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Run-off transcription in nuclei isolated from soybean seedlings was used to test the hypothesis that the expression of heat shock genes is controlled at the level of transcription. Only nuclei pretreated by a heat shock at 41°C prior to their isolation synthesized RNA from heat shock genes. The specificity of transcripts was determined by Southern blot hybridization of [32P]-labelled run-off RNA with DNA fragments from several heat shock and non-heat shock genes. The strand selectivity of heat shock gene transcription was exemplified by single stranded DNA probes. Low concentrations of α-amanitin completely inhibited the synthesis of heat shock specific RNA, but only partially inhibited the synthesis of ribosomal RNA. The overall transcription of nuclei isolated from heat shock tissue was reduced by more than 20% compared to that in nuclei from control tissue. This decline is consistent with a decrease in the transcriptional activity on non-heat shock genes transcribed by RNA polymearases I and II. Our results suggest that temperature stress induces the transcriptional activation of heat shock genes and has a negative effect on the transcription of other genes. [ABSTRACT FROM AUTHOR]

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) induce gastric ulcers. AIMS: To assess whether the somatostatin analogue octreotide prevents NSAID induced mucosal gastrointestinal damage in both animals and humans. The effect of octreotide on neutrophil adhesion to the endothelium was also evaluated. METHODS: Male Sprague-Dawley rats were pretreated either with saline (0.3 ml subcutaneously) or octreotide (0.001-1 ng/kg subcutaneously). After 30 minutes gastric ulcers were induced by the intragastric application of NSAIDs (20 mg/kg indomethacin, 200 mg/kg aspirin, 200 mg/kg ibuprofen, or 50 mg/kg diclofenac). Four hours later the rats were killed and gastric mucosal lesions were assessed by computed planimetry. To determine whether octreotide could prevent indomethacin induced injury in humans, 20 healthy volunteers were evaluated in a double blind, placebo controlled study. RESULTS: Octreotide prevented NSAID induced gastric mucosal lesions (p < 0.05). The dose response curve was U shaped and the most effective dose was 0.1 ng/kg. Leucocyte adherence in submucosal venules of the stomach was evaluated by in vivo microscopy. Octreotide (0.1 ng/kg subcutaneously) prevented indomethacin (20 mg/kg intragastric) induced leucocyte adherence in gastric submucosal venules (p < 0.05). Healthy human volunteers received 50 mg indomethacin orally thrice a day concomitantly with either an identical placebo or 0.01 microgram, 0.1 microgram, or 1 microgram octreotide subcutaneously thrice a day for three days. Injury was assessed by endoscopy. There was a negative correlation between the octreotide dose and injury score (p < 0.03 for gastric injury, p < 0.001 for duodenal injury). CONCLUSIONS: Octreotide protects the stomach from NSAID induced gastric injury, probably via its ability to reduce NSAID induced neutrophilic adhesion to the microvasculature. Octreotide also ameliorated indomethacin induced gastric and duodenal injury in humans. [ABSTRACT FROM PUBLISHER]

The localization of d-amino acid oxidase ( d-AAOX) in rat liver and kidney has been investigated using the cerium technique for electron microscopy and a recent modification of it for light microscopy. In the liver a mosaic pattern with strongly and weakly stained cells together with some completely negative hepatocytes is observed. The staining is stronger and more uniform in periportal than in perivenous regions of the liver lobule. In the kidney the reaction is confined to the proximal tubules of the renal cortex with the rest of the nephron being negative. At the ultrastructural level in both liver and kidney a marked heterogencity is obseved in the intensity of reaction in peroxisomes of some neighbouring cells. Moreover, in some cells heavily and weakly stained peroxisomes are seen side by side. When Pipes buffer is used in the incubation medium the d-AAOX reaction in kidney peroxiosomes is aggregated in the central region of the matrix with weaker staining of the periphery. A similar result is obtained when the enzyme is localized by immunocytochemistry confirming a recent report by Usuda et al. (1986). The heterogeneous staining of peroxisomes for d-AAOX suggests that subpopulation of this organelle with specialized functions may exist not only in different tissues and cells but even within the same cell. [ABSTRACT FROM AUTHOR]

