Electrofusion of protoplasts of anther-derived dihaploid lines of commercial potato cultivars

Selostus: Perunalajikkeiden ponsiviljelyllä tuotettujen dihaploidien protoplastien sähköfuusio

Sleep deprivation (SD) on focal brain ischemia in the rat : effects of different SD protocols

Sleep-wake disturbances are frequently observed in stroke patients and are associated with poorer functional outcome. Until now the effects of sleep on stroke evolution are unknown. The purpose of the present study was to evaluate the effects of three sleep deprivation (SD) protocols on brain damages after focal cerebral ischemia in a rat model. Permanent occlusion of distal branches of the middle cerebral artery was induced in adult rats. The animals were then subjected to 6h SD, 12h SD or sleep disturbances (SDis) in which 3 x 12h sleep deprivation were performed by gentle handling. Infarct size and brain swelling were assessed by Cresyl violet staining, and the number of damaged cells was measured by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining. Behavioral tests, namely tape removal and cylinder tests, were performed for assessing sensorimotor function. In the 6h SD protocol, no significant difference (P > 0.05) was found either in infarct size (42.5 ± 30.4 mm3 in sleep deprived animals vs. 44.5 ± 20.5 mm3 in controls, mean ± s.d.), in brain swelling (10.2 ± 3.8 % in sleep deprived animals vs. 11.3 ± 2.0 % in controls) or in number of TUNEL-positive cells (21.7 ± 2.0/mm2 in sleep deprived animals vs. 23.0 ± 1.1/mm2 in controls). In contrast, 12h sleep deprivation increased infarct size by 40 % (82.8 ± 10.9 mm3 in SD group vs. 59.2 ± 13.9 mm3 in control group, P = 0.008) and number of TUNEL-positive cells by 137 % (46.8 ± 15/mm in SD group vs. 19.7 ± 7.7/mm2 in control group, P = 0.003). There was no significant difference (P > 0.05) in brain swelling (12.9 ± 6.3 % in sleep deprived animals vs. 11.6 ± 6.0 % in controls). The SDis protocol also increased infarct size by 76 % (3 x 12h SD 58.8 ± 20.4 mm3 vs. no SD 33.8 ± 6.3 mm3, P = 0.017) and number of TUNEL-positive cells by 219 % (32.9 ± 13.2/mm2 vs. 10.3 ± 2.5/mm2, P = 0.008). Brain swelling did not show any difference between the two groups (24.5 ± 8.4 % in SD group vs. 16.7 ± 8.9 % in control group, p > 0.05). Both behavioral tests did not show any concluding results. In summary, we demonstrate that sleep deprivation aggravates brain damages in a rat model of stroke. Further experiments are needed to unveil the mechanisms underlying these effects.

Tumor cell budding and laminin-5 expression in colorectal carcinoma can be modulated by the tissue micro-environment.

Expression of laminin-5 alpha3, beta3 and gamma2 protein subunits was investigated in colorectal adenocarcinomas using immunostaining and confocal microscopy. The laminin-5 heterotrimer was found in basement membranes and as extracellular deposits in tumor stroma. In contrast to the alpha3 subunit, which was under-expressed, the gamma2 and beta3 subunits were detected in the cytoplasm of carcinoma cells dissociating (budding) from neoplastic tubules, suggestive of focal alterations in laminin-5 assembly and secretion. Laminin-5 gamma2 or beta3 subunit-reactive budding carcinoma cells expressed cytokeratins but not vimentin; they did not proliferate and were not apoptotic. Furthermore, expression of laminin-5 gamma2 and beta3 subunits in budding cells was associated with focal under-expression of the E-cadherin-beta-catenin complex. Results from xenograft experiments showed that budding activity in colorectal adenocarcinomas could be suppressed when these tumors grew at ectopic s.c. sites in nude mice. In vitro, cultured colon carcinoma cells, but not adenoma-derived tumor cells, shared the laminin-5 phenotype expressed by carcinoma cells in vivo. Using colon carcinoma cell lines implanted orthotopically and invading the cecum of nude mice, the laminin-5-associated budding was restored, indicating that this phenotype is not only determined by tumor cell properties but also dependent on the tissue micro-environment. Our results indicate that both laminin-5 alpha3 subunit expression and cell-cell cohesiveness are altered in budding carcinoma cells, which we consider to be actively invading. We propose that the local tissue micro-environment contributes to these events.

