Effect of calcium on brain metabolism in vitro

In attempts to distinguish between direct and indirect effects of Ca on brain cell metabolism, respiration, glycolysis, ATP, phosphocreatine, incorporation of [¹⁴C] leucine into protein, and accumulation of⁴⁵Ca was determined in brain slices. Incubation was carried out in normal salt-balanced medium, in high-potassiumor ouabain-containing medium under aerobic and anaerobic conditions. Calcium ions inhibited slightly glycolysis and respiration in normal medium and activated amino acid incorporation into proteins. Levels of ATP and phosphocreatine remained normal. These effects were interpreted as due to a stabilization of plasma membranes by Ca ions to prevent their spontaneous depolarization. Incubation of slices in high-potassium and ouabain media in aerobic conditions in the presence of Ca resulted in activation of respiration and glycolysis, decrease of ATP and phosphocreatine levels, and inhibition of amino acid incorporation into proteins. The disturbances in energy metabolism, caused by the respiration-linked Ca uptake in brain mitochondria and concomitant inhibition of oxidative phosphorylation, may lead to the inhibition of amino acid incorporation into proteins. An increase in Ca levels in the cytoplasm may only be expected in anaerobic conditions during the incubation in high-potassium and ouabain media. This is manifested by a direct inhibition of glycolysis by Ca ions and a drastic decrease of ATP and phosphocreatine in slices. The results suggest that stimulation of aerobic glycolysis and inhibition of anaerobic glycolysis by Ca may explain the unknown mechanism of the so-called reversed Pasteur effect of brain slices incubated in high-potassium media.     

Virus-modified homologus tumor-cell extract in the treatment of vulvar carcinoma

Summary Eight patients with squamous carcinoma of the vulva and two or more positive nodes have received adjunctive immunotherapy with a virus modified homologous cell extract. Seven of eight patients received radiation therapy in addition. Cells derived from the SW962 vulvar carcinoma cell line were infected with PR8/A/34 strain of influenza and a membrane extract was used for immunization. The extract was administered by the intradermal route weekly for three doses and then biweekly for up to 2 years. Each dose is equivalent to 1.5 mg protein. None of the patients have recurred and durations of remission are 24, 24, 22, 22, 21, 16, 7, and 2 months respectively. This compares favourably with similar groups of patients who were treated with surgery alone (22/33 recurred, median recurrence time 14.8 months) or surgery plus radiation therapy (8/9 recurrences, median recurrence time 11.0 months). No serious side effects have occurred with more than 200 doses of extract.Post immunization monitoring has indicated good in vitro and in vivo immunological responses and antibody titers to PR8 increased significantly in five of eight patients.Dr. Rutledge is Head of the Department of Gynecology     

Preparation of glial plasma membrane from a cell fraction enriched in astrocytes

A technique for obtaining glial plasma membrane has been developed, starting with a bulk-prepared glial cell-enriched fraction from rabbit cerebral cortex. The astrocytic-enriched fraction was hand-homogenized in isotonic sucrose media, and the crude membrane fraction sedimented at 3,000g. The isolation of a membrane-enriched fraction was accomplished with sucrose density gradient centrifugation. The plasma membrane fraction was collected at the interphase between 31.5% and 25.5% sucrose. Enzymatic and electron-microscopical analyses indicated a 4–7-fold enrichment in plasma membrane, and a 15–20% contamination with microsomal and mitochondrial material. Some multilaminar membrane structures were also seen in the fraction.     

POTASSIUM ACCUMULATION BY BULK PREPARED NEURONAL AND GLIAL CELLS

Abstract— Neuronal and glial cell enriched fractions were prepared by density gradient centrifugation of suspensions from rabbit cerebral cortex. The two cell types were incubated separately in media of extracellular ionic composition. The potassium accumulation was determined from analysis of potassium content of the cells by ultramicro flame photometry. Both neuronal and glial cells were capable of active potassium transport which was inhibited by ouabain (2 × 10⁻⁴ m ). The glial cells could accumulate potassium up to four to five times the concentration of the incubation medium and neurons up to one and a half to two times the medium concentration. The respiration in low potassium media was stimulated 15 per cent for neurons and 85 per cent for glia when potassium was added to a final concentration of 50 m m . The uptake by both neurons and glia showed temperature and sodium dependence. There was a definite magnesium requirement for the potassium uptake, particularly demonstrable for glial cells. Calcium inhibited potassium uptake by glia but stimulated slightly that by neurons.     