The substrate specificity and the intraperoxisomal localization of α-hydroxyacid oxidase in rat liver has been investigated cytochemically by the cerium technique and biochemically with a luminometric assay. Rat liver is fixed by perfusion with a low concentration (0.25%) of glutaraldehyde and vibratome sections are incubated for 60 min at 37°C in a medium containing 3 m M CeCl, 100 m M NaN and 5 m M of an α-hydroxyacid in 0.1 M of one of the following buffers: Pipes, Mops, Na-cacodylate, Tris-maleate, all adjusted to pH 7.8. Ten different α-hydroxyacids with a chain length between 2 and 8 carbon atoms were tested. The best results were obtained with glycolic, argininic and l-α-isocaproic acids. These cytochemical findings were confirmed also biochemically using purified peroxisomal fractions isolated by gradient centrifugation in metrizamide. The pattern of the intraperoxisomal localization of the enzyme was influenced markedly by the type of buffer used for the cytochemical incubation. Whereas in the Tris-maleate medium both the cores and the matrix stained with the same intensity, with all other buffers the reaction in cores was more prominent. The staining of cores was abolished by pretreating sections in Tris-maleate (pH 7.8) or alkaline pyrophosphate buffers. These observations establish the substrate specificity of α-hydroxyacid oxidase in rat liver and demonstrate the delicate association of this enzyme with the crystalline cores and the matrix of peroxisomes in rat liver. [ABSTRACT FROM AUTHOR]

The substrate specificity of α-hydroxyacid oxidase in the rat kidney has been investigated cytochemically by the cerium technique and biochemically with a luminometric assay applied to isolated renal peroxisomes. Rat kidneys were fixed by perfusion via the abdominal aorta with a low concentration (0.25%) of glutaraldehyde. Vibratome sections were incubated for 60 min at 37°C in a medium containing 3 m M CeCl, 100 m M NaN and 5 m M of an α-hydroxyacid in 0.1 M Pipes or 0.1 M Tris-maleate buffer both adjusted to pH 7.8. Ten aliphatic α-hydroxyacids with chain lengths between 2 and 8 carbon atoms and two aromatic substrates were tested. The α-hydroxyacid oxidase in the kidney exhibited a markedly different substrate specificity than the corresponding enzyme in the liver. Thus glycolate gave a negative reaction while two aromatic substrates, mandelic acid and phenyllactic acid, stained prommently. With aliphatic substrates a stronger reaction was obtained in Pipes than in the Tris-maleate buffered incubation media. The best reaction in the kidney was obtained with hydroxybutyric acid. These cytochemical findings were confirmed by the luminometric determination of the oxidase activity in isolated purified peroxisome fractions. By electron microscopy the electron dense reaction product of cerium perhydroxide was found in the matrix of peroxisomes in the proximal tubules. The intensity of reaction varied markedly in neighbouring epithelial cells but also in different peroxisomes within the same cell. Thus heavily stained particles were seen next to lightly reacted ones. These observations establish the substrate specificity of α-hydroxyacid oxidase in the rat kidney and demonstrate the marked heterogeneity in the staining of renal peroxisomes for this enzyme. [ABSTRACT FROM AUTHOR]

Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults in the Western world. B cell receptor (BCR) signaling is known to be crucial for the pathogenesis and maintenance of CLL cells which develop from mature CD5 + B cells. BCR signaling is regulated by the inhibitory co-receptor Siglec-G and Siglec-G-deficient mice have an enlarged CD5 + B1a cell population. Here, we determine how Siglec-G expression influences the severity of CLL. Our results show that Siglec-G deficiency leads to earlier onset and more severe course of the CLL-like disease in the murine Eμ-TCL1 model. In contrast, mice overexpressing Siglec-G on the B cell surface are almost completely protected from developing CLL-like disease. Furthermore, we observe a downmodulation of the human ortholog Siglec-10 from the surface of human CLL cells. These results demonstrate a critical role for Siglec-G in disease progression in mice, and suggest that a similar mechanism for Siglec-10 in human CLL may exist., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Siglec-15 is a conserved sialic acid-binding Ig-like lectin, which is expressed on osteoclasts. Deficiency of Siglec-15 leads to an impaired osteoclast development, resulting in a mild osteopetrotic phenotype. The role of Siglec-15 in arthritis is still largely unclear. To address this, we generated Siglec-15 knockout mice and analyzed them in a mouse arthritis model. We could show that Siglec-15 is directly involved in pathologic bone erosion in the K/BxN serum-transfer arthritis model. Histological analyses of joint destruction provided evidence for a significant reduction in bone erosion area and osteoclast numbers in Siglec-15 -/- mice, whereas the inflammation area and cartilage destruction was comparable to wild-type mice. Thus, Siglec-15 on osteoclasts has a crucial function for bone erosion during arthritis. In addition, we generated a new monoclonal anti-Siglec-15 Ab to clarify its expression pattern on immune cells. Whereas this Ab demonstrated an almost exclusive Siglec-15 expression on murine osteoclasts and hardly any other expression on various other immune cell types, human Siglec-15 was more broadly expressed on human myeloid cells, including human osteoclasts. Taken together, our findings show a role of Siglec-15 as a regulator of pathologic bone resorption in arthritis and highlight its potential as a target for future therapies, as Siglec-15 blocking Abs are available., (Copyright © 2020 by The American Association of Immunologists, Inc.)

A high proportion of human B cells carry B-cell receptors (BCRs) that are autoreactive. Inhibitory receptors such as CD22 can downmodulate autoreactive BCR responses. With its extracellular domain, CD22 binds to sialic acids in α2,6 linkages in cis, on the surface of the same B cell or in trans, on other cells. Sialic acids are self ligands, as they are abundant in vertebrates, but are usually not expressed by pathogens. We show that cis-ligand binding of CD22 is crucial for the regulation of B-cell Ca(2+) signaling by controlling the CD22 association to the BCR. Mice with a mutated CD22 ligand-binding domain of CD22 showed strongly reduced Ca(2+) signaling. In contrast, mice with mutated CD22 immunoreceptor tyrosine-based inhibition motifs have increased B-cell Ca(2+) responses, increased B-cell turnover, and impaired survival of the B cells. Thus, the CD22 ligand-binding domain has a crucial function in regulating BCR signaling, which is relevant for controlling autoimmunity.

Peroxisomes are a heterogeneous group of organelles fulfilling reactions in a variety of metabolic pathways. To investigate if functionally different subpopulations can be found within a single tissue, peroxisomes from the heavy mitochondrial fraction (HM-Po) of the rat liver were isolated and compared to "classic" peroxisomes from the light mitochondrial fraction (LM-Po) using iTRAQ tandem mass spectrometry. Peroxisomes represent only a minor although significant proportion of the heavy mitochondrial fraction (2700g(max)) precluding a straightforward isolation by standard protocols. Thus, a new fractionation scheme suitable for a subsequent mass spectrometrical analysis was developed using a combination of centrifugation techniques and zonal free flow electrophoresis. On the basis of the iTRAQ-measurement, a variation of the peroxisomal protein pattern between both fractions could be determined and further confirmed by immunoblotting and enzyme activity assays for selected proteins: whereas peroxisomes from the light mitochondrial fraction contain high amounts of beta-oxidation enzymes, peroxisomes from the heavy mitochondrial fraction were dominated by enzymes fulfilling other functions. Among other findings, HM-Po was characterized by a high abundance of D-amino acid oxidase. This observation can be mirrored at the ultrastructural level, where tissue sections of liver peroxisomes show a heterogeneous staining for the enzymes activity, when visualized by the cerium technique.