Glutamine synthesis rate in the hyperammonaemic rat brain using simultaneous localized in vivo H-1 and N-15 MRS

Objectives: Glutamine synthetase is a critical step in the glutamate-glutamine cycle, the major mechanism of glutamate neurotransmission and is implicated in the mechanism of ammonia toxicity. 15N MRS is an alternative approach to 13C MRS in studying glutamate- glutamine metabolism. 15N MRS studies allow to measure an apparent glutamine synthesis rate (Vsyn) which reflects a combination of the glutamate- glutamine cycle activity (Vnt) and net glutamine accumulation. The net glutamine synthesis (Vsyn-Vnt) can be directly measured from 1H NMR. Therefore, the aim of this study was to perform in vivo localized 1H MRS interleaved with 15N MRS to directly measure the net glutamine synthesis rate and the apparent glutamine synthesis rate under 15N labeled ammonia infusion in the rat brain, respectively. Methods: 1H and 15N MRS data were acquired interleaved on a 9.4T system (Varian/Magnex Scientific) using 5 rats. 15NH4Cl solution was infused continuously into the femoral vein for up to 10 h (4.5 mmol/h/kg).1 The plasma ammonia concentration was increased to 0.95±0.08 mmol/L (Analox GM7 analyzer). 1H spectra were acquired and quantified as described previously.2 15N unlocalized and localized spectra were acquired using the sequence;3 and quantified using AMARES and an external reference method.4 The metabolic model used to analyze the total Gln and 5-15N labeled Gln time courses is shown on Figure 1A. Results: Glutamine concentration increased from 2.5±0.3 to 15±3.3 mmol/kg whereas the total glutamate concentrations remained unchanged (Figure 1B). The linear fit of the time-evolution of the total Gln from the 1H spectra gave the net synthesis flux (Vsyn-Vnt), which was 0.021± 0.006 mmol/min per g (Figure 1D). The 5-15N Gln peak (_271 ppm) was visible in the first and all subsequent scans, whereas the 2-15N Gln/Glu peak (_342 ppm) appeared after B1.5 h (Figure 1C). From the in vivo 5-15N Gln time course, Vsyn = 0.29±0.1 mmol/min per g and a plasma NH3 fractional enrichment of 71%±6% were calculated. Vnt was 0.26±0.1 mmol/min/g, obtained assuming a negligible Gln efflux.5 Vsyn and Vnt were within the range of 13C NMR measurements.6 Conclusion: The combination of 1H and 15N NMR allowed for the first time a direct and localized measurement of Vnt and apparent glutamine synthesis rate. Vnt is approximately one order of magnitude faster than the net glutamine accumulation.

Evidence for catabolic pathway of propionate metabolism in CNS: expression pattern of methylmalonyl-CoA mutase and propionyl-CoA carboxylase alpha-subunit in developing and adult rat brain.

Methylmalonyl-CoA mutase (MCM) and propionyl-CoA carboxylase (PCC) are the key enzymes of the catabolic pathway of propionate metabolism and are mainly expressed in liver, kidney and heart. Deficiency of these enzymes leads to two classical organic acidurias: methylmalonic and propionic aciduria. Patients with these diseases suffer from a whole spectrum of neurological manifestations that are limiting their quality of life. Current treatment does not seem to effectively prevent neurological deterioration and pathophysiological mechanisms are poorly understood. In this article we show evidence for the expression of the catabolic pathway of propionate metabolism in the developing and adult rat CNS. Both, MCM and PCC enzymes are co-expressed in neurons and found in all regions of the CNS. Disease-specific metabolites such as methylmalonate, propionyl-CoA and 2-methylcitrate could thus be formed autonomously in the CNS and contribute to the pathophysiological mechanisms of neurotoxicity. In rat embryos (E15.5 and E18.5), MCM and PCC show a much higher expression level in the entire CNS than in the liver, suggesting a different, but important function of this pathway during brain development.

Inhibition of glutathione efflux in the perfused rat liver and isolated hepatocytes by organic anions and bilirubin. Kinetics, sidedness, and molecular forms.

