FORMATION OF ASPARAGINE FROM ASPARTIC ACID IN RAT BRAIN

The synthesis of asparagine in rat brain was studied both in vitro and in vivo. A conversion in vitro of about 2 per cent of the added l -[¹⁴C]aspartic acid into asparagine was found after a 2 h incubation with the 100,000 g supernatant fraction from brain. This corresponded to a formation of 4·8 nmol of asparagine/mg of protein/h. The reaction required ATP and glutamine, and was linear with time during the 2 h incubation. When the crude mitochondrial fraction was added to the incubation mixture the reaction was inhibited, probably because of the presence of ATPase activity in the mitochondrial preparation. Inhibition by the reaction product seemed unlikely since removal of endogenous asparagine did not stimulate the reaction; only when asparagine was added at levels of 0·5 or 1·0 mm was significant inhibition found. Ammonium chloride was less effective than glutamine as an amide donor. Endogenous asparaginase (EC 3.5.1.1.) activity was low in the in vitro preparation and did not significantly affect the conversion. Synthesis of asparagine from aspartic acid did not occur in slices of brain nor was there a significant conversion of aspartic acid or glucose to asparagine after their intracerebral administration in vivo.     

Cerebral lipid metabolism in experimental hyperphenylalaninaemia: incorporation of 14C-labelled glucose into total lipids

—1. Effects of the administration of phenylalanine to rats on incorporation in vivo or in vitro of [U-¹⁴C]glucose into cerebral lipids were studied during the first 5–10 days of postnatal development. In addition, the effects of added phenylalanine and its deaminated metabolites on incorporation of [U-¹⁴C]glucose by homogenates into lipids of developing rat brain were investigated. Hyperphenylalaninaemia reduced incorporation both in vivo and in vitro of [U-¹⁴C]glucose into cerebral lipids. 2. Phenylalanine or tyrosine added in vitro at concentrations equivalent to those in the brain of the hyperphenylalaninaemic rat (0-1 μmole/ml incubation medium) did not inhibit incorporation of [U-¹⁴C)glucose into lipids, although at much higher concentrations of phenylalanine (36 μumoles/ml incubation medium) slight inhibition (10 per cent) of incorporation of [U-¹⁴C]glucose into lipids was observed. 3. In contrast, the deaminated metabolites in general exerted greater inhibitory effects at lower concentrations. Phenyllactic acid, in comparison to phenylpyruvic and phenyl-acetic acid, was the most potent inhibitor of the incorporation in vitro of [U-¹⁴C]glucose into cerebral lipids. These results indicated that these metabolites of phenylalanine were the more potent inhibitors of cerebral lipid metabolism in immature animals.     

Hydroxyl containing seleno-imine compound exhibits improved anti-oxidant potential and does not inhibit thiol-containing enzymes

Design and synthesis of organoselenium compounds with high thiol peroxidase (TPx) and low thiol oxidase (TOx) activities have been a difficult task and remains a synthetic-activity relationship dilemma. In this regard we are reporting for the first time a detail experimental data (both in vitro and in vivo) about the anti-oxidant and toxicological profile of an Imine (–N) containing organoselenium compound (Compound A). The TPx activity of Compound A was significantly higher than diphenyl diselenide (DPDS). Both Compound A and DPDS protected sodium nitropruside (SNP) induced thiobarbituric acid reactive species (TBARS) production in rats tissue homogenate with significantly higher activity observed for Compound A than DPDS (p < 0.05). The Compound A also exhibited strong antioxidant activity in the DPPH and ABTS radical scavenging assays. This study reveals that an imine group close to selenium atom drastically enhances the catalytic activities in the aromatic thiol (PhSH) assay systems. The oxidation of biologically significant thiols reflects the toxicity of the compounds. However, the present data showed that treatment with Compound A at 0, 10, 25 or 50 mg/kg was not associated with mortality or body weight loss. Similarly it did not inhibit α-ALA-D and Na⁺¹/K⁺¹ ATPase (sulfhydryl group containing enzymes) activities after acute oral treatment; rather it enhanced non-protein thiols (NPSH) concentration. The Compound A did not cause any oxidative stress as measured by TBARS production in rat's tissue preparation. Our data also indicate that exposure to Compound A did not affect plasma transaminase activities or levels of urea and creatinine in rats. Ascorbic acid is always considered a marker of oxidative stress and the reduction of its content may indicate an increase in oxidative stress. Treatment with Compound A did not alter Ascorbic acid levels in rats. The conducted in vitro and in vivo tests show the versatile therapeutic potential of this compound in the area of free radical induced damages, will undoubtedly enhance our understanding of the mechanism of model compounds and may ultimately yield insights that result in improved GPx mimics.     