Oxidases generating and enzymes scavenging H2O2 predestine peroxisomes (PO) to a pivotal organelle in oxygen metabolism. Catalase, the classical marker enzyme of PO, exhibits both catalytic and peroxidatic activity. The latter is responsible for the staining with 3,3'-diamino-benzidine, which greatly facilitated the visualization of the organelle and promoted further studies on PO. D-Amino acid oxidase catalyzes with strict stereospecificity the oxidative deamination of D-amino acids. The oxidase is significantly more active in the kidney than in liver and more in periportal than pericentral rat hepatocytes. Peroxisomes in these tissues differ in their enzyme activity and protein concentration not only in adjacent cells but even within the same one. Moreover, the enzyme appears preferentially concentrated in the central region of the peroxisomal matrix compartment. Urate oxidase, a cuproprotein catalyzing the oxidation of urate to allantoin, is confined to the peroxisomal core, yet is lacking in human PO. Recent experiments revealed that cores in rat hepatocytes appear in close association with the peroxisomal membrane releasing H2O2 generated by urate oxidase to the surrounding cytoplasma. Xanthine oxidase is exclusively located to cores, oxidizes xanthine thereby generating H2O2 and O2(-) radicals. The latter are converted to O2 and H2O2 by CuZn superoxide dismutase, which has been shown recently to be a bona fide peroxisomal protein.

Peroxisomes (PO) are essential and ubiquitous single-membrane-bound organelles whose ultrastructure is characterized by a matrix and often a crystalloid core. A unique feature is their capacity to generate and degrade H(2)O(2) via several oxidases and catalase, respectively. Handling of H(2)O(2) within PO is poorly understood and, in contrast to mitochondria, they are not regarded as a default H(2)O(2) source. Using an ultrasensitive luminometric H(2)O(2) assay, we show in real time that H(2)O(2) handling by matrix-localized catalase depends on the localization of H(2)O(2) generation in- and outside the PO. Thus, intact PO are inefficient at degrading external but also internal H(2)O(2) that is generated by the core-localized urate oxidase (UOX). Our findings suggest that, in addition to the PO membrane, the matrix forms a significant diffusion barrier for H(2)O(2). In contrast, matrix-generated H(2)O(2) is efficiently degraded. We further show that the tubular structures in crystalloid cores of UOX are associated with and perpendicularly oriented toward the PO membrane. Studies on metabolically active liver slices demonstrate that UOX directly releases H(2)O(2) into the cytoplasm, with the 5-nm primary tubules in crystalloid cores serving as exhaust conduits. Apparently, PO are inefficient detoxifiers of external H(2)O(2) but rather can become an obligatory source of H(2)O(2)--an important signaling molecule and a potential toxin.

It has been proposed that the anti-double-stranded DNA (dsDNA) response in patients with systemic lupus erythematosus (SLE) is antigen driven and that DNA or nucleosomes select anti-DNA reactive, somatically mutated B cells. We have used site-directed mutagenesis to systematically revert the somatic mutations of two human anti-dsDNA antibodies from SLE patients to analyze the resulting changes in DNA binding as well as binding to other autoantigens. Our data demonstrate that high-affinity binding to dsDNA and nucleosomes is acquired by somatic replacement mutations in a stepwise manner. Reactivity to surface structures of apoptotic cells is acquired by the same somatic mutations that generate high-affinity dsDNA binding. Importantly, revertant antibodies with germ-line V regions did not show any measurable DNA reactivity. We propose that anti-DNA autoantibodies are generated from nonautoreactive B cells during a normal immune response. B cells may acquire autoreactivity de novo during the process of somatic hypermutation. Nucleosomes, if available in lupus patients because of defects in clearing of apoptotic debris, might subsequently positively select high affinity anti-DNA B cells.

