Role of sodium and potassium ions in regulation of glucose metabolism in cultured astroglia

Effects of increasing extracellular K+ or intracellular Na+ concentrations on glucose metabolism in cultures of rat astroglia and neurons were examined. Cells were incubated in bicarbonate buffer, pH 7.2, containing 2 mM glucose, tracer amounts of [14C]deoxyglucose ([14C]dGlc), and 5.4, 28, or 56 mM KCl for 10, 15, or 30 min, and then for 5 min in [14C]dGlc-free buffer to allow efflux of unmetabolized [14C]dGlc. Cells were then digested and assayed for labeled products, which were shown to consist of 96-98% [14C]deoxyglucose 6-phosphate. Increased K+ concentrations significantly raised [14C]deoxyglucose 6-phosphate accumulation in both neuronal and mixed neuronal-astroglial cultures at 15 and 30 min but did not raise it in astroglial cultures. Veratridine (75 microM), which opens voltage-dependent Na+ channels, significantly raised rates of [14C]dGlc phosphorylation in astroglial cultures (+20%), and these elevations were blocked by either 1 mM ouabain, a specific inhibitor of Na+,K(+)-ATPase (EC 3.6.1.37), or 10 microM tetrodotoxin, which blocks Na+ channels. The carboxylic sodium ionophore, monensin (10 microM), more than doubled [14C]dGlc phosphorylation; this effect was only partially blocked by ouabain and unaffected by tetrodotoxin. L-Glutamate (500 microM) also stimulated [14C]dGlc phosphorylation in astroglia--not through N-methyl-D-aspartate or non-N-methyl-D-aspartate receptor mechanisms but via a Na(+)-dependent glutamate-uptake system. These results indicate that increased uptake of Na+ can stimulate energy metabolism in astroglial cells.     

Practical model fitting approaches to the direct extraction of NMR parameters simultaneously from all dimensions of multidimensional NMR spectra

A maximum likelihood (ML)-based approach has been established for the direct extraction of NMR parameters (e.g., frequency, amplitude, phase, and decay rate) simultaneously from all dimensions of a D-dimensional NMR spectrum. The approach, referred to here as HTFD-ML (hybrid time frequency domain maximum likelihood), constructs a time-domain model composed of a sum of exponentially-decaying sinusoidal signals. The apodized Fourier transform of this time-domain signal is a model spectrum that represents the 'best-fit' to the equivalent frequency-domain data spectrum. The desired amplitude and frequency parameters can be extracted directly from the signal model constructed by the HTFD-ML algorithm. The HTFD-ML approach presented here, as embodied in the software package CHIFIT, is designed to meet the challenges posed by model fitting of D-dimensional NMR data sets, where each consists of many data points (10(8) is not uncommon) encoding information about numerous signals (up to 10(5) for a protein of moderate size) that exhibit spectral overlap. The suitability of the approach is demonstrated by its application to the concerted analysis of a series of ten 2D 1H-15N HSQC experiments measuring 15N T1 relaxation. In addition to demonstrating the practicality of performing maximum likelihood analysis on large, multidimensional NMR spectra, the results demonstrate that this parametric model-fitting approach provides more accurate amplitude and frequency estimates than those obtained from conventional peak-based analysis of the FT spectrum. The improved performance of the model fitting approach derives from its ability to take into account the simultaneous contributions of all signals in a crowded spectral region (deconvolution) as well as to incorporate prior knowledge in constructing models to fit the data.     