Using isolated, in situ, single-pass perfused rat livers, incubations of freshly isolated hepatocytes, and sinusoidal membrane-enriched vesicles, we and others have shown the saturability of transport (efflux) of hepatic glutathione (GSH). These observations have implicated a carrier mechanism. Our present studies were designed to provide further evidence in support of a carrier mechanism for hepatic GSH efflux by demonstrating competition by liver-specific ligands which are taken up by hepatocytes. Perfusing livers with different substances, we found that: (a) sulfobromophthalein-GSH (BSP-GSH) had a dose-dependent and fully reversible inhibitory effect on GSH efflux, while GSH alone did not have any effect; (b) taurocholate had no inhibitory effect; (c) all of the organic anions studied, i.e., BSP, rose bengal, indocyanine green, and unconjugated bilirubin (UCB), manifested potent, dose-dependent inhibitory effects, with absence of toxic effects and complete reversibility of inhibition in the case of UCB. The inhibitory effects of UCB could be overcome partially by raising (CoCl2-induced) hepatic GSH concentration. Because of the physiological importance of UCB, we conducted a detailed study of its inhibitory kinetics in the isolated hepatocyte model in the range of circulating concentrations of UCB. Studies with Cl- -free media, to inhibit the uptake of UCB by hepatocytes, showed that the inhibition of GSH efflux by UCB is apparently from inside the cell. This point was confirmed by showing that the inhibition is overcome only when bilirubin-loaded cells are cleared of bilirubin (incubation with 5% bovine serum albumin). Using Gunn rat hepatocytes and purified bilirubin mono- and diglucuronides, we found that both UCB and glucuronide forms of bilirubin inhibit GSH efflux in a dose-dependent manner. We conclude that the organic anions, although taken up by a mechanism independent of GSH, may competitively inhibit the carrier for GSH efflux from inside the hepatocyte.

Whole-rat protein content estimation: applicability of the Nx6.25 factor

The amino acid composition of the protein from three strains of rat (Wistar, Zucker lean and Zucker obese), subjected to reference and high-fat diets has been used to determine the mean empirical formula, molecular weight and N content of whole-rat protein. The combined whole protein of the rat was uniform for the six experimental groups, containing an estimate of 17.3% N and a mean aminoacyl residue molecular weight of 103.7. This suggests that the appropriate protein factor for the calculation of rat protein from its N content should be 5.77 instead of the classical 6.25. In addition, an estimate of the size of the non-protein N mass in the whole rat gave a figure in the range of 5.5 % of all N. The combination of the two calculations gives a protein factor of 5.5 for the conversion of total N into rat protein.

Glutamatergic and GABAergic energy metabolism measured in the rat brain by (13) C NMR spectroscopy at 14.1 T.

Energy metabolism supports both inhibitory and excitatory neurotransmission processes. This study investigated the specific contribution of astrocytic metabolism to γ-aminobutyric acid (GABA) synthesis and inhibitory GABAergic neurotransmission that remained to be ilucidated in vivo. Therefore, we measured (13) C incorporation into brain metabolites by dynamic (13) C nuclear magnetic resonance spectroscopy at 14.1 T in rats under α-chloralose anaesthesia during infusion of [1,6-(13) C]glucose. The enhanced sensitivity at 14.1 T allowed to quantify incorporation of (13) C into the three aliphatic carbons of GABA non-invasively. Metabolic fluxes were determined with a mathematical model of brain metabolism comprising glial, glutamatergic and GABAergic compartments. GABA synthesis rate was 0.11 ± 0.01 μmol/g/min. GABA-glutamine cycle was 0.053 ± 0.003 μmol/g/min and accounted for 22 ± 1% of total neurotransmitter cycling between neurons and glia. Cerebral glucose oxidation was 0.47 ± 0.02 μmol/g/min, of which 35 ± 1% and 7 ± 1% was diverted to the glutamatergic and GABAergic tricarboxylic acid cycles, respectively. The remaining fraction of glucose oxidation was in glia, where 12 ± 1% of the TCA cycle flux was dedicated to oxidation of GABA. 16 ± 2% of glutamine synthesis was provided to GABAergic neurons. We conclude that substantial metabolic activity occurs in GABAergic neurons and that glial metabolism supports both glutamatergic and GABAergic neurons in the living rat brain. We performed (13) C NMR spectroscopy in vivo at high magnetic field (14.1 T) upon administration of [1,6-(13) C]glucose. This allowed to measure (13) C incorporation into the three aliphatic carbons of GABA in the rat brain, in addition to those of glutamate, glutamine and aspartate. These data were then modelled to determine fluxes of energy metabolism in GABAergic and glutamatergic neurons and glial cells.

Differences in glucose transporter gene expression between rat pancreatic alpha- and beta-cells are correlated to differences in glucose transport but not in glucose utilization.

