Dynamic properties of cortical evoked (10 Hz) oscillations: theory and experiment

Experiments probed the dynamic properties of stimulus-evoked (10 Hz) oscillations in somatosensory cortex of anesthetized rats. Experimental paradigms and statistical time series analysis were based on theoretical ideas from a dynamic approach to temporal patterns of neuronal activity. From the results of a double-stimulus paradigm we conclude that the neuronal response contains two components with different dynamics and different coupling to the stimulus. Based on this result a quantitative dynamic model is derived, making use of normal form theory for bifurcating vector fields. The variables used are abstract, but measurable, dynamic components. The model parameters capture the dynamic properties of neuronal response and are related to experimental results. A structural interpretation of the model can be given in terms of the collective dynamics of neuronal groups, their mutual interaction, and their coupling to peripheral stimuli. The model predicts the stimulusdependent lifetime of the oscillations as observed in experiment. We show that this prediction relies on the basic concept of dynamic bistability and does not depend on the modeling details.     

Copper activation of superoxide dismutase 1 (SOD1) in vivo. Role for protein-protein interactions with the copper chaperone for SOD1

Insertion of copper into superoxide dismutase 1 (SOD1) in vivo requires the copper chaperone for SOD1 (CCS). CCS encompasses three protein domains: copper binding Domains I and III at the amino and carboxyl termini, and a central Domain II homologous to SOD1. Using a yeast interaction mating system, yeast CCS was seen to physically interact with SOD1, and this interaction required sequences at the predicted dimer interface of CCS Domain II. Interactions with SOD1 also required sequences of Domain III, but not Domain I. Mutations were introduced at the dimer interface of yeast SOD1, and the corresponding mutant failed to interact with CCS. When loaded with copper independent of CCS, this mutant SOD1 exhibited superoxide scavenging activity, but was normally inactive in vivo because CCS failed to recognize the enzyme. Activation of SOD1 by CCS was also examined using an in vivo assay for copper incorporation into SOD1. Yeast CCS was observed to insert copper into a pre-existing pool of apoSOD1 without the need for new SOD1 synthesis or for protein unfolding by the major SSA cytosolic heat shock proteins. Our data are consistent with a model in which prefolded dimers of apoSOD1 serve as substrate for the CCS copper chaperone.     

Sequential discharges of phasic activities (PGO waves) during paradoxical sleep after selective cortical lesions in the cat.

We investigated the role that cortical areas have upon the sequences of PGO waves and associated phasic activity in the lateral rectus muscles of the eyes (PALRE) during PS. We performed several combined type of lesions (bilateral ablations of frontal lobes carried out in two sessions, or occipital lesions combined with bifrontla lobotomies). We used statistical methods previously described for the normal cat (4). We showed that frontal lesions need to be bilateral in order to be effective, that thereafter there is a recovery in the complexity of the patterns in the survival period. We showed that occipital lobectomies "per se" do not affect the PGO pattern, but their combination with bifrontal lobotomies produce the most dramatic changes which lasted throughout the survival period. These findings are discussed on the light of the well known anatomical cortical projections to brain stem structures which participate in the determinism of PGO waves during paradoxical sleep.     

In vitro methylation of yeast tRNAAsp by rat brain cortical tRNA-(adenine-1) methyltransferase.

Rat brain cortices from young animals contain large amounts of tRNA (adenine-1)methyltransferase(s). The enzyme(s) can methylate E. coli tRNA and to a lower degree yeast tRNA. Among yeast tRNA species which can be methylated we have selected tRNAAsp as a substrate for the brain enzyme. The digestions of in vitro methylated [Me-3H]-tRNAAsp with pancreatic and/or T1 ribonucleases followed by chromatographies on DEAE-cellulose, 7 M urea, suggested that the methylation of tRNAAsp occurred at a single position within the D-loop. Further digestion of the radioactive oligonucleotide recovered after DEAE-cellulose chromatography by phosphomonoesterase and snake venom phosphodiesterase enzymes followed by bidimensional thin layer chromatography enabled us to determine the location of the adenine residue which becomes methylated by the brain enzyme. This one resulted to be the adenine 14 in the D-loop of yeast tRNAAsp.     

A gene for familial paroxysmal dyskinesia (FPD1) maps to chromosome 2q.

