PubMed Health⌕ Search

Biomedical subjects

M J de Groot

Publications and source records attributed to M J de Groot.

At least 37 records · Page 2Linked to original sources
PubMed1994

Non-recombinant background in gene targeting: illegitimate recombination between a hpt gene and a defective 5' deleted nptII gene can restore a Kmr phenotype in tobacco.

Previously we have demonstrated gene targeting in plants after Agrobacterium-mediated transformation. In these initial experiments a transgenic tobacco line 104 containing a T-DNA insertion with a defective neomycin phosphotransferase (nptII) gene was transformed with a repair construct containing an otherwise defective nptII gene. Homologous recombination between the chromosomally located target and the incoming complementary defective nptII construct generated an intact nptII gene and led to a kanamycin-resistant (Kmr) phenotype. The gene targeting frequency was 1 x 10(-5). In order to compare direct gene transfer and Agrobacterium-mediated transformation with respect to gene targeting we transformed the same transgenic tobacco line 104 via electroporation. A total of 1.35 x 10(8) protoplasts were transformed with the repair construct. Out of nearly 221,000 transformed cells 477 Kmr calli were selected. Screening the Kmr calli via PCR for recombination events revealed that in none of these calli gene targeting had occurred. To establish the origin of the high number of Kmr calli in which gene targeting had not occurred we analysed plants regenerated from 24 Kmr calli via PCR and sequence analysis. This revealed that in 21 out of 24 plants analysed the 5'-deleted nptII gene was fused to the hygromycin phosphotransferase (hpt) gene that was also present on the repair construct. Sequence analysis of 7 hpt/nptII gene fusions showed that they all contained a continuous open reading frame. The absence of significant homology at the fusion site indicated that fusion occurred via a process of illegitimate recombination. Therefore, illegitimate recombination between an introduced defective gene and another gene present on the repair construct or the chromosome has to be taken into account as a standard byproduct in gene targeting experiments.

Amino Acid Sequence↗
PubMed1994

The hydrolysis of glycerol-3-phosphate into glycerol in cardiac tissue: possible consequences for the validity of glycerol release as a measure of lipolysis.

Glycerol release has been generally accepted as an index of lipolysis in the intact heart. The glycerol moiety of glycerol-3-phosphate (glycerol-3-P) is incorporated into triacylglycerols, which are then hydrolysed with release of glycerol. This study investigates the possibility that glycerol may be derived directly from glycerol-3-P instead of passing through the triacylglycerol pool. The cardiac capacity for hydrolysis of glycerol-3-P into glycerol was determined in homogenates of rat hearts. Glycerol-3-P hydrolysis activity in homogenates increased with decreasing pH. The activity was approximately four times higher at pH 5.0 than at pH 7.2 (0.94 +/- 0.11 and 0.25 +/- 0.03 mumol.g wet weight-1.min-1 respectively). The substrate concentration at which half-maximal glycerol-3-P hydrolysis activity was reached did not significantly differ at pH 5.0 and pH 7.2 (4.2 +/- 1.1 mM and 2.9 +/- 1.0 mM respectively). In the intact heart, the pH and substrate conditions found under ischaemia are favourable for direct conversion of glycerol-3-P into glycerol. The glycerol-3-P hydrolysis activity measured in vitro was sufficiently high to account for glycerol production in the ischaemia heart. However, the lack of a stoichiometric relation between cardiac glycerol-3-P and glycerol levels in ischaemia indicates that production of glycerol cannot be explained solely by hydrolysis.

Animals↗
PubMed1993

The effects of exogenous lactate and pyruvate on the recovery of coronary flow in the rat heart after ischaemia.