The MAGlC project: from vision to reality

In the MAGIC project, three major components-an ATM internetwork, a distributed, network-based storage system; and a terrain visualization application-were designed, implemented, and integrated to create a testbed for demonstrating real-time, interactive exchange of data at high speeds distributed resources. The MAGIC internetwork, depicted includes six high-speed local area networks (LANs) interconnected by a wide area network (WAN) backbone. The testbed was developed as a system, with special consideration to how performance was affected by interactions among the components. This article presents an overview of the project, with emphasis on the challenges associated with implementing complex distributed system, and with coordinating a multi-organization collaborative project that relied on distributed development. System level design issues and performance measurements are described, as it is a tool that was developed for analyzing performance and diagnosing problems in a distributed system. The management challenges that were encountered and some of the lessons learned during the course of the three-year project are discussed, and a brief summary of MAGIC-II, a follow-on project, is given     

Cannabinoid-induced alterations in regional cerebral blood flow in the rat

A specific receptor for cannabinoids has been characterized at the pharmacological, molecular, and neuroanatomical level. However, less is known of the functional localization in the brain for the behavioral and physiological actions of these drugs. We have examined the effects of delta 9-tetrahydrocannabinol (THC) and its active metabolite 11-OH-THC on regional cerebral blood flow in the rat in order to determine functional CNS sites of action for the cannabinoids. Conscious rats were injected i.v. with one of four doses of THC (0.5, 1, 4, 16 mg/kg). 11-OH-THC (4 mg/kg), or vehicle 30 min prior to sacrifice. Regional cerebral blood flow was determined autoradiographically using the freely diffusible tracer method of Sakaruda et al. Changes in regional cerebral blood flow were observed in 16 of the 37 areas measured. Decreases in regional cerebral blood flow following THC were seen in such areas as the CA1 region of the hippocampus, frontal and medial prefrontal cortex, the nucleus accumbens, and the claustrum. Thresholds for these effects ranged from 0.5 to 16 mg/kg. Areas unaffected by THC include the medial septum, ventral tegmental area, caudate, temporal, parietal and occipital cortex, and cerebellum. These data indicate that THC and its active metabolite, 11-OH-THC, cause a heterogeneous alteration in the activity of specific CNS sites, many of which are involved in the characteristic behavioral actions of THC.     

Influence of rib spacing in proton-exchange membrane electrode assemblies

A two-dimensional design analysis of a membrane-electrode assembly for a proton-exchange membrane fuel cell is presented. Specifically, the ribs of the bipolar plates restrict the access of fuel and oxidant gases to the catalyst layer. The expected change in cell performance that results from the partial blocking of the substrate layer is studied by numerical simulation of the oxygen electrode and the membrane separator. The effects of rib sizing and the thickness of the gas-diffusion electrode on the current and water distributions within the cell are presented. For all of the cases considered, the two-dimensional effect only slightly alters the half-cell potential for a given applied current but has a significant influence on water management. Concentrated solution theory with variable transport properties is used in the membrane electrolyte to solve for the electrical potential and local water content. The Stefan-Maxwell equations are used in the gas-diffusion electrode to determine the local mole fractions of nitrogen, oxygen and water vapour. A control-volume formulation is used for the resolution of the coupled nonlinear differential equations. One advantage of the control-volume approach over finite-difference methods is the relative ease in which internal boundary points in fuel-cell and battery models are handled. This and other advantages are briefly discussed.     

Molecular and functional characterization of a calcium-sensitive chloride channel from mouse lung