Tumor necrosis factor-alpha (TNFalpha) induces apoptosis in hepatocytes only under transcriptional arrest induced by galactosamine (GalN). In this study we demonstrated the shuttle of the transcription factor NFkappaB (nuclear factor-kappa B) in the liver tissue of mice within 30 min-4.5 hr hours after GalN/TNFalpha treatment. NFkappaB translocation from cytoplasm to the nucleus is initiated by its separation from the inhibitory IkappaB proteins which include IkappaBalpha, IkappaBbeta, and IkappaB. Thirty minutes after GalN/TNFalpha administration, NFkappaBp65 in hepatocellular nuclei becomes increasingly detectable and reaches its highest level after 2.5 hr. Then export back into cytoplasm begins but, surprisingly, approximately 30% of NFkappaB remains in the nuclear fraction and appears as an immunoprecipitate in the nuclei of apoptotic hepatocytes. Non-apoptotic hepatocytes do not show any reaction product in the nuclei 4.5 hr after treatment. Correspondingly, the amount of dissociated IkappaBbeta decreases in the cytoplasm up to 2.5 hr and increases again afterwards, although it does not reach the level of the control samples. No evidence of IkappaBbeta in the nuclei was found either immunocytochemically or biochemically. Caspase-3 activity, which is responsible for apoptosis, increases significantly after 3.5 hr. At that time, apoptotic hepatocytes can occasionally be observed and, 4.5 hr after GalN/TNFalpha treatment, constitute approximately 30% of the hepatocytes.

Derivative chromosomes of 40 patients diagnosed with t(4;11) acute lymphoblastic leukemia (ALL) were analysed on the genomic DNA level. Chromosomal breakpoints were identified in most cases within the known breakpoint cluster regions of the involved MLL and AF4 genes. Due to our current knowledge of the primary DNA sequences of both breakpoint cluster regions, specific features were identified at the chromosomal fusion sites, including deletions, inversions and duplications of parental DNA sequences. After separation of all t(4;11) leukemia patients into two age classes (below and above 1 year of age), the analysis of chromosomal fusion sites revealed significant differences in the distribution of chromosomal breakpoints and led to the definition of two hotspot areas within the MLL breakpoint cluster region. This may point to the possibility of different age-linked mechanisms that were leading to t(4;11) chromosomal translocations.

During the screening for Rhodobacter capsulatus mutants defective in xanthine degradation, one Tn5 mutant which was able to grow with xanthine as a sole nitrogen source only in the presence of high molybdate concentrations (1 mM), a phenotype resembling Escherichia coli mogA mutants, was identified. Unexpectedly, the corresponding Tn5 insertion was located within the moeA gene. Partial DNA sequence analysis and interposon mutagenesis of regions flanking R. capsulatus moeA revealed that no further genes essential for molybdopterin biosynthesis are located in the vicinity of moeA and revealed that moeA forms a monocistronic transcriptional unit in R. capsulatus. Amino acid sequence alignments of R. capsulatus MoeA (414 amino acids [aa]) with E. coli MogA (195 aa) showed that MoeA contains an internal domain homologous to MogA, suggesting similar functions of these proteins in the biosynthesis of the molybdenum cofactor. Interposon mutants defective in moeA did not exhibit dimethyl sulfoxide reductase or nitrate reductase activity, which both require the molybdopterin guanine dinucleotide (MGD) cofactor, even after addition of 1 mM molybdate to the medium. In contrast, the activity of xanthine dehydrogenase, which binds the molybdopterin (MPT) cofactor, was restored to wild-type levels after the addition of 1 mM molybdate to the growth medium. Analysis of fluorescent derivatives of the molybdenum cofactor of purified xanthine dehydrogenase isolated from moeA and modA mutant strains, respectively, revealed that MPT is inserted into the enzyme only after molybdenum chelation, and both metal chelation and Mo-MPT insertion can occur only under high molybdate concentrations in the absence of MoeA. These data support a model for the biosynthesis of the molybdenum cofactor in which the biosynthesis of MPT and MGD are split at a stage when the molybdenum atom is added to MPT.

Some chromosomal translocations involved in the origin of leukemias and lymphomas are due to malfunctions of the recombinatorial machinery of immunoglobulin and T-cell receptor-genes. This mechanism has also been proposed for translocations t(4;11)(q21;q23), which are regularly associated with acute pro-B cell leukemias in early childhood. Here, reciprocal chromosomal breakpoints in primary biopsy material of fourteen t(4;11)-leukemia patients were analysed. In all cases, duplications, deletions and inversions of less than a few hundred nucleotides indicative of malfunctioning DNA repair mechanisms were observed. We concluded that these translocation events were initiated by several DNA strand breaks on both participating chromosomes and subsequent DNA repair by 'error-prone-repair' mechanisms, but not by the action of recombinases of the immune system.

