Biomedical subjects

W J Geerts

Publications and source records attributed to W J Geerts.

At least 19 recordsLinked to original sources

PubMed1999

High protein diet induces pericentral glutamate dehydrogenase and ornithine aminotransferase to provide sufficient glutamate for pericentral detoxification of ammonia in rat liver lobules.

The liver plays a central role in nitrogen metabolism. Nitrogen enters the liver as free ammonia and as amino acids of which glutamine and alanine are the most important precursors. Detoxification of ammonia to urea involves deamination and transamination. By applying quantitative in situ hybridization, we found that mRNA levels of the enzymes involved are mainly expressed in periportal zones of liver lobules. Free ammonia, that is not converted periportally, is efficiently detoxified in the small rim of hepatocytes around the central veins by glutamine synthetase preventing it from entering the systemic circulation. Detoxification of ammonia by glutamine synthetase may be limited due to a shortage of glutamate when the nitrogen load is high. Adaptations in metabolism that prevent release of toxic ammonia from the liver were studied in rats that were fed diets with different amounts of protein, thereby varying the nitrogen load of the liver. We observed that mRNA levels of periportal deaminating and transaminating enzymes increased with the protein content in the diet. Similarly, mRNA levels of pericentral glutamate dehydrogenase and ornithine aminotransferase, the main producers of glutamate in this zone, and pericentral glutamine synthetase all increased with increasing protein levels in the diet. On the basis of these changes in mRNA levels, we conclude that: (a) glutamate is produced pericentrally in sufficient amounts to allow ammonia detoxification by glutamine synthetase and (b) in addition to the catalytic role of ornithine in the periportally localized ornithine cycle, pericentral ornithine degradation provides glutamate for ammonia detoxification.

Ammonia↗

PubMed1997

Quantitative graphical description of portocentral gradients in hepatic gene expression by image analysis.

The liver consists of numerous repeating, randomly oriented, more or less cylindrical units, the lobules. Although enzyme-histochemical or microbiochemical assays accurately reflect zonal differences in lobular enzyme content, their results cannot be directly compared to biochemical assays. This is because section-based assays typically sample along a linear portocentral column of cells, even though periportal regions contribute substantially more to hepatic volume than pericentral regions. We have developed a time-efficient approach that depends on image analysis to determine the prevalence of hepatocytes (pixels) with a defined cellular concentration of a particular gene product (absorbance), and that generates a graph with the average absorbance per hepatocyte on the ordinate and the percentage of hepatocytes with absorbances in each of a predetermined range of absorbances incrementally summed on the abscissa. The direction of the gradient is read directly from the section. The gradient is a graphical representation of the two-dimensional distribution pattern of the gene product between the portal tracts and the central veins. The total surface area underneath the resulting graph represents the integrated absorbance and is equivalent to the outcome of a biochemical assay. The typical linear portocentral gradient can be derived from that representing the two-dimensional distribution if we assume that liver lobules are uniformly cylindrical or prismatic. The analysis, therefore, yields a quantitative description of the relation between the enzymatic phenotype of hepatocytes and their position on a normalized portocentral radius. We have used the procedure to compare portocentral gradients of different enzymes in the same liver and of the same enzyme in different livers. In addition, bipolar portocentral gradients of the same enzyme in the same liver were analyzed.

Animals↗

PubMed1997

Basic strategies for valid cytometry using image analysis.

The present review provides a starting point for setting up an image analysis system for quantitative densitometry and absorbance or fluorescence measurements in cell preparations, tissue sections or gels. Guidelines for instrumental settings that are essential for the valid application of image analysis in cytophotometry and cytofluorometry are described. The general principles of the working mechanism of CCD cameras in combination with general methods to improve the behaviour of the cameras are presented. Optimization of illumination of microscopical and macroscopical objects receives special attention because of its importance for valid cytometry. Sources of errors in quantitative measurements are listed and step-by-step charts for tuning the CCD camera, frame grabber and illumination for the optimal use of the systems are described. Suggestions are given for improvement of image arithmetic in difficult imaging situations, such as low fluorescence signals and high absorbance signals.

Absorption↗

PubMed1997

In situ measurement of glutamate concentrations in the periportal, intermediate, and pericentral zones of rat liver.

