The modular microarchitecture of human liver.

The morphological homogeneity of the liver parenchyma has represented a major obstacle in finding an acceptable definition of the structural/functional units of the liver. Concepts such as the "lobule," the "portal unit" and the "acinus" remain debatable. This study investigates the modular microarchitecture on the basis of the lobular concept.

Three-dimensional reconstruction of parenchymal units in the liver of the rat.

To investigate the parenchymal units in the liver of the rat three-dimensionally, 15 micrometer cryosections were used for the demonstration of glucose-6-phosphatase (G6Pase) activity to visualize the borders of the individual units. Together with the supplying and draining vessels, they were traced through a sequence of 146 sections and reconstructed. A cone-shaped secondary unit with a height of 2.

Purkinje cell lineage and the topographic organization of the cerebellar cortex: a view from X inactivation mosaics.

We utilized a strain of mice, derived from a radiation mutagenesis experiment and carrying an activity-attenuated allele of the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD), to analyze the development of the cell lineage leading to cerebellar Purkinje neurons. Due to random X inactivation during early embryonic development, X- linked genes can be used to distinguish between clonally related populations of cells in X inactivation mosaics. Following histochemical staining for G6PD activity, the numeric proportions of Purkinje cells expressing either the wild-type or the mutant enzyme and the spatial distribution of these cellular phenotypes and their relation to anatomically and genetically defined cerebellar compartments were analyzed.

Glucose levels and succinate and lactate dehydrogenase activity in EMT6/Ro tumor spheroids.

To evaluate the effects of glucose on the development of cell heterogeneity and the occurrence of necrotic areas in the center of tumor spheroids, a procedure (combining microdissection of small tissue samples from frozen-dried cryosections and microchemical analysis) was developed to measure glucose in distinct, concentrically arranged, microregions of tumor spheroids: the outermost area of proliferating cells, the area of nonproliferating cells and 2 central "necrotic" areas, with either abundant or little intercellular space. Since glucose levels, for analytical reasons, had to be expressed on a dry weight basis, and because of the morphological heterogeneity of the microregions of tumor spheroids, it was necessary to measure and take into account the regional differences in cell density (water content), in order to obtain adequate estimates of the glucose levels in the various microregions. At glucose concentrations of 5.

Characterization of recombinant plant cinnamate 4-hydroxylase produced in yeast. Kinetic and spectral properties of the major plant P450 of the phenylpropanoid pathway.

Helianthus tuberosus cinnamate 4-hydroxylase (CYP73 or CA4H), a member of the P450 superfamily which catalyses the first oxidative step of the phenylpropanoid pathway in higher plants by transforming cinnamate into p-coumarate, was expressed in the yeast Saccharomyces cerevisiae. The PCR-amplified CA4H open reading frame was inserted into pYeDP60 under the transcriptional control of a galactose-inducible artificial promoter. Engineered S.

Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway.

Cinnamate 4-hydroxylase [CA4H; trans-cinnamate,NADPH:oxygen oxidoreductase (4-hydroxylating), EC 1.14.13.

Distribution of 3-hydroxybutyrate dehydrogenase in primary lobules of rat liver.

In an attempt to establish the functional organization of the hepatic parenchymal unit, we used histo- and microchemical procedures to assess metabolic liver cell heterogeneity at the level of the primary lobule. Because of the close interrelation of glucogenesis and ketone body formation, and in view of the distinct regional differences of the in vivo activity of glucose-6-phosphatase (G6Pase), these techniques were used on livers from male rats to investigate the distribution of the ketogenic enzyme, 3-hydroxybutyrate dehydrogenase (3-HBDH), during the post-resorptive phase. A close reciprocity was found between the general increase in the activity of 3-HBDH and the decrease of the in vivo activity of G6Pase along the sinusoidal axis, and also with regard to enzyme gradients along sinusoids of different origin.

Glycerol allows low-temperature phase separation of membrane proteins solubilized in Triton X-114: application to the purification of plant cytochromes P-450 and b5.