Objective/methods: In order to investigate whether selective hypertriglyceridemia impairs endothelium-dependent vasodilatation in the rat hindlimb, rats were selectively bred to establish two strains, one with a pronounced hypertriglyceridemia (HT) and the other with normal plasma levels of triglycerides (LT)., Results: Carotid arteries and aortae removed from 3, 6, 9 and 12 month old LT- and HT-rats exhibited a normal morphology. However, marked morphological differences were observed between vessels from 18-20 month old HT- and LT-rats. The endothelium-dependent vasodilator acetylcholine (2 to 50 micrograms/kg), administered into the iliac artery, elicited a concentration-dependent increase in hindlimb blood flow which was not different in 3, 6 and 9 month old LT- or HT-rats but was impaired in 12 and 18-20 month old HT-rats. In contrast the endothelium-independent vasodilator sodium nitroprusside enhanced blood flow in both strains to a similar extent. Neither administration of the nitric oxide (NO) synthase (NOS) substrate, L-arginine, nor the NOS inhibitor NGnitro-L-arginine, affected the responsiveness to endothelium-dependent vasodilators in 12 month old HT-rats. These attenuated responses could not be attributed to a decrease in endothelial NOS expression as Western blot analysis revealed identical levels of this enzyme in the aortae and carotid arteries from LT- and HT-rats. Determination of superoxide anion (O2-) formation however, demonstrated a markedly elevated production of O2- in aortae from HT-rats., Conclusion: We conclude that chronic selective hypertriglyceridemia, an independent risk factor in the development and progression of atherosclerosis, leads to an endothelial dysfunction which is associated with an increased vascular O2- production and a subsequent decrease in bioavailable NO.

The electron microscopical studies presented here show that characteristic morphological alterations in mitochondria are a very early hallmark of the hepatocellular apoptotic program. Before chromatin condensation occurs, the outer mitochondrial membrane is focally disrupted and the inner membrane protrudes through this gap forming a hernia. The demonstration of cytochrome oxidase in mitochondria revealed a very strong activity in pre-apoptotic and apoptotic cells as well as in apoptotic bodies.

The most potent virulence factor of Pseudomonas aeruginosa, its exotoxin A (PEA), inhibits protein synthesis, especially in the liver, and is a weak T cell mitogen. This study was performed to correlate hepatotoxic and possible immunostimulatory features of PEA in vivo. Injection of PEA to mice caused hepatocyte apoptosis, an increase in plasma transaminase activities, and the release of TNF, IFN-gamma, IL-2, and IL-6 into the circulation. Most strikingly, liver damage depended on T cells. Athymic nude mice or mice depleted of T cells by anti-Thy1.2 mAb pretreatment failed to develop acute hepatic failure, and survival was significantly prolonged following T cell depletion. Neutralization of TNF or lack of TNF receptors prevented liver injury. In the liver, TNF was produced by Kupffer cells before hepatocellular death occurred. After T cell depletion, Kupffer cells failed to produce TNF. Transaminase release was significantly reduced in perforin knockout mice, and it was even elevated in lpr/lpr mice. These results demonstrate that PEA induces liver damage not only by protein synthesis inhibition but also by TNF- and perforin-dependent, Fas-independent, apoptotic signals.

AIDS is characterized by a progressive decrease of CD4(+) helper T lymphocytes. Destruction of these cells may involve programmed cell death, apoptosis. It has previously been reported that apoptosis can be induced even in noninfected cells by HIV-1 gp120 and anti-gp120 antibodies. HIV-1 gp120 binds to T cells via CD4 and the chemokine coreceptor CXCR4 (fusin/LESTR). Therefore, we investigated whether CD4 and CXCR4 mediate gp120-induced apoptosis. We used human peripheral blood lymphocytes, malignant T cells, and CD4/CXCR4 transfectants, and found cell death induced by both cell surface receptors, CD4 and CXCR4. The induced cell death was rapid, independent of known caspases, and lacking oligonucleosomal DNA fragmentation. In addition, the death signals were not propagated via p56(lck) and Gialpha. However, the cells showed chromatin condensation, morphological shrinkage, membrane inversion, and reduced mitochondrial transmembrane potential indicative of apoptosis. Significantly, apoptosis was exclusively observed in CD4(+) but not in CD8(+) T cells, and apoptosis triggered via CXCR4 was inhibited by stromal cell-derived factor-1, the natural CXCR4 ligand. Thus, this mechanism of apoptosis might contribute to T cell depletion in AIDS and might have major implications for therapeutic intervention.