We developed a quantitative histochemical assay for measurement of local glutamate concentrations in cryostat sections of rat liver. Deamination of glutamate by glutamate dehydrogenase (GDH) was coupled to the production of formazan and formazan precipitation was used for colorimetric visualization. The method was tested and validated with gelatin model sections with known glutamate concentrations. Calibration graphs showed linear relationships with high correlation coefficients (> 96%) between glutamate concentrations or section thickness and absorbance values. The method was reproducible, with a constant percentage of 60 +/- 5% of glutamate being converted in gelatin model sections containing glutamate concentrations of 2 mM and higher. Glutamate concentrations were estimated in periportal, intermediate, and pericentral zones of liver lobules that contain low, intermediate, and high GDH activity, respectively. In fed adult male rat livers, periportal zones contained the highest concentrations of glutamate (approximately 14 mM) and intermediate and pericentral zones approximately 13 and 9 mM, respectively. On starvation, glutamate concentrations increased only in the small rim of pericentral cells that express glutamine synthetase, to approximately 15 mM. In livers of fetal and newborn rats, glutamate was homogeneously distributed, with a concentration of approximately 5 mM. In suckling rat liver, distribution of glutamate was still homogeneous but the concentration was increased to approximately 8 mM. These glutamate distribution patterns were in agreement with those detected immunohistochemically.

Age Factors↗

PubMed1996

The dynamics of local kinetic parameters of glutamate dehydrogenase in rat liver.

Kinetic parameters of glutamate dehydrogenase (GDH, EC 1.4.1.2) for glutamate were determined in periportal and pericentral zones of adult male and female rat liver lobules under normal fed conditions and after starvation for 24 h. GDH activity was measured as formazan production over time against a range of glutamate concentrations in serial cryostat sections using image analysis. Captured gray value images were transformed to absorbance images and local initial velocities (Vini) were calculated. A hyperbolic function was used to describe the relationship between substrate concentration and local Vini. Under fed conditions, Vmax values were similar in male and female rats (8 +/- 2 and 16 +/- 2 mumol min-1 cm-3 liver tissue in periportal and pericentral zones, respectively). Starvation increased Vmax, especially in pericentral zones of females (to 27 +/- 1 mumol min-1 cm-3 liver tissue). Under fed conditions, the affinity of GDH for glutamate was similar in male and female rats (2.5 +/- 0.5 mM and 3.5 +/- 0.8 mM in periportal and pericentral zones, respectively). Starvation had no effect on K(m) values in male rats, but in female rats affinity for glutamate decreased significantly in both zones (K(m) values of 4.0 +/- 0.1 mM and 8.6 +/- 0.8 mM, respectively). These local changes in the kinetic parameters of GDH indicate that conversion of glutamate to alpha-oxoglutarate cannot be predicted on the basis of GDH concentrations or zero-order activity in the different zones of liver lobules alone.

Animals↗

PubMed1996

Involvement of methyltransferase-activating protein and methyltransferase 2 isoenzyme II in methylamine:coenzyme M methyltransferase reactions in Methanosarcina barkeri Fusaro.

The enzyme systems involved in the methyl group transfer from methanol and from tri- and dimethylamine to 2-mercaptoethanesulfonic acid (coenzyme M) were resolved from cell extracts of Methanosarcina barkeri Fusaro grown on methanol and trimethylamine, respectively. Resolution was accomplished by ammonium sulfate fractionation, anion-exchange chromatography, and fast protein liquid chromatography. The methyl group transfer reactions from tri- and dimethylamine, as well as the monomethylamine:coenzyme M methyltransferase reaction, were strictly dependent on catalytic amounts of ATP and on a protein present in the 65% ammonium sulfate supernatant. The latter could be replaced by methyltransferase-activating protein isolated from methanol-grown cells of the organism. In addition, the tri- and dimethylamine:coenzyme M methyltransferase reactions required the presence of a methylcobalamin:coenzyme M methyltransferase (MT2), which is different from the analogous enzyme from methanol-grown M. barkeri. In this work, it is shown that the various methylamine:coenzyme M methyltransfer steps proceed in a fashion which is mechanistically similar to the methanol:coenzyme M methyl transfer, yet with the participation of specific corrinoid enzymes and a specific MT2 isoenzyme.

Adenosine Triphosphate↗

PubMed1996

Gender-dependent regulation of glutamate dehydrogenase expression in periportal and pericentral zones of rat liver lobules.

We studied the level(s) at which glutamate dehydrogenase (GDH; EC 1.4.1.2) expression is regulated in the livers of fed male and female rats. The cellular content of GDH mRNA, protein, and enzyme activity was determined quantitatively using image analysis for measurement of the absorbance in consecutive serial sections that were processed for in situ hybridization, immunohistochemistry, and enzyme histochemistry. In both males and females, GDH protein and activity patterns were similar, with pericentral values being twice as high as periportal values. GDH mRNA distribution patterns in female liver lobules reflected those of GDH protein and activity, but GDH mRNA distribution patterns in male rat livers were found to be homogeneous owing to a more than twofold lower cellular mRNA content in pericentral zones than in female rats. We conclude that gender affects GDH expression selectively in pericentral zones at posttranscriptional and pretranslational levels.