Monosialoganglioside Increases Catalase Activity in Cerebral Cortex of Rats

Monosialoganglioside (GM1) is a neuroprotective agent that has been reported to scavenge free radicals generated during reperfusion and to protect receptors and enzymes from oxidative damage. However, only a few studies have attempted to investigate the effects of GM1 on enzymatic antioxidant defenses of the brain. In the present study, we evaluate the effects of the systemic administration of GM1 on the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), and on spontaneous chemiluminescence and total radical-trapping potential (TRAP) in cerebral cortex of rats ex vivo. The effects of GM1 on CAT activity and spontaneous chemiluminescence in vitro were also determined.

Animals received two injections of GM1 (50?mg/kg, i.p.) or saline (0.85% NaCl, i.p.) spaced 24?h apart. Thirty minutes after the second injection the animals were sacrificed and enzyme activities and spontaneous chemiluminescence and TRAP were measured in cell-free homogenates. GM1 administration reduced spontaneous chemiluminescence and increased catalase activity ex vivo, but had no effect on TRAP, SOD or GSH-Px activities. GM1, at high concentrations, reduced CAT activity in vitro. We suggest that the antioxidant activity of GM1 ganglioside in the cerebral cortex may be due to an increased catalase activity.     

In vivo and in vitro effects of copper and cadmium on the plasma membrane H+-ATPase from cucumber (Cucumis sativus L.) and maize (Zea mays L.) roots

The plasmalemma vesicles isolated from cucumber and maize roots were used to study the effect of Cu²⁺ and Cd²⁺ on the hydrolytic and proton pumping activities of ATPase. In vivo application of metal ions to the plant growth solutions resulted in stimulation of the proton transport in maize. In cucumber roots the action of metals was not the same: cadmium stimulated the H⁺ transport through plasmalemma whereas Cu²⁺ almost completely inhibited it. Copper ions decreased the hydrolytic activity of H⁺-ATPase in cucumber, without any effect on this activity in membranes isolated from maize roots. The effect of cadmium on the hydrolytic activities was opposite: ATP-hydrolysis activity in plasmalemma was not altered in cucumber, whereas in maize its stimulation was observed. The amount of accumulated metals was not the main reason of different influence of metals on H⁺-ATPase activity in tested plants. In in vitro experiments Cu²⁺ inhibited H⁺ transport in the cucumber, to a higher degree than Cd²⁺ and both metals did not change this H⁺-ATPase activity of plasmalemma isolated from corn roots. Cu²⁺ added into the incubation medium reduced the hydrolytic activity of ATPase in the plasma membrane isolated from cucumber as well as from corn roots. Cd²⁺ diminished the hydrolytic activity of ATPase in cucumber, and no effect of Cd²⁺ in the plasmalemma isolated from corn roots was found. Our results indicated different in vitro and in vivo action of both metals on H⁺-ATPase and different response of this enzyme to Cu²⁺ and Cd²⁺ in maize and cucumber.     