Structural basis of DNA polymerase θ mediated DNA end joining

DNA polymerase θ (Pol θ) plays an essential role in the microhomology-mediated end joining (MMEJ) pathway for repairing DNA double-strand breaks. However, the mechanisms by which Pol θ recognizes microhomologous DNA ends and performs low-fidelity DNA synthesis remain unclear. Here, we present cryo-electron microscope structures of the polymerase domain of Lates calcarifer Pol θ with long and short duplex DNA at up to 2.4 Å resolution. Interestingly, Pol θ binds to long and short DNA substrates similarly, with extensive interactions around the active site. Moreover, Pol θ shares a similar active site as high-fidelity A-family polymerases with its finger domain well-closed but differs in having hydrophilic residues surrounding the nascent base pair. Computational simulations and mutagenesis studies suggest that the unique insertion loops of Pol θ help to stabilize short DNA binding and assemble the active site for MMEJ repair. Taken together, our results illustrate the structural basis of Pol θ-mediated MMEJ.     

Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca) BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome

Background  

Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer.     

Quantitative immunochemistry on neuronal loss, reactive gliosis and BBB damage in cortex/striatum and hippocampus/amygdala after systemic kainic acid administration

Cell specific markers were quantified in the hippocampus, the amygdala/pyriform cortex, the frontal cerebral cortex and the striatum of the rat brain after systemic administration of kainic acid. Neuron specific enolase (NSE) reflects loss of neurons, glial fibrillary acidic protein (GFAP) reflects reactive gliosis, and brain levels of serum proteins measures blood-brain-barrier permeability. While the concentration of NSE remained unaffected in the frontal cerebral cortex and the striatum, their GFAP content increased during the first three days. In the hippocampus and amygdala, NSE levels decreased significantly. GFAP levels in the hippocampus were unaffected after one day and decreased in the amygdala/pyriform cortex. After that, GFAP increased strikingly until day 9 or, in the case of amygdala/pyriform cortex, even longer. This biphasic time course for GFAP was accompanied by a decrease of S-100 during days 1-9 followed by a significant increase at day 27 above the initial level. The regional differences in GFAP and S-100 could result from the degree of neuronal degeneration, the astrocytic receptor set-up and/or effects on the blood-brain barrier.     

N-WASP has the ability to compensate for the loss of WASP in macrophage podosome formation and chemotaxis

Wiskott-Aldrich syndrome protein (WASP) and its homologue neural-WASP (N-WASP) are nucleation promoting factors that integrate receptor signaling with actin cytoskeleton rearrangement. While hematopoietic cells express both WASP and N-WASP, WASP deficiency results in altered cell morphology, loss of podosomes and defective chemotaxis. It was determined that cells from a mouse derived monocyte/macrophage cell line and primary cells of myeloid lineage expressed approximately 15-fold higher levels of WASP relative to N-WASP. To test whether N-WASP can compensate for the loss of WASP and restore actin cytoskeleton integrity, N-WASP was overexpressed in macrophages, in which endogenous WASP expression was reduced by short hairpin RNA (shWASP cells). Many of the defects associated with the loss of WASP, such as podosome-dependent matrix degradation and chemotaxis were corrected when N-WASP was expressed at equimolar level to that of the wild-type WASP. Furthermore, the ability of N-WASP to partially compensate for the loss of WASP may be physiologically relevant since activated murine WASP-deficient peritoneal macrophages, which show enhanced N-WASP expression, also show an increase in matrix degradation. Our study suggests that expression levels of WASP and N-WASP may influence their roles in actin cytoskeleton rearrangement and shed light to the complex intertwining roles WASP and N-WASP play in macrophages.