Animals↗

PubMed1995

Image analysis and image processing as tools to measure initial rates of enzyme reactions in sections: distribution patterns of glutamate dehydrogenase activity in rat liver lobules.

To analyze regional differences in the activity of glutamate dehydrogenase in rat liver in situ, we developed an image recording and processing system for monitoring the formation of a colored final reaction product in time. All absorbance measurements of test and control reactions in time in consecutive sections were used to fit the data to a quadratic curve, with the derivative at t = 0 representing the initial velocity of formazan formation. The images of sections incubated for test and control reactions were topographically matched with an affine transformation using the positions of vessels as fiducials. Specific enzyme activity was calculated by subtracting the coefficients representing the initial velocity at corresponding locations in the test and control reactions and appeared to be 8 and 4 mumoles glutamate converted per min per cm3 of tissue at 20 degrees C in pericentral and periportal zones of fasted female rats, respectively. Those values are in agreement with biochemical data. The ability to construct two-dimensional images of cellular distribution patterns of enzyme activity in liver lobules is particularly useful for the study of metabolic zonation in this organ.

Animals↗

PubMed1994

Lobular patterns of expression and enzyme activities of glutamine synthase, carbamoylphosphate synthase and glutamate dehydrogenase during postnatal development of the porcine liver.

Carbamoylphosphate synthase and glutamine synthase show a complementary distribution in the liver lobule of the rat. In the human liver lobule, which is approximately 2-fold larger than that of the rat, an intermediate, 'empty' zone is present between the periportal carbamoylphosphate synthase-positive and the pericentral glutamine synthase-positive zone. To investigate whether these differences in gene expression can be attributed to the size of the liver lobule, we investigated the patterns of expression of carbamoylphosphate synthase, glutamine synthase and glutamate dehydrogenase during postnatal development of the pig, a species in which the total number of lobules does not increase after birth. We demonstrate that lobular size increases 3-fold between 1 week and 8 months after birth. In the same developmental period the number of hepatocytes on the porto-central axis increases 2-fold, resulting in a 3-fold increase in cellular volume. However, the lobular patterns of expression of carbamoylphosphate synthase, glutamate dehydrogenase and glutamine synthase do not change anymore after 1 month, i.e., when lobular diameter is comparable to that in rat liver, showing that lobular size is not a major determinant of the heterogeneous patterns of expression of these enzymes. The adult patterns of expression of glutamine synthase, glutamate dehydrogenase and, in particular carbamoylphosphate synthase in the porcine liver resemble those of man. Changes in the enzyme activities of glutamate dehydrogenase and carbamoylphosphate synthase are not related to the lobular size. However, the 70% decrease of GS activity in the 8-month-old pigs corresponds with the gradual 2-3-fold decrease in the size of the GS-positive compartment during postnatal development. During adulthood GS activity increases again to values observed 1 week after birth demonstrating a 2-fold increase in cellular glutamine synthase content. The present data show that the pig is an excellent model to study the regulation and functional implication of zonation of gene expression in the human liver.

Animals↗

PubMed1993

Differences in erythropoiesis in normal chicken and quail embryos.

Using antibodies against the fetal and adult forms of alpha- and beta-globin, it has been shown that erythropoiesis in the para-aortic foci (PAF) constitutes a major species-specific difference between chicken and quail embryos. In quail embryos, para-aortic foci are rare, small and rather heterogeneous with regard to their erythropoietic and haemopoietic cell composition. In contrast, the PAFs in chicken embryos are abundant and consist of large numbers of erythropoietic cells. In both species a time difference (approximately 1 day) is observed between the first expression of the fetal alpha- and beta-globin and the adult alpha- and beta-globin in erythropoietic cells. Adult erythropoiesis in both species can be detected first in the stalk of the yolk sac; this is similar to the situation in mammalian and amphibian species. From this time onward the number of circulating adult erythrocytes increases steadily. Whereas in chicken, large intraembryonic foci that can serve as sources for these adult cells arise concomitantly, no such foci can be detected in quail embryos, suggesting that the quail yolk sac is a major source for these adult red blood cells.

Animals↗

PubMed1991

cDNA sequence of the long mRNA for human glutamine synthase.