Fluid transport, ATP level and ATPase activities in isolated rabbit corneal endothelium

The effect of certain biochemical parameters on transendothelial fluid transport has been studied. Cellular ATP level and (Na⁺ + K⁺)-activated as well as Mg²⁺-activated ATPase activities were measured by ultramicrotechniques using individual rabbit corneal endothelium after they had been subjected to in vitro perfusion with solutions fully supplemented or deficient singly or severally in glucose, adenosine and glutathione (GSH). With the complete medium, the transport system operates in vitro for approx. 6 h. Deletion of glucose alone, glucose and adenosine or glucose, adenosine and GSH brings about a cessation of fluid transport after 3.5 h, 2 to 2.5 h and 0.5 to 1 h, respectively. A marked decrease (62%) of the endothelial ATP level, however, occurs only when all metabolites are omitted. The favorable effect of GSH on transport activity is attributable to its capacity to sustain cellular ATP rather than to protect the functionality of (Na⁺ + K⁺)-activated ATPase. Adenosine, in the presence of GSH, maintains normal ATP levels and, additionally, exerts a protective effect on Mg²⁺-activated ATPase and possibly also on (Na⁺ + K⁺)-activated ATPase.     

Salt-stimulated Phosphoenolpyruvate Carboxylase in Cakile maritima

The effects of NaCl and other salts, in vivo and in vitro, on the activity of phosphoenolpyruvate carboxylase from the coastal C₃ halophyte Cakile maritima Scop, were investigated. Plants grown with 100 mM NaCl in their growth medium yielded some 30% higher rates of phosphoenolpyruvate carboxylase activity than did salt-depleted plants. Activity of the enzyme was stimulated when NaCl was added to the reaction mixture in concentrations of up to 200 mM. The magnitude of this in vitro stimulation was similar for plants grown in the presence or absence of NaCl. The effect seems to be caused by chloride rather than by sodium ions.     

METHYL MERCURY INHIBITION OF SYNAPTOSOME AND BRAIN SLICE PROTEIN SYNTHESIS: IN VIVO AND IN VITRO STUDIES

Subacute methyl mercury (MeHg) intoxication was induced in adult rats following the daily intragastric administration of 1 mg MeHg/100 g body weight. Decreased [¹⁴C]leucine incorporation into cerebral and cerebellar slice protein was found. Weight loss occurred during the latent and neurotoxic phases but pair feeding did not reveal a significant defect in amino acid incorporation into slice protein. There was no decline in synaptosome protein synthesis in vitro during the latent phase but a significant decline in cerebellar and cerebral synaptosome synthesis was found during the neurotoxic phase. MeHg in vitro inhibited cerebral slice and synaptosome protein synthesis at half maximal concentrations of 7.5 and 12.5 μM respectively. Inhibition of synthesis in synaptosomes was non-competitive with K₁ of 4 × 10^?6M. MeHg had no effect on [¹⁴C]leucine or [¹⁴C]proline uptake into synaptosomes. There was no significant inhibition of synaptosome basal ATPase or Na + K ATPase at concentrations of MeHg (12 μM) giving half maximal inhibition of protein synthesis. No preferential inhibition of the chloramphenicol (55S) or cycloheximide sensitive components of synaptosome fraction protein synthesis was found, suggesting that the inhibition is common to both mitochondrial and extramitochondrial protein synthesizing systems. Addition of nucleotides and/or atractylate failed to influence protein synthesis and did not reverse the MeHg inhibition. Mannitol, as a replacement for the predominant cation species of the incubation medium, gave 40% inhibition of protein synthesis in the control but protected against further inhibition by MeHg.     