The potentiality of the Triton X-114 phase separation technique for the purification of proteins from plant microsomal membranes has been investigated. It was shown that glycerol significantly lowers the cloud point of Triton X-114 solutions in water and of Triton X-114 solubilizates from microsomal membranes. It was also established that solubilized membrane components decrease the temperature of Triton X-114 micellar aggregation.

Purification and immunocharacterization of a plant cytochrome P450: the cinnamic acid 4-hydroxylase.

Cinnamic acid 4-hydroxylase (CA4H) was purified from microsomes of manganese-induced Jerusalem artichoke (Helianthus tuberosus L.) tuber tissues. The three-step purification procedure involved solubilization and phase partitioning in Triton X-114, followed by chromatography on DEAE-Trisacryl and hydroxylapatite columns.

Two cytochrome P-450 isoforms catalysing O-de-ethylation of ethoxycoumarin and ethoxyresorufin in higher plants.

The O-dealkylating activities of 7-ethoxycoumarin O-de-ethylase (ECOD) and 7-ethoxyresorufin O-de-ethylase (EROD) have been fluorimetrically detected in microsomes prepared from manganese-induced Jerusalem artichoke tubers. Cytochrome P-450 dependence of the reactions was demonstrated by light-reversed CO inhibition, NADPH-dependence, NADH-NADPH synergism and by use of specific inhibitors: antibodies to NADPH-cytochrome P-450 reductase, mechanism-based inactivators and tetcyclasis. Apparent Km values of 161 microM for 7-ethoxycoumarin and 0.

Regionality of glucose-6-phosphate hydrolysis in the liver lobule of the rat: metabolic heterogeneity of "portal" and "septal" sinusoids.

To investigate intercellular compartmentation of liver metabolism, we have recently introduced new procedures for quantitative assessment of metabolic liver cell heterogeneity both along sinusoids of portal and septal origins as well as at the level of the parenchymal unit, and also for three-dimensional imaging of enzyme and metabolite distribution. As part of the evaluation of the role of metabolic liver cell heterogeneity for the regulation of net substrate flux in the glucose-6-phosphatase/glucokinase system, and for the reduction of of these antagonistic enzymes, these techniques were used on livers from male rats. They served to obtain distribution data on glucose-6-phosphatase (the hydrolytic component of the glucose-6-phosphatase/glucokinase system) and its substrate, glucose-6-P, during the postresorptive phase (i.

A new sample isolation procedure for microchemical analysis of functional liver cell heterogeneity.

In conjunction with the investigation of intercellular compartmentation of liver carbohydrate metabolism, a new procedure for isolation of tissue samples from freeze-dried cryosections was developed. It was designed to permit assessment of functional differences between sinusoids of portal and septal origin, and to extend investigation of liver cell heterogeneity along sinusoids to the level of the structural-functional unit. Application of this procedure, together with microchemical assays of high analytical sensitivity, enabled measurement of 50 individual glucose and glucose-6-P values in a single cross-sectional area of about 0.

Use of 3-D-computer graphics for imaging of distribution of hepatic metabolites.

In conjunction with the investigation of intercellular compartmentation of liver metabolism and as a logical further step, following the introduction of a new sample isolation procedure for microchemical analysis of functional liver cell heterogeneity, the possible benefit of computer-assisted three-dimensional imaging procedures for the reconstruction of hepatic metabolite distribution was investigated. In this intent, we elected to access a central computer facility by means of a small microcomputer system which, nevertheless, permitted to take full advantage of a large capacity main-frame computer and a high quality graphics plotter, at comparatively low overall costs. Commercially available software (SAS/GRAPH) was tailored to the specific requirements of this application.

Quantitative histochemical assessment of regional differences in hepatic glucose uptake and release.