OBJECTIVE: The effect of exogenous lactate and pyruvate on the recovery of coronary flow (total, regional) after ischaemia as a function of the duration of ischaemia was evaluated. METHODS: Isolated, ejecting rat hearts were subjected to ischaemia for 15, 30, or 45 minutes. Glucose (11 mM) was present as the basal substrate in the perfusion medium and lactate (5 mM) or pyruvate (5 mM) was added as the cosubstrate. Flow variables were measured by the timed collection of coronary effluents and by the radioactive microsphere technique. RESULTS: In the lactate perfused hearts, reactive hyperaemia was present after 15 minutes but absent after 30 and 45 minutes of ischaemia. Total coronary flow was significantly reduced after 45 minutes of ischaemia. Transmural flow was impaired after 15, 30, and 45 minutes of ischaemia in the lactate perfused hearts, -that is, flow in the inner layers of the left ventricle was transiently reduced after 15 minutes and remained continuously depressed after 30 and 45 minutes of ischaemia. The pyruvate perfused hearts showed reactive hyperaemia after 15 and 30 minutes of ischaemia. After 45 minutes total coronary flow was reduced below the value before ischaemia, and in particular, the inner layers of the left ventricle were severely deprived of flow as in lactate perfused hearts. When neither lactate nor pyruvate was added to the perfusion medium (containing glucose), impairment of coronary flow in the inner layers of the left ventricle was only transiently obvious after 30 and 45 minutes of ischaemia. Impaired perfusion of the inner layers during reperfusion resulted in delayed washout of lactate dehydrogenase. CONCLUSIONS: Exogenous substrates modify the recovery of flow after ischaemia. In the presence of exogenous lactate, severe disturbances of flow are already obvious after 30 minutes of ischaemia in the inner layers of the left ventricle. Exogenous pyruvate delays impairment of flow in the inner layers compared with exogenous lactate.

Animals↗
PubMed1993

Substrate-induced changes in the lipid content of ischemic and reperfused myocardium. Its relation to hemodynamic recovery.

To investigate the effect of lactate, pyruvate, and glucose on the endogenous levels of lipids in the normoxic, ischemic, and reperfused myocardium, isolated working rat hearts were exposed to various grades of ischemic insult (15, 30, or 45 minutes). Glucose was present as the basal substrate in the perfusion medium, and lactate (5 mM) or pyruvate (5 mM) was added as the cosubstrate. Lipid metabolism was evaluated by fatty acid accumulation, triacylglycerol turnover, and phospholipid homeostasis. Exogenous lactate significantly increased fatty acid content above preischemic levels after 45 minutes of ischemia. In glucose-perfused hearts, fatty acid levels were even slightly higher than in lactate-perfused hearts, whereas pyruvate-perfused hearts demonstrated less accumulation of fatty acids. By reperfusion, fatty acid levels in glucose-perfused hearts returned to control values. In lactate- and pyruvate-perfused hearts, fatty acid accumulation was further enhanced by reperfusion. When the fatty acid content exceeded 400 nmol/g dry wt during reperfusion, hemodynamic function was impaired, whereas fatty acid levels below 400 nmol/g dry wt did not correlate with hemodynamic recovery. The total triacylglycerol content did not change during ischemia and reperfusion. However, accumulation of glycerol was remarkable during the first 15 minutes of ischemia in all hearts, and release of glycerol by reperfusion was considerable in lactate-perfused hearts after 30 minutes of ischemia and in all groups of hearts after 45 minutes of ischemia. Release of glycerol in association with maintained levels of triacylglycerols suggests turnover of the triacylglycerol pool. The rate of triacylglycerol cycling correlated poorly with hemodynamic recovery. Accumulation of arachidonic acid revealed disturbances in phospholipid turnover. Arachidonic acid accumulation during reperfusion demonstrated a strong relation with impairment of cardiac function. Hence, derangements in phospholipid homeostasis during reperfusion might be involved in myocardial damage, which is influenced by the substrates available.

Animals↗
PubMed1992

The nucleotide metabolism in lactate perfused hearts under ischaemic and reperfused conditions.

It was examined whether lactate influences postischaemic hemodynamic recovery as a function of the duration of ischaemia and whether changes in high-energy phosphate metabolism under ischaemic and reperfused conditions could be held responsible for impairment of cardiac function. To this end, isolated working rat hearts were perfused with either glucose (11 mM), glucose (11 mM) plus lactate (5 mM) or glucose (11 mM) plus pyruvate (5 mM). The extent of ischaemic injury was varied by changing the intervals of ischaemia, i.e. 15, 30 and 45 min. Perfusion by lactate evoked marked depression of functional recovery after 30 min of ischaemia. Perfusion by pyruvate resulted in marked decline of cardiac function after 45 min of ischaemia, while in glucose perfused hearts hemodynamic performance was still recovered to some extent after 45 min of ischaemia. Hence, lactate accelerates postischaemic hemodynamic impairment compared to glucose and pyruvate. The marked decline in functional recovery of the lactate perfused hearts cannot be ascribed to the extent of degradation of high-energy phosphates during ischaemia as compared to glucose and pyruvate perfused hearts. Glycolytic ATP formation (evaluated by the rate of lactate production) can neither be responsible for loss of cardiac function in the lactate perfused hearts. Moreover, failure of reenergization during reperfusion, the amount of nucleosides and oxypurines lost or the level of high-energy phosphates at the end of reperfusion cannot explain lactate-induced impairment. Alternatively, the accumulation of endogenous lactate may have contributed to ischaemic damage in the lactate perfused hearts after 30 min of ischaemia as it was higher in the lactate than in the glucose or pyruvate perfused hearts. It cannot be excluded that possible beneficial effects of the elevated glycolytic ATP formation during 15 to 30 min of ischaemia in the lactate perfused hearts are counterbalanced by the detrimental effects of lactate accumulation.