Dyskinesias are hyperkinetic and involuntary movements that may result from any of a number of different genetic, infectious, and drug-induced causes. Some of the hereditary dyskinetic syndromes are characterized by paroxysmal onset of the abnormal movements. The classification of the familial paroxysmal dyskinesias (FPD) recognizes several distinct, although overlapping, phenotypes. Different forms of the disorder include attacks that are (1) induced by sudden movement (kinesiogenic); (2) spontaneous (non-kinesiogenic); and (3) induced by prolonged periods of exertion. Linkage analysis was pursued in a family segregating an autosomal dominant allele for non-kinesiogenic FPD. The disease allele was mapped to a locus on chromosome 2q31-36 (LOD score 4.64, theta = 0). Identification of distinct genetic loci for the paroxysmal dyskinesias will lead to a new genetic classification and to better understanding of these disorders.     

In vivo topography of Rap1p-DNA complex at Saccharomyces cerevisiae TEF2 UAS(RPG) during transcriptional regulation

We have analyzed in detail the structure of RAP1-UAS(RPG) complexes in Saccharomyces cerevisiae cells using multi-hit KMnO(4), UV and micrococcal nuclease high-resolution footprinting. Three copies of the Rap1 protein are bound to the promoter simultaneously in exponentially growing cells, as shown by KMnO(4) multi-hit footprinting analysis, causing extended and diagnostic changes in the DNA structure of the region containing the UAS(RPG). Amino acid starvation does not cause loss of Rap1p from the complex; however, in vivo UV-footprinting reveals the occurrence of structural modifications of the complex. Moreover, low-resolution micrococcal nuclease digestion shows that the chromatin of the entire region is devoid of positioned nucleosomes but is susceptible to changes in accessibility to the nuclease upon amino acid starvation. The implications of these results for the mechanism of Rap1p action are discussed.     

In vivo footprinting of a low molecular weight glutenin gene (LMWG-1D1) in wheat endosperm.

The quality of the wheat grain is determined by the quantity and composition of storage proteins (prolamins) which are synthesized exclusively in endosperm tissue. We are investigating the mechanisms underlying the regulation of expression of a prolamin gene, the low molecular weight glutenin gene LMWG-1D1. The LMWG-1D1 promoter contains the endosperm box, a sequence motif highly conserved in the promoter region of a large number of storage protein genes, which is thought to confer endosperm-specific expression of prolamin genes. Here we show by in vivo DMS footprinting of wheat endosperm tissue that the endosperm box becomes occupied by putative trans-acting factors during grain ripening. During early stages of development the endosperm motif within the 5' half of the endosperm box becomes occupied first, followed by binding of a second activity to a GCN4/jun-like motif in the 3' half just prior to the stage of maximum gene expression. Occupancy of the endosperm box is highly tissue-specific: no protection was observed in husk and leaf tissues. Several binding activities were identified in vitro from nuclear protein extracts of wheat endosperm which bind specifically to the endosperm and GCN4/jun motifs identified by in vivo footprinting.     

In vivo expression of IL-1 beta and IL-6 genes during viral infections in human.

Macrophage infiltration is a constant feature of human virus-infected tissues. However, the in situ functional status of these cells remains undetermined. In order to document an activation of macrophages in virus-infected tissues, the expression of IL-1 beta and IL-6 genes was analyzed using in situ hybridization. Several tissues were studied, as well as infections induced by different viruses: lymph nodes infected by HIV-1 (9 cases) or EBV (one case), lungs infected by CMV (5 cases) or adenovirus (1 case), livers infected by HBV, either chronically (2 cases) or acutely (7 cases presenting a fulminant hepatitis). With the exception of fulminant HBV hepatitis, IL-1 beta and IL-6 genes were expressed in all cases. IL-1 beta and IL-6 genes were usually coordinately regulated, as cells containing IL-1 beta or IL-6 mRNA were present in identical amounts and displayed a similar distribution. Analysis of the location and the morphology of monokine gene-expressing cells indicated that both small macrophages and endothelial cells expressed IL-1 beta and IL-6 genes. However, neither tingible body macrophages present in lymph node follicles nor Kupffer cells expressed these genes at a detectable level. Infected cells themselves were also negative for monokine gene expression. These findings indicate that expression of IL-1 beta and IL-6 genes by reactive cells may play a role in viral spreading limitation as well as virus-induced tissue damage.     