Glucose exerts inverse effects upon the secretory function of islet alpha- and beta-cells, suppressing glucagon release and increasing insulin release. This diverse action may result from differences in glucose transport and metabolism between the two cell types. The present study compares glucose transport in rat alpha- and beta-cells. beta-Cells transcribed GLUT2 and, to a lesser extent, GLUT 1; alpha-cells contained GLUT1 but no GLUT2 mRNA. No other GLUT-like sequences were found among cDNAs from alpha- or beta-cells. Both cell types expressed 43-kDa GLUT1 protein which was enhanced by culture. The 62-kDa beta-cell GLUT2 protein was converted to a 58-kDa protein after trypsin treatment of the cells without detectable consequences upon glucose transport kinetics. In beta-cells, the rates of glucose transport were 10-fold higher than in alpha-cells. In both cell types, glucose uptake exceeded the rates of glucose utilization by a factor of 10 or more. Glycolytic flux, measured as D-[5(3)H]glucose utilization, was comparable in alpha- and beta-cells between 1 and 10 mmol/liter substrate. In conclusion, differences in glucose transporter gene expression between alpha- and beta-cells can be correlated with differences in glucose transport kinetics but not with different glucose utilization rates.

High-resolution ultrasonography of subretinal structure and assessment of retina degeneration in rat.

The purpose of this work was to evaluate the ability of 80 MHz ultrasonography to differentiate intra-retinal layers and quantitatively assess photoreceptor dystrophy in small animal models. Four groups of 10 RCS rats each (five dystrophic and five controls) were explored at 25, 35, 45 and 55 days post-natal (PN). A series of retina cross-sections were obtained ex vivo from outside intact eyes using an 80 MHz three-dimensional ultrasound backscatter microscope (20-microm-axial resolution). Ultrasound features of normal retina were correlated to those of corresponding histology and thickness measurements of photoreceptor segment and nuclear layers were performed on all groups. To show the ability of 80 MHz ultrasonography to distinguish the retinal degeneration in vivo, one RCS rat was explored at 25 and 55 days post-natal. Ultrasound image of normal retina displayed four distinct layers marked by reflections at neurites/nuclei interfaces and permitted to differentiate the photoreceptor segment and nuclear layers. The backscatter level from the retina was shown to be related to the size, density and organization of the intra-layer structure. Ultrasound thickness measurements highly correlated with histologic measurements. A thinning (p<0.05) of outer nuclear layer (ONL) was detected over time for controls and was thought to be assigned to retina maturation. Retinal degeneration started at PN35 and resulted in a more pronounced ONL thinning (p<0.05) over time. ONL degeneration was accompanied by segment layer thickening (p<0.05) at PN35 and thinning thereafter. These changes may indicate accumulation of outer segment debris at PN35 then progressive destruction. In vivo images of rat intra-retinal structure showed the ability of the method to distinguish the photoreceptor layer changes. Our results indicate that 80 MHz ultrasonography reveals intra-retinal layers and is sensitive to age and degenerative changes of photoreceptors. This technique has great potential to follow-up retinal dystrophy and therapeutic effects in vivo.

Na(+)-K(+)-ATPase gene expression during in vitro development of rat fetal forebrain.

The existence of at least three isoforms of Na(+)-K(+)-ATPase in adult brain tissues [alpha 1, kidney type; alpha 2 [or alpha(+)]; alpha 3] suggests that these genes might be regulated in a cell-specific and time-dependent manner during development. We have studied this question in serum-free aggregating cell cultures of mechanically dissociated rat fetal telencephalon. At the protein level, the relative rate of synthesis of the pool of alpha 1-, alpha 2-, and alpha 3-subunits increased approximately twofold over 15 days of culture, leading to a marked increase in the immunochemical pool of alpha-subunits as measured by a panspecific polyclonal antibody. Concomitantly, Na(+)-K(+)-ATPase enzyme-specific activity increased three- (lower forebrain) to sixfold (upper forebrain). The transcripts of all three alpha-isoforms and beta-subunit were detected in vitro in similar proportion to the level observed in vivo. alpha 3-mRNA (3.7 kb) was more abundant than alpha 1 (3.7 kb) or alpha 2 (5.3 and 3.4 kb). Cytosine arabinoside (0.4 microM) and cholera toxin (0.1 microM) were used to selectively eliminate glial cells or neurons, respectively. It was found that alpha 2-mRNA is predominantly transcribed in glial cell cultures, whereas alpha 3- and beta 1-mRNA (2.7, 2.3, and 1.8 kb) are predominant in neuronal cultures.

Molecular identification and characterization of the Arabidopsis delta(3,5),delta(2,4)-dienoyl-coenzyme A isomerase, a peroxisomal enzyme participating in the beta-oxidation cycle of unsaturated fatty acids.