Adenosine↗
PubMed1992

Interrelationship between lactate and cardiac fatty acid metabolism.

This overview is presented, in the main, to summarize the following aspects of lactate and cardiac fatty acid metabolism: 1. The utilization of exogenous carbohydrates and fatty acids by the heart. 2. The competition between lactate and fatty acids in cardiac energy metabolism. 3. The effect of lactate on endogenous triacylglycerol homeostasis. 4. Lactate-induced impairment of functional recovery of the post-ischemic heart. 5. The effect of lactate on lipid metabolism in the ischemic and post-ischemic heart. 6. The consequences of hyperlactaemia for cardiac imaging.

Animals↗
PubMed1992

Mechanisms of intermolecular homologous recombination in plants as studied with single- and double-stranded DNA molecules.

To elucidate the mechanism for intermolecular homologous recombination in plants we cotransformed Nicotiana tabacum cv Petit Havana SR1 protoplasts with constructs carrying different defective derivatives of the NPTII gene. The resulting kanamycin resistant clones were screened for possible recombination products by PCR, which proved to be a valuable technique for this analysis. Our results show that the double-stranded circular DNA molecules used in this study recombine predominantly via a pathway consistent with the single-strand annealing (SSA) model as proposed for extrachromosomal recombination in mammalian cells. In the remaining cases recombination occurred via a single reciprocal recombination, gene conversion and possibly double reciprocal recombination. Since single-stranded DNA is considered to be an important intermediate in homologous recombination we also established the recombination ability of single-stranded DNA in intermolecular recombination. We found that single-stranded DNA enters in recombination processes more efficiently than the corresponding double-stranded DNA. This was also reflected in the recombination mechanisms that generated the functional NPTII gene. Recombination between a single-stranded DNA and the complementing DNA duplex occurred at similar rates via a single reciprocal recombination and the SSA pathway.

Base Sequence↗
PubMed1991

A quick method to estimate the T-DNA copy number in transgenic plants at an early stage after transformation, using inverse PCR.

Agrobacterium-mediated transformation of plants is known to result in transgenic plants with a variable number of integrated T-DNA copies. Our aim was to obtain transgenic tobacco plants containing one integrated T-DNA copy per genome. Therefore, a quick method was developed to estimate the T-DNA copy number of young transgenic plantlets within 10 weeks after transformation. Inverse polymerase chain reaction (IPCR) was used to amplify junction fragments, i.e. plant genomic DNA sequences flanking the known T-DNA sequences.

Base Sequence↗
PubMed1990

Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation.

We determined whether T-DNA molecules introduced into plant cells using Agrobacterium are suitable substrates for homologous recombination. For the detection of such recombination events different mutant versions of a NPTII construct were used. In a first set of experiments protoplasts of Nicotiana tabacum SR1 were cocultivated with two Agrobacterium tumefaciens strains. Each strain contained a different T-DNA, one carrying a 5' deleted NPTII gene and the other a NPTII gene with a 3' deletion. A restored NPTII gene was found in 1-4% of the protoplasts that had been cotransformed with both T-DNAs. Restoration of the NPTII gene could only be the consequence of homologous recombination between the two different T-DNAs in the plant cell, since the possibility of recombination in Agrobacterium was excluded in control experiments. In subsequent experiments was investigated the potential use of Agrobacterium for gene targeting in plants. A transgenic tobacco line with a T-DNA insertion carrying a defective NPTII gene with a 3' deletion was transformed via Agrobacterium with a T-DNA containing a defective NPTII repair gene. Several kanamycin resistant plant lines were obtained with an intact NPTII gene integrated in their genome. In one of these lines the defective NPTII gene at the target locus had been properly restored. Our results show that in plants recombination can occur between a chromosomal locus and a homologous T-DNA introduced via A. tumefaciens. This opens the possibility of using the Agrobacterium transformation system for site directed mutagenesis of the plant genome.