A protein (mCLCA1) has been cloned from a mouse lung cDNA library that bears strong sequence homology with the recently described bovine tracheal, Ca2+-sensitive chloride channel protein (bCLCA1), bovine lung endothelial cell adhesion molecule-1 (Lu-ECAM-1), and the human intestinal Ca2+-sensitive chloride channel protein (hCLCA1). In vitro, its 3.1-kilobase message translates into a 100-kDa protein that can be glycosylated to an approximately 125-kDa product. SDS-polyacrylamide gel electrophoresis from lysates of mCLCA1 cDNA-transfected transformed human embryonic kidney cells (HEK293) reveals proteins of 130, 125, and 90 kDa as well as a protein triplet in the 32-38 kDa size range. Western analyses with antisera raised against Lu-ECAM-1 peptides show that the N-terminal region of the predicted open reading frame is present only in the larger size proteins (i.e. 130, 125, and 90 kDa), whereas the C-terminal region of the open reading frame is observed in the 32-38 kDa size proteins, suggesting a posttranslational, proteolytic processing of a precursor protein (125/130 kDa) into 90 kDa and 32-38 kDa components similar to that reported for Lu-ECAM-1. Hydrophobicity analyses predict four transmembrane domains for the 90-kDa protein. The mCLCA1 mRNA is readily detected by Northern analysis and by in situ hybridization in the respiratory epithelia of trachea and bronchi. Transient expression of mCLCA1 in HEK293 cells was associated with an increase in whole cell Cl- current that could be activated by Ca2+ and ionomycin and inhibited by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, dithiothreitol, and niflumic acid. The discovery of mCLCA1 opens the door for further investigating the possible contribution of a Ca2+-sensitive chloride conductance to the pathogenesis of cystic fibrosis.     

Protein organization on the PHA inclusion cytoplasmic boundary

Biotransformation of ethanol by liver nuclei was studied. The formation of acetaldehyde was determined by GC/FID. The 1-hydroxyethyl (1HEt) formation was established by spin trapping of the radical with N-t-butyl-alpha-phenylnitrone (PBN) followed by GC/MS. Liver nuclei, free of endoplasmic reticulum, cytosol or mitochondria, were able to biotransform ethanol to acetaldehyde in the presence of NADPH under air. Only 22% activity was observed in the absence of the cofactor. Twenty-six percent of the NADPH-dependent activity and 47% of the NADPH-independent activity were observable under nitrogen. Aerobic biotransformation was inhibited by CO, SKF 525A, 4-methylpyrazole and by diethyldithiocarbamate. This suggests that CYP2E1 is involved in the process. However, the formation of acetaldehyde was able to proceed under a pure CO atmosphere. The lack of inhibitory effects of 2-mercapto-1-methylimidazol and thiobenzamide excludes the potential participation of the NADPH flavin monooxigenase system. The formation of hydroxyl radicals in the process is suggested by the partial inhibitory effect of 5 mM mannitol and 5 mM sodium benzoate and by the fact that the 1HEt was detected. The NADPH-dependent anaerobic ethanol biotransformation pathway was stimulated by FAD and inhibited to some extent by iron chelators. The relevance of a liver nuclear ethanol biotransformation, generating reactive metabolites, such as acetaldehyde and free radicals, nearby DNA, nuclear proteins and lipids is discussed.     

Case of the month: May 1997--a 31 year old woman with seizures, fatigue and attention deficits

A 31-year-old woman with a five year history of seizures complained of worsening fatigue and attention deficits. At age 26, neuroradiological imaging studies had shown a small, non-enhancing hypodense lesion involving the left frontobasal and subcallosal region. Follow-up CT and MRI images showed that the lesion had not changed. A stereotactic biopsy showed a dysembryoplastic neuroepithelial tumor (DNT). The lesion was subtotally resected and the diagnosis of DNT was fully confirmed. However, the distinction between oligodendroglioma, ganglioglioma, focal dysplastic cerebral cortex, and DNT was only possible with knowledge of the clinical and neuroradiological data.     

Stress increases the population of splenic macrophages, permissive for Langat virus, in mice

Cytogenetic study conducted after a single intraperitoneal injection of cyclophosphane in doses of 10, 20, and 40 mg/kg showed in 1.5-2-month-old male mice that the level of bone marrow cells damaged by the mutagen was significantly higher in NZW animals than in C57Bl/6 animals. The ability to produce active oxygen forms in response to addition of opsonized zymosan and phorbol myristate acetate was also higher in bone marrow suspensions of NZW mice. The higher sensitivity of NZW animals to pro-oxidant and clastogenic effects may be related to the genetically determined decrease of antioxidant protection in NZW animals.