In vivo selection for the directed evolution of L-rhamnulose aldolase from L-rhamnulose-1-phosphate aldolase (RhaD)

Dihydroxyacetone phosphate (DHAP)-dependent aldolases have been widely used for organic synthesis. The major drawback of DHAP-dependent aldolases is their strict donor substrate specificity toward DHAP, which is expensive and unstable. Here we report the development of an in vivo selection system for the directed evolution of the DHAP-dependent aldolase, L-rhamnulose-1-phosphate aldolase (RhaD), to alter its donor substrate specificity from DHAP to dihydroxyacetone (DHA). We also report preliminary results on mutants that were discovered with this screen. A strain deficient in the L-rhamnose metabolic pathway in Escherichia coli (DeltarhaDAB, DE3) was constructed and used as a selection host strain. Co-expression of L-rhamnose isomerase (rhaA) and rhaD in the selection host did not restore its growth on minimal plate supplemented with L-rhamnose as a sole carbon source, because of the lack of L-rhamnulose kinase (RhaB) activity and the inability of WT RhaD aldolase to use unphosphorylated L-rhamnulose as a substrate. Use of this selection host and co-expression vector system gives us an in vivo selection for the desired mutant RhaD which can cleave unphosphorylated L-rhamnulose and allow the mutant to grow in the minimal media. An error-prone PCR (ep-PCR) library of rhaD gene on the co-expression vector was constructed and introduced into the rha-mutant, and survivors were selected in minimal media with l-rhamnose (MMRha media). An initial round of screening gave mutants allowing the selection strain to grow on MMRha plates. This in vivo selection system allows rapid screening of mutated aldolases that can utilize dihydroxyacetone as a donor substrate.     

In vitro and in vivo studies of F(0)F(1)ATP synthase regulation by inhibitor protein IF(1) in goat heart

A method has been developed to allow the level of F(0)F(1)ATP synthase capacity and the quantity of IF(1) bound to this enzyme be measured in single biopsy samples of goat heart. ATP synthase capacity was determined from the maximal mitochondrial ATP hydrolysis rate and IF(1) content was determined by detergent extraction followed by blue native gel electrophoresis, two-dimensional SDS-PAGE and immunoblotting with anti-IF(1) antibodies. Anaesthetized open-chest goats were subjected to ischemic preconditioning and/or sudden increases of coronary blood flow (CBF) (reactive hyperemia). When hyperemia was induced before ischemic preconditioning, a steep increase in synthase capacity, followed by a deep decrease, was observed. In contrast, hyperemia did not affect synthase capacity when applied after ischemic preconditioning. Similar effects could be produced in vitro by treatment of heart biopsy samples with anoxia (down-regulation of the ATP synthase) or high-salt or high-pH buffers (up-regulation). We show that both in vitro and in vivo the same close inverse correlation exists between enzyme activity and IF(1) content, demonstrating that under all conditions tested the only significant modulator of the enzyme activity was IF(1). In addition, both in vivo and in vitro, 1.3-1.4 mol of IF(1) was predicted to fully inactivate 1 mol of synthase, thus excluding the existence of significant numbers of non-inhibitory binding sites for IF(1) in the F(0) sector.     

Peripheral benzodiazepine binding sites: effect of PK 11195, 1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3 isoquinolinecarboxamide. II. In vivo studies

Peripheral type of benzodiazepine binding sites were labelled in the kidney, the heart and the brain with [3H] RO5-4864 following intravenous injection in mice. The regional distribution of this in vivo binding parallels the in vitro binding: heart and kidney were more labelled than brain. Benzodiazepine potencies in reducing [3H] RO5-4864 binding in vivo parallel relative affinities for [3H] RO5-4864 binding sites in isolated organs membranes: RO5-4864 greater than diazepam greater than clonazepam. PK 11195 a new compound, chemically unrelated to benzodiazepines, which is a potent inhibitor of [3H] RO5-4864 in vitro is also very effective (more than RO5-4864) after I.P. injection and oral administration. These results emphasize the feasibility of using this technique to examine the effects on various pharmacological and physiological manipulations of these binding sites in vivo. Moreover the fact that PK 11195 binds to these sites in vivo might indicate that this compound could help to elucidate the physiological relevance of the peripheral type of benzodiazepine binding sites.     

Vascular adhesion protein 1 (VAP-1) functions as a molecular brake during granulocyte rolling and mediates recruitment in vivo.