ATPase Activities and Membrane Fine Structure of Rhizodermal Cells from Sorghum and Spartina Roots Grown Under Mild Salt Stress

The study aims at correlations between ultrastructure of rhizodermal membranes and ATPase activities of two gramineous C₄-species effected by salinization of the growth medium. Comparative investigations were done with drought-resistant Sorghum and drought-sensitive, salt tolerant Spartina plants grown under steady-state conditions without or with 40 mol m^?3 NaCl. Both species sustained this treatment and showed marked influences of salt on growth but not on root respiration. Generally, the intramembraneous particle (IMP) frequencies, found on freeze-fracture replicates of rhizodermal plasmic fracture faces of the plasmalemma (P_PF) and tonoplast (T_PF), were higher in Sorghum than in Spartina. NaCl leads in both species to an increase of the rhizodermal IMP frequency in the T_PF (～150%) and P_PF (～120%). The activities of T- and P-ATPase were determined for membrane vesicles from crude extracts and from isolated protoplasts of roots, respectively; the results from both preparations were the same. The vanadate-sensitive tissue ATPase activities increased under salt stress ～ 5 times in Sorghum and ～ 2 times in Spartina whilst the nitrate and azide-sensitive tissue ATPase activities increased ～ 6 times only in Sorghum. The combination of the results from the membrane fine structures with the biochemical tests points out that salt-effected increases of ATPase activities of Sorghum roots were ～4 times higher than the increases of IMP frequencies on rhizodermal membranes; in Spartina this effect is less intense. It is concluded that a salty environment leads to a higher ATPase activity per IMP in both species. The stronger reaction of Sorghum is explainable by the increase in K⁺/Na⁺ selectivity of the plasma membrane under salt stress. This is not the case for Spartina because the roots of this species include salt which subsequently is sequestered by the salt glands of the leaves.     

FURTHER EVALUATION OF POLYPEPTIDE SYNTHESIS IN CEREBRAL ANOXIA, HYPOXIA AND ISCHEMIA

The alteration of polypeptide synthesis was evaluated with microsomes isolated from anoxic rabbit, hypoxic rat and ischemic gerbil brains to estimate the extent of functional or structural changes in polyribosomes in situ and the extent of artifact during tissue preparation. By using two-stage experimentation with combination of control and pathological microsomes and supernatant, it was found that the previously observed effects on microsomal or polyribosomal polypeptide synthesis in the above pathophysiological conditions were mainly the reflection of the alteration of polyribosomes in situ rather than the artifact during tissue preparation by degradative processes. In support of this finding. the use of inhibitors of degradative enzymes did not significantly protect microsomes either in normal or in pathological conditions. It was noted that the decline of tissue pH, to a certain extent, could be correlated with dysfunction of polyribosomes both in situ and during tissue preparation in cerebral hypoxia and anoxia. Since there is little change in ATP level, it was postulated that the alteration of pH in situ is responsible for the observed suppression of polypeptide synthesis in vitro at least in cerebral hypoxia. This hypothesis was supported by the subsequent experiments with incubation of brain slices and homogenization of brain tissue under various pH. It was emphasized that the environmental biochemical elements surrounding polyribosomes in cytoplasm should be evaluated as possible contributing factors for polyribosomal dysfunction in such pathological conditions as cerebral anoxia, hypoxia or ischemia if the alteration of energy state does not explain the phenomenon entirely.     

D- AND L-STEREOISOMERS OF ALLYLGLYCINE: CONVULSIVE ACTION AND INHIBITION OF BRAIN L-GLUTAMATE DECARBOXYLASE

DL-Allylglycine was resolved into the L- and D-stereoisomers using hog kidney acylase. Both isomers were active as convulsants after administration to mice. The dose of D-allylglycine required to induce convulsions was greater than that of the L-isomer. Studies on the concentration of the two isomers in brain suggest that the lower effectiveness of D-allylglycine is partially due to its slower penetration into the brain through the blood-brain barrier. Both isomers of allylglycine inhibited brain glutamate decarboxylase in vitro to approximately the same extent, however, in vivo L-allylglycine inhibited the enzyme more strongly than the D isomer. Concentrations of allylglycine which caused a significant inhibition of L-glutamate decarboxylase in vivo were ineffective in inhibiting the enzyme in vitro. Oxidation products derived from L- or D-allylglycine by the action of either L- or D-amino acid oxidase caused an almost complete inhibition of the enzyme in vitro. It is suggested that a common intermediate derived from the two isomers (possibly 2-keto-4-pentenoic acid) is responsible for the in vivo inhibition of L-glutamate decarboxylase and possibly also for the induction of convulsions.     