Degradation of unsaturated fatty acids through the peroxisomal beta-oxidation pathway requires the participation of auxiliary enzymes in addition to the enzymes of the core beta-oxidation cycle. The auxiliary enzyme delta(3,5),delta(2,4)-dienoyl-coenzyme A (CoA) isomerase has been well studied in yeast (Saccharomyces cerevisiae) and mammals, but no plant homolog had been identified and characterized at the biochemical or molecular level. A candidate gene (At5g43280) was identified in Arabidopsis (Arabidopsis thaliana) encoding a protein showing homology to the rat (Rattus norvegicus) delta(3,5),delta(2,4)-dienoyl-CoA isomerase, and possessing an enoyl-CoA hydratase/isomerase fingerprint as well as aspartic and glutamic residues shown to be important for catalytic activity of the mammalian enzyme. The protein, named AtDCI1, contains a peroxisome targeting sequence at the C terminus, and fusion of a fluorescent protein to AtDCI1 directed the chimeric protein to the peroxisome in onion (Allium cepa) cells. AtDCI1 expressed in Escherichia coli was shown to have delta(3,5),delta(2,4)-dienoyl-CoA isomerase activity in vitro. Furthermore, using the synthesis of polyhydroxyalkanoate in yeast peroxisomes as an analytical tool to study the beta-oxidation cycle, expression of AtDCI1 was shown to complement the yeast mutant deficient in the delta(3,5),delta(2,4)-dienoyl-CoA isomerase, thus showing that AtDCI1 is also appropriately targeted to the peroxisome in yeast and has delta(3,5),delta(2,4)-dienoyl-CoA isomerase activity in vivo. The AtDCI1 gene is expressed constitutively in several tissues, but expression is particularly induced during seed germination. Proteins showing high homology with AtDCI1 are found in gymnosperms as well as angiosperms belonging to the Monocotyledon or Dicotyledon classes.

Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection.

Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the proportion of GFP-positive cells as determined by flow cytometry was fourfold higher for the PEI transfection as compared to the CaPO4 transfection. However, the mean level of transient GFP expression for the cells with the highest level of fluorescence was twofold greater for the CaPO4 transfection. Fluorescence in situ hybridization on metaphase chromosomes from pools of cells grown under selective pressure demonstrated that plasmid integration always occurred at a single site regardless of the transfection method. Importantly, the copy number of integrated plasmids was measurably higher in cells transfected with CaPO4. The efficiency of recombinant cell line recovery under selective pressure was fivefold higher following PEI transfection, but the average specific productivity of a recombinant antibody was about twofold higher for the CaPO4-derived cell lines. Nevertheless, no difference between the two transfection methods was observed in terms of the stability of protein production. These results demonstrated the feasibility of generating recombinant CHO-derived cell lines by PEI transfection. However, this method appeared inferior to CaPO4 transfection with regard to the specific productivity of the recovered cell lines.

Differential regulation of Na-K-ATPase isoform gene expression by T3 during rat brain development.

A fetal rat telencephalon organotypic cell culture system was found to reproduce the developmental pattern of Na-K-adenosinetriphosphatase (ATPase) gene expression observed in vivo [Am. J. Physiol. 258 (Cell Physiol. 27): C1062-C1069, 1990]. We have used this culture system to study the effects of triiodothyronine (T3; 0.003-30 nM) on mRNA abundance and basal transcription rates of Na-K-ATPase isoforms. Steady-state mRNA levels were low at culture day 6 (corresponding to the day of birth) but distinct for each isoform alpha 3 much greater than beta 1 = beta 2 greater than alpha 2 greater than alpha 1. At culture day 6, T3 did not modify mRNA abundance of any isoform. At culture day 12 (corresponding to day 7 postnatal), T3 increased the mRNA level of alpha 2 (4- to 7-fold), beta 2 (4- to 5-fold), alpha 1 (3- to 6-fold), and beta 1 (1.5-fold), whereas alpha 3 mRNA levels remained unchanged. Interestingly, the basal transcription rate for each isoform differed strikingly (alpha 2 greater than alpha 1 much greater than beta 1 = beta 2 greater than alpha 3) but remained stable throughout 12 days of culture and was not regulated by T3. Thus we observed an inverse relationship between rate of transcription and rate of mRNA accumulation for each alpha-isoform, suggesting that alpha 1- and alpha 2-mRNA are turning over rapidly whereas alpha 3-mRNA is turning over slowly. Our data indicate that one of the mechanisms by which T3 selectively controls Na-K-ATPase gene expression during brain development in vitro occurs at the posttranscriptional level.