-grade astrocytomas. All studied were performed on a 1.5-T Signa unit. Average voxel sizes were 6-8 cm3. A corresponding point-resolved spectroscopy spectrum was obtained from normal-appearing brain parenchyma in each patient for comparison with the spectra from known areas of pathology. RESULTS: In patients with low-grade gliomas (grades 1 and 2), we observed decreased N-acetylaspartate (12 of 12) and slightly increased choline (11 of 12) when we compared these metabolites with those in the spectra of patients' normal brains. This comparison in patients with GBM yielded markedly decreased N-acetylaspartate (29 of 29) and prominently increased choline (27 of 29). In the short TE spectra, we frequently saw lipid signal in high-grade tumors, especially in GBMs (12 of 20). We identified lactate peaks in high-grade tumors (anaplastic astrocytoma and GBM, 29 of 34) and also in low-grade tumors (four of 12). The creatine signal in all gliomas was slightly less than that of healthy brain tissue. The lowest N-acetylaspartate, choline, and creatinine levels in conjunction with the highest lactate levels were usually found in necrotic portions of high-grade tumors. CONCLUSION: PROBE/SV is a simplified MR spectroscopy technique that reduces setup time and provides automatic on-line data processing and display. The voxel location can be selected to focus on the area of interest and to minimize voxel contamination from unwanted tissue. The results from our experimentation with PROBE/SV in patients with brain tumors generally concur with published reports of tumor spectra obtained by conventional MR spectroscopic techniques. The ease and accuracy of this new technique make it a useful clinical tool in differentiating human brain tumor grades.

Chorea-ballismus with nonketotic hyperglycemia in primary diabetes mellitus.

PURPOSE: To describe the neuroimaging (Ct, MR, and single-photon emission CT [SPECT]) findings in a series of patients with chorea-ballismus associated with nonketotic hyperglycemia in primary diabetes mellitus and to correlate the imaging findings with the clinical presentation. METHODS: The neuroimaging and clinical data from 10 patients with chorea-ballismus associated with nonketotic hyperglycemia in primary diabetes mellitus were evaluated. Family and drug histories, as well as other causes of chorea, were excluded. All 10 patients had CT, 5 also had MR imaging, and 3 had SPECT examinations. Three had follow-up CT and MR imaging studies, and MR findings were correlated with CT findings in 5 cases. Two experienced neuroradiologists, aware of the diagnosis but blinded to the clinical status of the patients, evaluated all images and reached a consensus as to the final interpretation. RESULTS: CT studies in 9 of 10 patients showed a hyperdense putamen and/or caudate nucleus; in 1, the CT findings were normal. T1-weighted MR images in all 5 patients who had MR imaging (including the patient with a normal CT study) showed hyperintense lesions without significant T2 signal alternation at the basal ganglia. In all 3 of the patients who had SPECT studies of the brain, the scans revealed hypoperfusion at corresponding areas. All 3 follow-up studies depicted resolution of the lesions in the abnormal basal ganglia. Increased hypointensity on T2-weighted and gradient-echo T2*-weighted images was also observed in the sequential MR images. In all patients, the initial side of involvement correlated well with the neuroimaging findings. The chorea resolved within 2 days after treatment of the hyperglycemia in 9 patients. CONCLUSION: In patients with chorea-ballismus associated with nonketotic hyperglycemia in primary diabetes mellitus, CT and T1-weighted MR images show unilateral or bilateral lesions of the putamen and/or caudate. SPECT scans show hypoperfusion. These findings may be related to petechial hemorrhage and/or myelin destruction. Early recognition of these imaging characteristics may facilitate diagnosis of primary diabetes mellitus with hyperglycemia and prompt appropriate therapy.

Treatment of patients with pineoblastoma with high dose cyclophosphamide.

The outcome for patients with pineoblastoma has historically been very poor, with most patients dying of disseminated disease despite irradiation. Furthermore, the low incidence of this tumor has hindered progress toward defining better treatment strategies. Here we report the activity and toxicity of cyclophosphamide administered as a single agent at a dose schedule of 2 g/m2/day for 2 successive days at monthly intervals for a maximum of four courses. Eight patients were evaluated, six newly diagnosed and two recurrent. Amongst the six newly diagnosed patients, there were three patients demonstrating partial responses, and three had stable disease throughout the cyclophosphamide treatment period. All six patients are alive and disease free after further therapy. One patient with recurrent disease demonstrated tumor progression on cyclophosphamide, and the other had stable disease throughout the cyclophosphamide treatment period. Both patients subsequently died of progressive disease. The major toxicity of high dose cyclophosphamide was hematopoietic, with one patient requiring a dose reduction after three courses due to prolonged thrombocytopenia. One patient was also withdrawn from treatment with cyclophosphamide due to impaired pulmonary function. This study demonstrates the activity of high dose cyclophosphamide in the treatment of pineoblastoma and may serve as basis for the design of future studies of this tumor.

Intrathecal 131I-labeled antitenascin monoclonal antibody 81C6 treatment of patients with leptomeningeal neoplasms or primary brain tumor resection cavities with subarachnoid communication: phase I trial results.

We aimed to determine the maximum tolerated dose (MTD) of 131I-labeled 81C6 in patients with leptomeningeal neoplasms or brain tumor resection cavities with subarachnoid communication and to identify any objective responses. 81C6 is a murine IgG monoclonal antibody that reacts with tenascin in gliomas/carcinomas but does not react with normal adult brain. 131I-labeled 81C6 delivers intrathecal (IT) radiation to these neoplasms. This study was a Phase I trial in which patients were treated with a single IT dose of 131I-labeled 81C6. Cohorts of three to six patients were treated with escalating doses of 131I (starting dose, 40 mCi; 20 mCi escalations) on 10 mg 81C6. MTD is defined as the highest dose resulting in serious toxicity in no more than two of six patients. Serious toxicity is defined as grade III/IV nonhematological toxicity or major hematological toxicity. We treated 31 patients (8 pediatric and 23 adult). Eighteen had glioblastoma multiforme. Patients were treated with 131I doses from 40 to 100 mCi. Hematological toxicity was dose limiting and correlated with the administered 131I dose. No grade III/IV nonhematological toxicities were encountered. A partial response occurred in 1 patient and disease stabilization occurred in 13 (42%) of 31 patients. Twelve patients are alive (median follow-up, > 320 days); five are progression free >409 days median posttreatment. The MTD of a single IT administration of 131I-labeled 81C6 in adults is 80 mCi 131I-labeled 81C6. The MTD in pediatric patients was not reached at 131I doses up to 40 mCi normalized for body surface area.