Gap junctions in human glioblastomas: implications for suicide gene therapy.

Glioblastoma is a very aggressive astrocytic tumor and most patients have 1-year survival time after diagnosis. A promising therapeutic strategy is the local delivery of the herpes simplex virus thymidine kinase gene in the tumor bed followed by ganciclovir treatment. The presence of functional gap junctions is highly relevant for the success of suicide gene therapy. Connexins are expressed in practically all tissues and form gap junctions that allow intercellular communication. Connexin 43 (Cx43) is the major connexin member being expressed in astrocytes but its status in glioblastoma is not well defined. We have investigated by immunofluorescence the presence of Cx43 in 74 human glioblastoma samples; its expression was detected in 77% of the samples analyzed. We report here that glioblastoma is a heterogenous disease as regards Cx43 expression with presentations, in which Cx43 expression is unaltered, reduced or totally lost. A predominant Cx43 cytoplasmic localization was observed in four out of eight primary glioblastoma cultures that we have established. This aberrant localization reduced gap junctionnal intercellular communication by 50 to 75% as compared with primary cell cultures displaying gap junctional plaques. However, the bystander effect evaluated after lentiviral delivery of the herpes simplex virus thymidine kinase gene and ganciclovir treatment was detected in all Cx43-positive primary cell cultures, and it was independant of the Cx43 localization. These findings may have important clinical implications for the design of anticancer cytotoxic therapies that rely on the gap junction-mediated bystander effect for their success.

Calretinin-immunoreactive neurons in the human thalamus.

The distribution of the calcium-binding protein calretinin in the thalamus of normal human individuals was studied with immunohistochemistry. Calretinin immunoreactivity was weak in the geniculate bodies and in nuclei of the ventral and posterior groups, moderate in the reticular nucleus and in nuclei of the anterior, medial, and lateral groups, and strong in nuclei of the midline group and anterior intralaminar nuclei. The mediodorsal nucleus was unique among thalamic nuclei because it contained a wide variety of intensely immunostained perikarya embedded in a moderately-labelled neuropil. The reticular nucleus displayed several small and uniformly distributed neuronal clusters composed of immunostained perikarya lying in a moderately-labelled neuropil. Intense and uniform immunostaining was observed in all midline nuclei and in the anterior intralaminar nuclei, including the paracentral and central lateral nuclei. These nuclei, which harboured numerous intensely-stained perikarya lying in a dense immunoreactive neuropil, were the most strongly-immunoreactive structures of the entire human thalamus. At the level of the posterior intralaminar nuclei, the central median nucleus was virtually free of immunostaining whereas the parafascicular nucleus was moderately labelled. The nucleus submedius located just beneath the central median/parafascicular complex displayed a very intense calretinin immunostaining. This study has provided evidence for the presence of the protein calretinin in the human thalamus. The pattern of distribution of calretinin, as delineated in the present study, suggests that this calcium-binding protein may participate in various subcortical and cortical thalamic systems involved in the modulation of emotional and motivational states.

Recent studies on oculopharyngeal muscular dystrophy in Québec.

In 1990, we launched a major study to ascertain the clinical picture of OPMD in Québec and to identify large families for linkage analysis. In 14 patients, the chromosomes were karyotyped to eliminate any deletion or translocation. Relevant family information and clinical data were computerized and correlations were sought for the age of onset, the identification of the first symptom and the distribution of weakness. A simple test to detect dysphagia was validated. Twenty-one families have taken part in the study, which led to our localization of the gene in 1995 [Brais B, Xie Y-G, Sanson M, et al. Hum Mol Genet 1995; 4:429-434]. At least one case in each family underwent muscle biopsy to confirm the presence of the typical nuclear filaments found in OPMD. Electrodiagnostic and pathologic studies were also conducted to better understand the disease process. An illustrative case is presented.

Sparing of striatal neurons coexpressing calretinin and substance P (NK1) receptor in Huntington's disease.

Immunohistochemical studies of the striatum in normal human subjects with a double-antigen localization method have revealed the presence of large and medium-sized aspiny neurons displaying immunoreactivity for both the calcium-binding protein calretinin and substance P (neurokinin-1) receptor. These large and medium-sized cells from two distinct classes of striatal interneurons, which together represent less than 3% of the total neuronal population of the human striatum. Observations made in four cases of Huntington's disease revealed that such doubly labeled interneurons are still present in the striatum of these patients, despite the marked atrophy of the structure. This study provides the first evidence for the existence of interneurons containing calretinin and expressing tachykinin receptors in the human striatum. It also demonstrates the selective sparing of these chemospecific striatal neurons in Huntington's disease.

[Multiple intracerebral granulocytic sarcomas in a patient with chronic myeloid leukemia]. / Sarcomes granulocytaires intracérébraux multiples chez un patient atteint de leucémie myéloïde chronique.

The authors describe a young man with known chronic myeloid leukemia who sought medical attention after a first episode of seizure. The hematologic investigation revealed hyperleukocytosis without evidence of blastic transformation in peripheral blood or bone marrow. Numerous intracerebral nodules, characterized pathologically as granulocytic sarcomas, were demonstrated by computed tomography and magnetic resonance imaging (MRI). The appearance of a granulocytic sarcoma in chronic myeloid leukemia is unusual, and location of the lesion in the central nervous system is rare. Follow-up MRI after treatment showed almost complete regression of all lesions. The radiologic features of this case are discussed with reference to others reported in the literature.

Interaction of 5-bromocamphor with cytochrome P-450 cam. Production of 5-ketocamphor from a mixed spin state hemoprotein.

Camphor is stereospecifically hydroxylated by the soil bacterium Pseudomonas putida at the 5-exo position by a cytochrome P-450 mixed function oxidase system consisting of a flavoprotein reductase; putidaredoxin, an iron-sulfur oxidation-reduction transport-effector protein; and the P-450 hemoprotein. We have studied the interaction of a substrate analog of camphor, 5-exo-bromocamphor, with this cytochrome P-450 mixed function oxidase system in order to probe the molecular mechanisms of electron transport and catalytic substrate oxygenation. 5-exo-Bromocamphor was found to bind tightly in a 1:1 complex with P-450 with a dissociation constant of 2.9 microM, very near that for the normal camphor substrate. Contrary to camphor, however, the 5-exo-bromocamphor-bound material exhibits only a 46% population of the high spin form of the macromolecule. Measurement of the oxidation-reduction potential of the bromocamphor-P-450 complex yields a value of E0' = -246 mV, intermediate between that of substrate-free (-300 mV) and camphor-bound (-173 mV) cytochrome. These data are interpreted in terms of a thermodynamic linkage model relating spin, substrate, and oxidation-reduction equilibria in the P-450 monoxygenase system. 5-exo-Bromocamphor is readily metabolized by the P-450cam mixed function oxidase to 5-ketocamphor at rates and yields similar to that of the normal substrate, camphor, suggesting abstraction of the endo-hydrogen of 5-exo-bromocamphor and oxygen addition to produce a transient 5-bromo-5-hydroxycamphor intermediate. Implications of these chemical findings for the chemical mechanisms of P-450-catalyzed oxygenations and the flexibility of substrates at the active site of the macromolecule are discussed.