Blotting, Southern↗
PubMed1990

Ischemia and reperfusion induced multilamellar vesicles in isolated rabbit hearts: time correlation between morphometric data and metabolic alterations.

In normoxic hearts a limited number of multilamellar vesicles was found in both endothelial cells and myocytes. The total number of multilamellar vesicles observed in myocytes, particularly those extruded from mitochondria, significantly increased in hearts rendered ischemic for at least 60 mins. The number of multilamellar vesicles extruded from sarcolemma was increased in hearts reperfused after this period of ischemia. The number of multilamellar vesicles in or adjacent to lipid droplets was independent of the duration of ischemia. Multilamellar vesicles were similar in size and periodicity of the lamellae. It is proposed that the number of multilamellar vesicles can be used to quantitate ischemic membrane injury. The formation of multilamellar vesicles was significantly related in time to (a) the accumulation of arachidonic acid and total fatty acids; (b) a decrease in the tissue content of ATP and (c) the release of lactate dehydrogenase (LDH). No significant alterations in the total tissue content of triacylglycerols and phospholipids were detected. The amount of arachidonic acid accumulated in the hearts reflects the degradation of only a minor fraction of the phospholipid pool. Assuming a close relationship between phospholipid degradation, induction of multilamellar vesicles and loss of cellular integrity, the present findings might indicate that the loss of a small part of phospholipids might have serious pathophysiological consequences, as indicated by the morphological changes in cellular membranes and the release of cytoplasmic macromolecules.

Adenosine Triphosphate↗
PubMed1989

Lactate-induced stimulation of myocardial triacylglycerol turnover.

The addition of lactate (5.6 mM) to a perfusion medium containing glucose (11 mM) stimulated the turnover of the cardiac triacylglycerol pool throughout the perfusion period as indicated by increased glycerol release in association with maintained levels of triacylglycerols. Attenuation of feedback inhibition of triacylglycerol lipase by fatty acids as a possible cause of the elevated triacylglycerol turnover rate should be ruled out, since tissue fatty acid levels were 3-times higher in glucose plus lactate perfused hearts than in hearts perfused with glucose as the sole substrate. The present findings are in favor of the notion that lactate enhances triacylglycerol turnover through increased glycerol 3-phosphate levels.

Animals↗
PubMed1989

Single-stranded DNA used as an efficient new vehicle for transformation of plant protoplasts.

In relation to the question which DNA form (single- or double-stranded) is transferred by Agrobacterium tumefaciens to plant cells, we studied the behaviour of single-stranded DNA, as compared to double-stranded DNA, when it is introduced into plant protoplasts by electroporation. To this end, we cloned a construct with a plant NPTII gene as well as a CAT gene in the M13 vectors tg130 and tg131. We found that both complementary single-stranded molecules gave rise to substantial CAT activity in plant protoplasts, suggesting that single-stranded DNA is converted into double-stranded DNA by the plant cell replication machinery. Unexpectedly, we found that single-stranded DNA leads to a 3-10-fold higher frequency of stable transformation (selection for kanamycin resistance) than double-stranded DNA. These results indicate that the use of single-stranded DNA might be considered in experiments in which optimal transformation frequencies are needed, e.g. with protoplasts from recalcitrant plant species.

Cloning, Molecular↗
PubMed1989

Effects of monounsaturated fatty acids v complex carbohydrates on serum lipoproteins and apoproteins in healthy men and women.