Granulocyte extravasation from the blood into tissues is a prerequisite for a proper inflammatory response. It is regulated by a multistep adhesion cascade consisting of successive contacts between leukocyte surface receptors and their endothelial ligands on vessels. Vascular adhesion protein 1 (VAP-1) is an endothelial surface glycoprotein with two functions. It is an enzyme (monoamine oxidase) and an adhesion molecule for lymphocytes. Its function in binding of granulocytes or in leukocyte trafficking into sites of inflammation in vivo has remained unknown. Here we show that treatment of rabbits with anti-VAP-1 monoclonal antibodies abrogates approximately 70% of granulocyte extravasation into a site of an experimental inflammation. Using intravital microscopy, VAP-1 blockade is shown to increase the velocity of the rolling granulocytes and the frequency of their jerky skippings during the rolling. In addition, the number of firmly bound leukocytes decreased by 44% when VAP-1 was rendered nonfunctional. Our results suggest that VAP-1 functions as a molecular brake early in the adhesion cascade and consequently decreases the firm adherence; it may also directly influence the transmigration step. These data elucidate a new interplayer in the granulocyte extravasation process and provide a novel physiological function for a member of the monoamine oxidase family.     

In vivo control of gluconeogenesis in wild-type Neurospora crassa and in the adenylate cyclase-deficient cr-1 (crisp) mutant.

The rate of cycloheximide-resistant incorporation of carbon from [14C]alanine and [14C]acetate into polysaccharidic material was used to study gluconeogenic activity in wild-type Neurospora crassa and in the adenylate cyclase-deficient cr-1 (crisp-1) mutant. The wild-type efficiently utilized alanine and acetate as gluconeogenic substrates, whereas the mutant used acetate efficiently but was unable to use alanine. Cycloheximide-resistant 14C-incorporating activity was sensitive to carbon catabolite effects (repression and inactivation) in the two strains, which suggested that cyclic AMP metabolism was not involved in these regulatory responses. In the wild type, gluconeogenesis was induced by incubation of the cells in the absence of a carbon source. In contrast, cr-1 required supplementation with acetate. This finding suggested that induction of gluconeogenesis in N. crassa could be mediated by metabolites formed in carbon-starved cells. The cr-1 mutant seemed to be deficient in this process and to depend on an exogenous effector to induce gluconeogenesis. Incubation of cr-1 with cyclic AMP partially overcame the acetate requirement for induction of gluconeogenesis.     

Cell-specific in vivo DNA-protein interactions at the proximal promoters of the pro alpha 1(I) and the pro alpha2(I) collagen genes.

We performed in vivo dimethylsulfate footprinting of the 220 bp mouse proximal proalpha1(I) collagen promoter and the 350 bp mouse proximal proalpha2(I) collagen promoter in BALB/3T3 fibroblasts, primary mouse skin fibroblasts, S-194 B cells, NMuLi liver epithelial cells and RAG renal adenocarcinoma cells and in vitro DNase I footprinting of these promoters using nuclear extracts of these different cell types. Whereas proalpha1(I) and proalpha2(I) collagen RNAs were present in BALB/3T3 fibroblasts and primary fibroblasts, these RNAs could not be detected in the three other cell lines. Comparison of in vitro DNase I footprints for each of the two proximal collagen promoters indicated that the patterns of protection were very similar with the different nuclear extracts, suggesting that the DNA binding proteins binding to these promoters were present in all cell types tested. In contrast, in vivo footprints over these proximal promoters were cell-specific, occurring only in fibroblast cells and not in the other three cell types. The in vivo footprints were generally located within the in vitro footprinted regions. Our results suggest that although all cell types tested contained nuclear proteins that can bind to the proximal proalpha1(I) and proalpha2(I) collagen promoters in vitro , it is only in fibroblasts that these proteins bind to their cognate sites in vivo . We discuss possible regulatory mechanisms in type I collagen genes that can contribute to the cell-specific in vivo protein-DNA interactions at the proximal promoters.     

Investigating cryoinjury using simulations and experiments: 2. TF-1 cells during graded freezing (interrupted slow cooling without hold time)

Cryopreservation plays a key role in the long-term storage of native and engineered cells and tissues for research and clinical applications. The survival of cells and tissues after freezing and thawing depends on the ability of the cells to withstand a variety of stresses imposed by the cryopreservation protocol. A better understanding of the nature and kinetics of cellular responses to temperature-induced conditions is required to minimize cryoinjury. An interrupted freezing procedure that allows dissection of cryoinjury was used to investigate the progressive damage that occurs to cells during cryopreservation using slow cooling. Simulations of cellular osmotic responses were used to provide interpretation linking states of the cell with events during the freezing procedure. Simulations of graded freezing (interrupted slow cooling without hold time) were correlated with cell recovery results of TF-1 cells. Calculated intracellular supercooling and osmolality, were used as indicators of the probability of cryoinjury due to intracellular ice formation and solution effects, providing direct links of cellular conditions to events in the freezing process. Using simulations, this study demonstrated that both intracellular supercooling and osmolality are necessary to explain graded freezing results.