Screening a human liver cDNA library in lambda ZAP revealed several clones for the mRNA of glutamine synthase. The longest clone was completely sequenced and consists of a 109 bp 5' untranslated region, a 1119 bp protein coding region, a 1498 bp 3' untranslated region and a poly(A) tract of 12 bp.

Base Sequence↗

PubMed1991

Purification and properties of 5,10-methylenetetrahydromethanopterin dehydrogenase and 5,10-methylenetetrahydromethanopterin reductase, two coenzyme F420-dependent enzymes, from Methanosarcina barkeri.

5,10-Methylenetetrahydromethanopterin dehydrogenase and 5,10-methylenetetrahydromethanopterin reductase have been purified to homogeneity by a factor of 86 and 68, respectively, from methanol-grown Methanosarcina barkeri cells. The dehydrogenase was isolated as a hexamer of a single 35 kDa subunit, whereas the reductase was composed of four identical 38 kDa subunits. The purified oxygen-stable enzymes catalyzed the oxidation of 5,10-methylenetetrahydromethanopterin and methyltetrahydromethanopterin with Vmax values of 3000 and 200 mumol min-1 mg-1, respectively. The methanogenic electron carrier coenzyme F420 was a specific electron acceptor for both enzymes. Steady state kinetics for the two enzymes were in agreement with ternary complex (sequential) mechanisms. Methylene reductase and methylene dehydrogenase are proposed to function in the methanol oxidation step to CO2.

Chromatography, Gel↗

PubMed1990

Isomyosin expression pattern during formation of the tubular chicken heart: a three-dimensional immunohistochemical analysis.

Three-dimensional (3-D) distribution of atrial and ventricular isomyosins is analysed immunohistochemically during the formation of the tubular chicken heart (stage 7 to 12 [H/H]) using antibodies specific for adult chicken atrial and ventricular myosin heavy chains, respectively. This analysis revealed that both types of isomyosins can be first detected at stage 8 (H/H, possessing four pairs of somites), i.e., when the heart primordium still exists as two separate cardiogenic plates. The ventricular type of isomyosin is initially expressed in those areas of cardiogenic plates in the vicinity of the anterior intestinal portal. The atrial type of isomyosin is initially expressed in zones caudal and lateral to the areas of ventricular isomyosin expression. Medial to the atrial isomyosin-expressing areas, cardiogenic plate areas exist that initially lack myosin expression. Those parts of the cardiogenic plates that fuse in front of the anterior intestinal portal, thereby forming the heart tube, are characterized by the expression of both isomyosins; however, the caudolateral parts of the heart primordium maintain their single atrial isomyosin expression during further development. Cardiac contractions are therefore first observed at stage 10 (H/H, possessing ten pairs of somites) in myocardium that coexpresses both isomyosins.

Animals↗

PubMed1990

Distribution pattern of acetylcholinesterase in early embryonic chicken hearts.

To study the developmental appearance of acetylcholinesterase in early embryonic hearts, an enzyme-histochemical study was carried out in chicken embryos ranging from cardiogenic plate to late tubular stages. Initially acetylcholinesterase is present in all cells of the (future) myocardium. When 13-14 pairs of somites have developed, i.e., shortly before blood propulsion starts, acetylcholinesterase selectively disappears from the ventral and lateral wall of the developing ventricle. Slightly later, when 18-19 pairs of somites have developed, acetylcholinesterase also disappears from the dorsal and anterior wall of the atrium. High concentrations of acetylcholinesterase remain present in the outflow tract and lower concentrations in a continuous tract along the lesser curvature of the heart, the atrial side of the atrioventricular canal, and the left wall of the atrium. In late tubular stages of heart development, acetylcholinesterase is reexpressed in the inner myocardial layer of the ventricle, i.e., in the developing trabeculae and the ventricular side of the atrioventricular canal, where it is continuous with the acetylcholinesterase-expressing cells of the atrial side of the atrioventricular canal. The expression pattern of acetylcholinesterase in early embryonic chick hearts coincides with that of areas that control the conduction of the impulse and may reveal a cholinergic signal transduction system that is responsible for a coordinated contraction pattern of the myocardium prior to the development of the definitive conductive system.

Acetylcholinesterase↗

PubMed1990

Purification and properties of 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanosarcina barkeri.