Subcellular Distribution of Basal and Cation-sensitive ATPases in Roots of Phaseolus vulgaris

A smooth microsomal fraction isolated from homogenates of Pbaseolus vulgaris root tissue has been found to possesss a highly active basal ATPase (measured in the absence of added cations). The microsomal membranes also feature a cation-sensitive ATPase which responds to Mg²⁺, Na⁺ and K⁺, but in a manner that is highly variable with pH. In contrast, membrane fragments prepared by a technique designed to yield purified plasma membrane were capable of little or no hydrolysis of ATP either in the presence or absence of added cations. This suggests that the microsomal activity is a reflection of membrane-bound ATPase which has been derived from cytoplasmic membranes, possibly the tonoplast, rather than plasma membrane.     

Relationship between mechanical dysfunction and depression of sarcolemmal Ca2+-pump activity in hearts perfused with oxygen free radicals

Although in vitro studies have shown that oxygen free radicals depress the sarcolemmal Ca²⁺-pump activity and thereby may cause the occurrence of intracellular Ca²⁺ overload for the genesis of contractile failure, the exact relationship between changes in sarcolemmal Ca²⁺-pump activity and cardiac function due to these radicals is not clear. In this study we examined the effects of oxygen radicals on sarcolemmal Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities as well as contractile force development by employing isolated rat heart preparations. When hearts were perfused with medium containing xanthine plus xanthine oxidase, the sarcolemmal Ca²⁺-stimulated ATPase activity and ATP-dependent Ca²⁺ accumulation were depressed within 1 min whereas the developed contractile force, rate of contraction and rate of relaxation were increased at 1 min and decreased over 3–20 min of perfusion. The resting tension started increasing at 2 min of perfusion with xanthine plus xanthine oxidase. Catalase showed protective effects against these alterations in heart function and sarcolemmal Ca²⁺-pump activities upon perfusion with xanthine plus xanthine oxidase whereas superoxide dismutase did not exert such effects. The combination of catalase and superoxide dismutase did not produce greater effects in comparison to catalase alone. These results are consistent with the view that the depression of heart sarcolemmal Ca²⁺ pump activities may result in myocardial dysfunction due to the formation of hydrogen peroxide and/or hydroxyl radicals upon perfusing the hearts with xanthine plus xanthine oxidase.     

Improvement of <Emphasis Type="Italic">ex vitro</Emphasis> transfer of tobacco plantlets by addition of abscisic acid to the last subculture

Tobacco (Nicotiana tabacum L.) plantlets were grown on Murashige and Skoog medium in ventilated Magenta boxes and for the last subculture 10 μM ABA was added to the medium. After three weeks plantlets were transferred into pots with Perlite moistened with water and grown in controlled conditions (16-h photoperiod, day/night temperature 25/20 °C, air humidity about 45 %) either under low or high irradiance of 150 (LI) and 700 (HI) μmol m⁻² s⁻¹, respectively. Content of endogenous ABA was 271.7 pmol g⁻¹(f.m.) in ABA treated plantlets, while in control plantlets it was only 53.3 pmol g⁻¹(f.m.). After ex vitro transfer, stomatal conductance and transpiration rate decreased considerably in comparison with in vitro grown plantlets and remained lower also 7 d after ex vitro transfer, especially in ABA-treated plants and so wilting of plants was practically eliminated. Net photosynthetic rate also decreased 1 d after ex vitro transfer but after 7 d it was mostly higher than that of in vitro grown plantlets. Water use efficiency significantly increased in ABA-treated plants. Chlorophyll a+b content did not change immediately after ex vitro transfer, nevertheless, after 7 d chlorophyll content was higher in ABA-treated plants. Pool of xanthophyll cycle pigments (XCP) and the degree of their deepoxidation (DEPS), which are connected with harmless dissipation of light energy, increased under high irradiance. Contents of XCP and ABA precursors (neoxanthin and violaxanthin) were lower in ABA-treated plants than in control plants indicating less stress in these plants. Most chlorophyll a fluorescence parameters did not change considerably after ex vitro transfer and so the photoinhibition was not observed even under HI. Slight increase in non-photochemical quenching under HI in ABA-treated plants suggested their better photoprotection. Thus application of ABA to the last subculture can improve acclimatization of in vitro grown plants to ex vitro conditions     