The effects of a high-carbohydrate, high-fiber diet and an olive-oil-rich diet on the distribution of cholesterol over the various lipoproteins, on serum apolipoproteins, and on the composition of HDL2 and HDL3 were studied under strict dietary control. Forty-eight healthy subjects first consumed a high-saturated-fat diet [proportion of energy, en%] (saturated fat 20 en%, total fat 38 en%) for 17 days. For the next 36 days, 24 subjects consumed a diet high in complex carbohydrates (monounsaturated fat 9 en%, total fat 22 en%) and the other 24 consumed a high-fat, olive-oil-rich diet (monounsaturated fat 24 en%, total fat 41 en%). The amounts of protein (12% to 14 en%), polyunsaturated fat (4 to 5 en%), and cholesterol (31 to 35 mg/MJ) were similar in all three diets. Serum cholesterol levels fell by 0.44 mmol/L in subjects consuming the carbohydrate diet and by 0.52 mmol/L for those receiving the olive-oil-rich diet. VLDL-cholesterol levels rose by 0.08 mmol/L in the carbohydrate group and fell by 0.08 mmol/L in the olive oil group (P less than .05 for difference between test diets). HDL2 and LDL cholesterol levels fell to the same extent on both diets. HDL3 cholesterol fell by 0.09 mmol/L on the high-carbohydrate diet and increased by 0.01 mmol/L on the olive oil diet (P less than .05). There was no change in the composition of HDL3, suggesting that the fall was due to a decrease in the total number of circulating particles.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

Degradation of adenine nucleotides in ischemic and reperfused rat heart.

Complete cessation of flow in isolated rat hearts for 90 min resulted in a gradual breakdown of ATP and concomitant accumulation of degradation products, such as adenosine, inosine (major break-down product), hypoxanthine, and, to a lesser extent, xanthine. After 45 min of ischemia, the content and relative composition of purines hardly changed, whereas the AMP content continued to rise. This finding points to constraints on AMP degradation and flux through the degradation pathway from adenosine to uric acid in the ischemic heart. In myocardial preparations, the cells of which were deliberately disrupted by freezing and thawing before anoxic incubation, AMP did not accumulate and was finally converted to hypoxanthine. These results indicate that compartmentalization of substrates and enzymes is responsible for the observed preferential accumulation of AMP and inosine in the ischemic heart. Inhibition of hypoxanthine degradation is explained by the absence of oxygen. Restoration of flow and oxygen supply abolished the inhibition of metabolic flux. Accumulated purines were released into the coronary effluent and, concomitantly, further metabolized. Comparison of tissue levels of hypoxanthine, xanthine, and uric acid before reperfusion and the amounts released during reperfusion indicates that in rat myocardium substantial amounts of potentially hazardous xanthine oxidase-derived reactive oxygen species are likely to be formed during the early reperfusion phase.

Adenine Nucleotides↗
PubMedDate not supplied

Degradation of phospholipids and triacylglycerol, and accumulation of fatty acids in anoxic myocardial tissue, disrupted by freeze-thawing.

The degradation of lipids by endogenous hydrolytic activity has been studied in rat cardiac tissue deliberately damaged by freezing and thawing prior to storage under anoxic conditions. Aliquots of the freeze-thawed material were kept at 37 degrees C under an atmosphere of N2 up to 120 minutes. Triacylglycerol was hydrolyzed at a rate of 0.14 mumol fatty acids per minute per gram dry weight of tissue. Hydrolysis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was associated with proportional production of lyso PC and lyso PE, respectively. This finding indicates that the activity of lysophospholipase is negligible in autolyzing cardiac tissue. The rate of hydrolysis of PC and PE was found to be 0.10 and 0.06 mumol per minute per gram dry weight of tissue. The observation that lyso PC and lyso PE mainly contained saturated and mono-unsaturated fatty acids indicates that phospholipase A2 rather than A1 is active in autolyzing cardiac tissue. The accumulation of fatty acids corresponded with the loss of triacylglycerol and phospholipids from the tissue during 120 minutes of autolysis.

Animals↗
PubMedDate not supplied

Metabolite predictions for para-substituted anisoles based on ab initio complete active space self-consistent field calculations.

The cytochrome P450 mediated oxidative metabolism of a series of para-substituted anisoles has been examined using ab initio CASSCF (complete active space self-consistent field) calculations. On the basis of these calculations, oxidative metabolites were rationalized using the concept of hydrogen atom abstraction, spin delocalization, and hydroxyl radical recombination, which is believed to govern part of the oxidation and oxygenation reactions catalyzed by cytochrome P450. Spin distributions and energy differences between substrates, metabolic intermediates, and products were calculated. A comparison of the predictions with recent experimental findings from other laboratories supports the applicability of the currently used computational model for predicting qualitatively the oxidative metabolism by cytochrome P450.

Anisoles↗