Tumor necrosis factor (TNF) induces apoptotic death of hepatocytes in the galactosamine (GalN)-sensitized mouse liver after 5 hr. In our study, the most remarkable sign of the early stage of apoptosis was the focal rupture of the outer mitochondrial membrane. Parts of the inner membrane extended through the gap of the outer membrane, whereas the rest of the inner membrane still formed the cristae. This feature appeared in hepatocytes before chromatin condensation. With the diaminobenzidine technique for localization of cytochrome oxidase activity, the reaction product was detectable by light and electron microscopy. Ten percent of the hepatocytes were apoptotic, with condensed chromatin and high enzyme activity, 37% were pre-apoptotic, without chromatin condensation but high enzyme activity, and 53% had neither condensed chromatin nor a remarkable reaction product of cytochrome oxidase activity. Fas (APO-1, CD95) molecules on the plasma membrane of hepatocytes increased and were represented immunohistochemically in cells without chromatin condensation. DNA strand breaks were also detectable before chromatin aggregation. The results of this study indicate that mitochondria play a pivotal role in pre-apoptotic hepatocytes, together with an increase of the Fas molecule on the plasma membrane and with the occurrence of DNA strand breaks in the nucleus.

Background: During allograft rejection, cytokines and lipid mediators contribute to cell injury and organ failure. Peroxisomes play a crucial role in lipid metabolism, including the degradation of lipid mediators by peroxisomal beta-oxidation. Therefore, we investigated the alterations of hepatic peroxisomes after allogeneic rat liver transplantation., Methods: MHC-incompatible Dark Agouti (RT1a) donor rats and Lewis (RT1(1)) recipient rats were used for allogeneic transplantation. For immunosuppression, a group of these animals received cyclosporine (CsA) intraperitoneally (1 mg/kg body weight per day). Lewis rats were used for isogeneic transplant combination. Ten days after transplantation, livers were investigated using morphometrical methods for determination of peroxisomal diameter and volume density. The activities of peroxisomal catalase (CAT) and acyl-coenzyme A oxidase (AOX) were determined, and the corresponding proteins were evaluated by quantitative immunocytochemistry and immunoblotting. The expressions of mRNAs encoding CAT and AOX were investigated by Northern blotting., Results: The volume density and diameter of peroxisomes were significantly decreased in allogeneic transplanted livers but were unchanged in CsA-treated animals. Both the activities of CAT and AOX and their protein levels were significantly reduced in liver allografts. Moreover, the corresponding mRNA levels of CAT and AOX were decreased significantly in liver allografts, whereas CsA treatment led to an increase of those mRNAs. Isogeneic transplanted livers showed only a slight reduction of the corresponding enzyme values., Conclusions: Peroxisomes are severely affected both morphologically and functionally after allogeneic liver transplantation. These results suggest that impairment of peroxisomal lipid beta-oxidation could contribute to the pathogenesis of the rejection process by decreased catabolism of lipid mediators involved in the regulation of the inflammatory response. CsA, in addition to its immunosuppressive effects, may contribute to allograft survival by maintenance of those important peroxisomal functions.