Acclimation of tobacco plantlets to ex vitro conditions as affected by application of abscisic acid

Plantlets of Nicotiana tabacum L. cv. Petit Havana SR1 were grown in vitro on Murashige and Skoog medium containing 2% saccharose, and then transplanted ex vitro into pots with coarse sand and Hewitt nutrient solution. In the first day after transplantation, the anti-transpirant abscisic acid (ABA; 0.01, 0.05 or 0.10 mM) was added to the substrate. Leaf stomatal conductance (gs), which was high in plants during the first days after transplantation similarly as in plantlets grown in vitro, was considerably decreased by ABA-treatment. However, in the further days gs decreased more quickly in control than in ABA-treated plants, and after 2 or 3 weeks gs was significantly lower than that of plantlets grown in vitro but similar in control and ABA-treated plants. Two weeks after transplantation, net photosynthetic rate, chlorophyll a + b content, maximal photochemical efficiency, and actual quantum yield of photosystem II in plant leaves were higher in comparison with those in plantlets grown in vitro. ABA-treatment had slight positive or insignificant effect on photosynthetic parameters and enhanced plant growth. Thus ABA application can alleviate 'transplant shock' and speed up acclimation of plantlets to ex vitro conditions.     

Comparison of gene expression under <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">ex vitro</Emphasis> conditions during rooting of grape cuttings through mRNA differential display

An mRNA differential display (DD) analysis during rooting in grape cuttings was carried out to determine whether gene expression patterns differed under in vitro and ex vitro conditions. The four tissue samples for differential display and subsequent Northern hybridization analyses included control stem tissue from in vitro and ex vitro sources, microcuttings planted in MS based in vitro rooting medium and softwood cuttings planted in ex vitro soil medium, both collected 48 h after planting. DD autoradiographs showed gross similarity in banding pattern between in vitro and ex vitro stem tissue, whether constitutive or induced. Northern blot analysis of a few bands that appeared to be differentials did not indicate them as true positives. The results suggested that gene expression pattern during physiological processes such as rooting may be identical under in vitro and ex vitro conditions.     

Photosynthetic pigments and gas exchange during ex vitro acclimation of tobacco plants as affected by CO2 supply and abscisic acid

Nicotiana tabacum L. plantlets were grown in glass vessels or in Magenta boxes with better CO₂ supply. To improve the ex vitro transfer we tested application of abscisic acid and elevated CO₂ concentration. In the first two weeks after transfer, net photosynthetic rate, chlorophyll a+b content, and Chl a/b ratio were higher, and content of xanthophyll cycle pigments lower in M-plants than in G-plants, but during further growth the differences almost disappeared. ABA application alleviated the risk of wilting because it decreased stomatal conductance. The effect of ABA was enhanced under CE (28 days after transfer). In situ, P_N was always higher at CE than at CA, but when measured under CA, positive effect of CE was found 2 and 16 days after transfer in M-plants and only 16 days after transfer in G-plants. Slightly increased Chl a content was found in all ABA-treated plants, and in M-plants grown under CE. The content of xanthophyll cycle pigments was lower under CE compared to CA, and the lowest one was found in ABA-treated M-plants grown under CE. On the contrary, the degree of their deepoxidation (DEPS) was slightly higher in plants grown under CE. No significant effects of ABA-treatment or growth under CE on fluorescence kinetic parameters were found and inconsistent effects on photochemical activities. The photochemical efficiency of PS2 (variable to maximum fluorescence ratio, F_v/F_m) after ex vitro transfer was similar to that in in vitro grown plants. This together with the values of DEPS indicated that no photodamage during ex vitro transfer occurred. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of Dionaea muscipula extract and plumbagin on maceration of potato tissue by Pectobacterium atrosepticum