The 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanosarcina barkeri was purified 313-fold to a specific activity of 470 mumol min-1 mg-1 at 37 degrees C and pH 7.8. At this stage, the enzyme was pure as judged from polyacrylamide gel electrophoresis. The monofunctional enzyme was oxygen stable, but the presence of a detergent proved to be essential for its stability. Like the cyclohydrolase purified from Methanobacterium thermoautotrophicum (A. A. Dimarco, M. I. Donnelly, and R. S. Wolfe, J. Bacteriol. 168:1372-1377, 1986), the protein showed an apparent Mr of 82,000, and it is composed of two identical subunits as was concluded from nondenaturating and denaturating polyacrylamide gel electrophoresis. The enzymes from M. thermoautotrophicum and M. barkeri markedly differ with respect to the hydrolysis product of 5,10-methenyltetrahydromethanopterin: 5-formyl- and 10-formyltetrahydromethanopterin, respectively. The apparent Km for 5,10-methenyltetrahydromethanopterin was 0.57 mM at 37 degrees C and pH 7.8.

Aminohydrolases↗

PubMed1989

Creatine kinase isozyme expression in embryonic chicken heart.

The distribution pattern of creatine kinase (EC 2.7.3.2) isozymes in developing chicken heart was studied by immunohistochemistry. Creatine kinase M, which is absent from adult heart, is transiently expressed between 4 and 11 days of incubation. During that period, numerous muscular cells in the roof and septum of the atrium, in the interventricular septum and on top of the trabeculae cordis and at the rim of the outflow tract stain strongly with a polyclonal antibody that is specific for the M subunit. In the ventricle and outflow tract, the M-positive cells are found mainly subendocardially and in the right half, at the transition of conducting and working myocytes. Creatine kinase B, which is the predominant adult isozyme, is initially expressed to a high concentration in a small group of disperse myocardial cells in upstream part of the inflow tract. When compared to the expression pattern of cardiac myosin heavy chains, the observed creatine kinase expression pattern suggests that M-positive cells are mainly found in areas that participate in the formation of cardiac conductive tissue, whereas B-positive cells are first found in areas that are involved in the generation of cardiac rhythm.

Animals↗

PubMed1988

Complementary distribution of carbamoylphosphate synthetase (ammonia) and glutamine synthetase in rat liver acinus is regulated at a pretranslational level.

We studied the distribution of the mRNAs for carbamoylphosphate synthetase (ammonia) and glutamine synthetase in frozen sections of adult rat liver by in situ hybridization to [35S]-labeled cDNA probes. The density of silver grains resulting from hybridization to the labeled cDNA probe for carbamoylphosphate synthetase is highest around the portal venules, decreases towards the central venule, and is virtually absent from an area two to three cells wide that lines the central venules in which mRNA for glutamine synthetase is predominantly localized. Therefore, both mRNAs show the same complementary distribution within the liver acinus that was found for the proteins they encode, demonstrating that compartmentalization of the expression of these enzymes is controlled at a pretranslational level. In addition, we found that carbamoylphosphate synthetase mRNA is present mainly in the epithelium of the crypts of the proximal part of the small intestine, whereas carbamoylphosphate synthetase protein is present in the epithelium of both crypts and villi.

Animals↗

PubMed1987

Isomyosin expression patterns in tubular stages of chicken heart development: a 3-D immunohistochemical analysis.

The 3-D distribution of atrial and ventricular isomyosins is analysed in tubular chicken hearts (stage 12+ to 17 (H/H)) using antibodies specific for adult chicken atrial and ventricular myosin heavy chains, respectively. At stage 12+ (H/H) all myocytes express the atrial isomyosin; furthermore, all myocytes except those originally situated in the dorsolateral wall of the sinu-atrium coexpress the ventricular isomyosin as well. Moreover, it appears that recently incorporated myocardial cells at both ends of the heart tube start with a coexpression of both isomyosins. From stage 14 (H/H) onwards a regional loss of expression of one of either isomyosins is observed in the atrial and ventricular compartment. In this way the single isomyosin expression types that are characteristic for the adult working myocardium of the atria and ventricles arise. So, the isomyosin expression patterns are, unexpectedly, hardly useful to discriminate the different heart parts of the tubular heart. The ventricle, defined by its adult type of isomyosin expression, is even not detectable before stage 14 (H/H). Interestingly, interconnected coexpression areas, which may be precursor conductive tissues, are still present at stage 17 (H/H) in the outflow tract, the ventricular trabeculae, the atrio-ventricular transitional zone and in the sinu-atrium. The pattern of isomyosin coexpression was found to correlate with a peristaltoid contraction and a slow conduction velocity, whereas single expression areas correlate with a synchronous contraction and a relatively fast conduction velocity. The possible implications of the changing isomyosin pattern for the differentiation of the tubular myocardium, in particular in relation to the development of the conductive tissues, will be discussed.

Animals↗