Background/aims: In cholestatic liver disease, bile acids may initiate or aggravate hepatocellular damage. Cellular necrosis and cell death may be due to detergent effects of bile acids, but apoptosis may also play a role. In cholestasis, the conditions determining either apoptotic or cytolytic cell death are still unclear. Primary rat hepatocytes in culture represent a suitable model to study bile-acid-induced liver damage., Methods: Glycochenodeoxycholic acid, a hydrophobic bile acid, was used to induce cell damage. Tauroursodeoxycholic acid, a hydrophilic bile acid, served as substrate to study possible protective effects of such compounds. To study the time and concentration dependency of bile-acid-induced cytolysis and apoptosis, morphologic alterations, hepatocellular enzyme release and nucleosomal DNA fragmentation were evaluated., Results: Bile-acid-induced cytolysis, as indicated by hepatocellular enzyme release and by morphologic signs of membrane destruction, increased with concentration and time. Addition of tauroursodeoxycholic acid to the incubation medium reduced cytolysis significantly, indicating a direct hepatoprotective effect of this bile acid against the detergent action of hydrophobic bile acids. In contrast to cytolysis, apoptosis with DNA fragmentation was induced by low concentrations of glycochenodeoxycholic acid a few hours after incubation. Coincubation with tauroursodeoxycholic acid in equimolar concentrations significantly reduced apoptosis, indicating another direct hepatoprotective effect of tauroursodeoxycholic acid., Conclusions: It seems likely that in severe cholestasis, bile-acid-induced injury of hepatocytes is due mainly to cytolysis, whereas in moderately severe cholestasis apoptosis represents the predominant mechanism of bile acid toxicity. Tauroursodeoxycholic acid may reduce both bile-acid-induced apoptosis and cytolysis.

Background: Cholestasis is a complication that occurs during the rejection of liver transplants. The aim of this study was to investigate the association of activated Kupffer cells (KCs) and Na+,K+-ATPase activity for taurocholate cotransport and bile canalicular (BC) Mg++-ATPase activity for hepatobiliary excretion in rat liver allograft., Methods: Quantitative analyses of KC number and size in relationship to enzyme activity of Na+,K+-ATPase and of BC Mg++-ATPase were conducted in rejected liver after allogenic transplantation and after prevention of rejection using cyclosporine., Results: The animals were examined on the 10th postoperative day. In the rejection group, the number of KCs significantly increased more than fourfold in comparison with the number of KCs in the control livers. Some KCs were found in the sinusoids, but the majority were located in the space of Disse. Na+,K+-ATPase activity vanished from the basolateral plasma membrane, whereas BC Mg++-ATPase activity was restored in the apical domain. With immunosuppression, KCs showed the same behavior as in the control group, and activity of both ATPases was observed as strong electron-dense precipitates in basolateral and apical plasma membrane domains., Conclusions: In this study, we demonstrate that activated KCs migrate into the donor liver and release cytokines, which leads to the loss of Na+,K+-ATPase activity in the rejection group. BC Mg++-ATPase activity was not influenced by these mediators of activated macrophages. Since Na+,K+-ATPase is the cotransporter for hepatocyte taurocholate uptake, these data may contribute to understanding the mechanisms for cholestasis during hepatic allograft rejection.

Calcium channel blockers protect cells against ischaemia-reperfusion injury. In the present study, the effect of verapamil on mitochondrial calcium content was investigated in situ in normoxic, hypoxic and reoxygenated rat liver. Subcellular distribution of exchangeable calcium ions, which form an electron-dense precipitate with antimonate, was demonstrated with the glutaraldehyde-osmium antimonate technique. Calcium precipitates were quantified morphometrically using automatic image analysis. In normoxic liver, the mitochondrial calcium content formed a gradient decreasing from the periportal to perivenous regions. The low mitochondrial calcium content in perivenous regions remained unaffected in all experimental conditions. In hypoxic and reoxygenated liver, the calcium content in mitochondria of the periportal areas was significantly reduced. Verapamil pretreatment levelled the calcium gradient in normoxic liver by reducing the periportal calcium content. Verapamil had no effect on the mitochondrial calcium content in hypoxic liver. In contrast, in verapamil-pretreated reoxygenated liver, the mitochondrial calcium content in periportal mitochondria increased significantly, thus restoring the zonal calcium gradient. In conclusion, these data suggest that modulations of mitochondrial calcium content in the periportal region of the liver lobule may play an important role in the protective effects of verapamil against ischaemia-reperfusion injury.