As a further step in the investigation of the heterogeneity of liver cells in general and regionality of glucose metabolism in particular, requirements for isolation of appropriate tissue samples were defined and procedures for measurement of the biochemical parameters responsible for glucose uptake and release developed and tested. By using enzymatic cycling for chemical amplification, in conjunction with the oil-well technique, sufficient analytical sensitivity was provided to assay samples averaging 20 ng dry weight. Microchemical data on the distribution of glucokinase and glucose-6-phosphatase and of their substrates, glucose and glucose-6-P, were used to, first calculate in vivo rates of these catalytic steps by means of the Michaelis-Menten equation, and then, to determine the direction and rate of net glucose flux, as well as, the rate of substrate cycling between glucose and glucose-6-P.

Sex-specific regionality of liver metabolism during starvation; with special reference to the heterogeneity of the lobular periphery.

Staining procedures for glucose-6-phosphatase and 3-hydroxybutyrate dehydrogenase activity and for glycogen were used to investigate adaptive changes in the regionality of hepatic gluconeogenesis and ketogenesis in fasting male and female rats. A reciprocal distribution of gluconeogenic and ketogenic capacities was found in both sexes, but male and female animals were different with respect to: a) the time necessary for full induction of glucose-6-phosphatase activity (24 h in females, 48 h in males); b) the overall activity of 3-hydroxybutyrate dehydrogenase; and c) glycogen content. The activity of the latter enzyme and the glycogen content did increase with time of starvation, but at all times, were higher in males, than in females.

High activity of glucose-6-phosphate dehydrogenase in Kupffer cells isolated from rat liver.

Sinusoidal cells in the rat liver react intensively for G6DPH activity after appropriate incubation (Rieder et al. 1978). After isolation and purification of the sinusoidal Kupffer and endothelial cells, it was demonstrated that Kupffer cells exhibit a 5-8 times higher G6PDH activity on a per cell basis by comparison with endothelial cells, while the specific G6PDH activity was 3-4 times higher in Kupffer cells.

Metabolic zonation in thioacetamide-induced liver cirrhosis.

After TAA administration to rats a central part may be distinguished histochemically from a marginal part in most of the cirrhotic nodules. The centre is characterized by a high glycogen content and by high activity of phosphorylase, G6Pase and SDH; the maxima of which are situated around the larger blood vessels. The vasculatory periphery, however, shows moderate G6PDH-activity.

The development of functional heterogeneity in the liver parenchyma of the golden hamster.

Prenatal and postnatal stages of the development of golden hamsters were studied histochemically and biochemically. It was shown that, beginning with the 12th gestational day, the fetal liver starts to store glycogen, and that this process reaches its maximum a birth. Glycogen phosphorylase and glucose-6-phosphatase (G6Pase)-activity increased drastically in the last two days before birth, glycogen phosphorylase preceding G6Pase.

Histochemical studies on carbohydrate metabolism in rat liver after galactosamine administration.

The development of hepatitis, induced in 48 rats by the administration of galactosamine (GalN) in varying doses, was studied with the use of substrate and enzyme histochemical techniques. The so-called atypical glycogen, which is at first highly resistant to diastase, was shown to be digestible after deamination. The increasing accumulation of atypical glycogen during the course of GalN-hepatitis conceals the loss of normal glycogen when the PAS-reaction is used.

NADP-dependent dehydrogenases in rat liver parenchyma. II. Comparison of qualitative and quantitative G6PDH distribution patterns with particular reference to sex differences.

Qualitative histochemical G6PDH distribution patterns obtained in the liver acinus of adult male and female rats with an improved method (Rieder et al., 1978) served as a basis for the isolation by microdissection of tissue samples of defined zonal affiliation. G6PDH activity was assayed quantitatively in tissue samples of zones 1 and 3 by a microfluorometric method, using the oil well technique and enzymatic cycling (Burch et al.

Identification of G6PDH-active sinusoidal cells as Kupffer cells in the rat liver.

The aim of this study was to identify the G6PDH-active sinusoidal cells in the rat liver described by Rieder et al. (1978). Because of their number and distribution in the liver parenchyma, endothelial cells and pit cells could be excluded.