Pectobacterium atrosepticum (Pba) is a plant pathogen that causes major crop losses. Dionaea muscipula extracts and their antibacterial constituent, plumbagin, inhibit Pba growth in vitro. However, this effect is reduced when the extracts are added to bacterial cultures present on potato tubers or suspended in potato tuber filtrate (PF). To explain this, we examined the response mechanism of Pba cells to Dionaea extract and plumbagin and compared it with the effect of a bactericidal peptide – CAMEL. The addition of the extract and plumbagin to a Pba1043 culture in stationary phase increased the extracellular pectate lyase (Pel) activity in the presence of PF. While the addition of the Dionaea extract and plumbagin caused a dramatic reduction in RNA and protein synthesis in Pba1043, it did not result in cellular damage. PF alone increased the expression of Pba genes encoding protein components of cellular efflux pump systems: ompX, acrA and emrA. Application of both PF and plumbagin resulted in a synergistic stimulation of acrA gene expression. Plumbagin added to potato tubers inoculated with a field isolate Pba5A/1/2005 increased extracellular Pel activity and reduced tissue maceration but did not affect bacterial counts per gram of tissue. These results show that plumbagin in the presence of compounds from potato tuber stimulates Pel production/secretion in Pba cells and increases the expression of the acrA gene. This may be the molecular basis for the less pronounced effects of Dionaea extract on Pba in planta relative to those observed in vitro.     

What is the correct value for Na,K-ATPase activity in homogenates of cerebral cortex?

Activity of Na, K-ATPase in homogenates of fresh cerebral cortex of rats was compared with that of cortex frozen under different conditions. Activity yields after rapid in situ freezing of the exposed cerebral cortex were twice, higher (26.1 U) than in homogenates of the fresh cortex (13.3 U). Fresh brain kept on ice for 60 and 300 s and subsequently frozen in liquid nitrogen yielded activities comparable to those of the tissue frozen in situ (24.1 U and 24.9 U for 60 s and 300 s periods, respectively). Inhibition of Na, K-ATPase by 10⁻⁷ M vanadate was significantly stronger (38%) in homogenates of the fresh brain then in those of the cortex frozen in situ (28%). High Na, K-ATPase activity (47.6 U) in suspensions of synaptosomal membranes (SM) prepared from fresh cortical homogenates was only slightly inhibited by 10⁻⁷ M vanadate (12%). Various treatments of homogenates or SM suspensions, like increase of piston rotation speed, repeated freezing and thawing procedure or vigorous shaking did not significantly affect the enzyme activity. Mg-ATPase activity and its sensitivity to vanadate was also modified by tissue treatment but the effect was much less pronounced.     

Transient reporter gene (GUS) expression in creeping bentgrass (Agrostis palustris) is affected by in vivo nucleolytic activity

Leaf and callus tissues of a creeping bentgrass cultivar (Penn A4) had high nuclease activities that degraded exogenously added plasmid DNA. When callus tissue was incubated for 24 h with heparin, spermidine, aurintricarboxylic acid or polyethylene glycol, only heparin and spermidine were effective as in vitro nuclease inhibitors, protecting exogenously added plasmid DNA from degradation. When -glucuronidase (GUS) reporter gene activity was evaluated in heparin-treated (0.6%), 14-month old callus following microprojectile bombardment, GUS activity increased 1000-fold compared to equivalent aged untreated Penn A4 callus. Similar enhancement from heparin pretreatment (0.6% or 1.2%) was not observed in 6-month old callus. This is likely due to much higher activities of nuclease in the younger callus.