Biased distribution of chromosomal breakpoints involving the MLL gene in infants versus children and adults with t(4;11) ALL.

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.

Effect of tauroursodeoxycholic acid on bile acid-induced apoptosis in primary human hepatocytes.

BACKGROUND/AIMS: The accumulation of endogenous bile acids contributes to hepatocellular damage during cholestatic liver disease. To evaluate the potential role of apoptotic cell death due to increased concentrations of bile acids, primary human hepatocytes were treated with hydrophobic and hydrophilic bile acids. Because the Fas receptor-ligand system may mediate apoptosis in human liver cells, the effect of toxic bile acids on hepatocellular Fas receptor expression was evaluated. MATERIALS AND METHODS: Primary human hepatocytes were incubated with 50 and 100 microM glycochenodeoxycholic acid (GCDCA) and co-incubated with equimolar concentrations of tauroursodeoxycholic acid (TUDCA). To evaluate cytolytic and apoptotic effects, morphological alterations, hepatocellular enzyme release, nuclear DNA fragmentation and hepatocellular Fas receptor expression were evaluated. RESULTS: Apoptotic cell death was significantly increased after exposure to 50 microM GCDCA. Bile acid-induced apoptosis was not accompanied by hepatocellular Fas receptor overexpression. Tauroursodeoxycholic acid reduced apoptosis, as indicated by a significant reduction of oligonucleosomal DNA cleavage. Fas receptor expression was not significantly affected by tauroursodeoxycholic acid. At higher concentrations, direct cytolytic cell destruction was observed. CONCLUSION: Primary human hepatocytes represent a suitable model to study bile acid-induced apoptotic cell death. In these hepatocytes, already low bile acid concentrations might induce apoptotic cell death, which is not triggered by hepatocellular Fas receptor overexpression. Apoptotic DNA fragmentation was significantly reduced by co-incubation with tauroursodeoxycholic acid. The reduction of bile acid-induced apoptosis by ursodeoxycholic acid and its conjugates may contribute to the beneficial effects of these hydrophilic bile acids used for medical treatment of several cholestatic liver diseases.

Chronic selective hypertriglyceridemia impairs endothelium-dependent vasodilatation in rats.

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.

Activity of the molybdopterin-containing xanthine dehydrogenase of Rhodobacter capsulatus can be restored by high molybdenum concentrations in a moeA mutant defective in molybdenum cofactor biosynthesis.

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.

A DNA damage repair mechanism is involved in the origin of chromosomal translocations t(4;11) in primary leukemic cells.

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.

Ultrastructural alterations of mitochondria in pre-apoptotic and apoptotic hepatocytes of TNF alpha-treated galactosamine-sensitized mice.

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.

Acute hepatotoxicity of Pseudomonas aeruginosa exotoxin A in mice depends on T cells and TNF.

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.

CXCR4 and CD4 mediate a rapid CD95-independent cell death in CD4(+) T cells.

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.

Pre-apoptotic alterations in hepatocytes of TNFalpha-treated galactosamine-sensitized mice.

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.

Effect of S-adenosylmethionine versus tauroursodeoxycholic acid on bile acid-induced apoptosis and cytolysis in rat hepatocytes.

BACKGROUND: S-adenosylmethionine (SAMe) increases survival in alcoholic liver cirrhosis and may have a beneficial effect in cholestatic liver disease. SAMe repletes glutathione stores and protects tissue from oxygen free radicals. The effect of SAMe on bile acid-induced apoptosis is unknown. In the present study the possible hepatoprotective effect of SAMe was evaluated and compared with that of tauroursodeoxycholic acid (TUDCA). METHODS: Primary rat hepatocytes treated with glycochenodeoxycholic acid (GCDCA) were used as a model for cholestasis-induced hepatocellular damage, which served to study the effects of SAMe and TUDCA on bile acid-induced apoptosis and cytolysis. RESULTS: SAMe reduced bile acid-induced apoptosis but did not prevent bile acid-induced cytolysis. Compared with SAMe, TUDCA was more efficient in reducing apoptosis due to toxic bile acids. The combination of SAMe and TUDCA had additive effects in reducing apoptosis. CONCLUSION: The reduction in bile acid-induced apoptosis by SAMe may represent one of the factors responsible for its beneficial effects in the treatment of liver diseases.

Impairment of peroxisomal structure and function in rat liver allograft rejection: prevention by cyclosporine.

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.

Expression, distribution, and activity of Na+,K+-ATPase in normal and cholestatic rat liver.

Hepatocellular Na+,K+-ATPase is an important driving force for bile secretion and has been localized to the basolateral plasma membrane domain. Cholestasis or impaired bile flow is known to modulate the expression, domain specificity, and activity of various transport systems involved in bile secretion. This study examined Na+, K+-ATPase after ethinylestradiol (EE) treatment and after bile duct ligation (BDL), two rat models of cholestasis. It applied quantitative immunoblotting, biochemical and cytochemical determination of enzyme activity, and immunocytochemistry to the same livers. The data showed a good correlation among the results of the different methods. Neither EE nor BDL induced alterations in the subcellular distribution of Na+,K+-ATPase, which was found in the basolateral but not in the canalicular (apical) plasma membrane domain. Protein expression and enzyme activity showed a small (approximately 10%) decrease after EE treatment and a similar increase after BDL. These modest changes could not be detected by immunofluorescence, immuno EM, or cytochemistry. The data, therefore, demonstrate that Na+,K+-ATPase is only slightly affected by EE and BDL. They suggest that other components of the bile secretory apparatus that take effect downstream of the primary basolateral driving force may play a more prominent role in the pathogenesis of cholestasis.

Effect of tauroursodeoxycholic acid on bile-acid-induced apoptosis and cytolysis in rat hepatocytes.

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.

Significant increase of Kuppfer cells associated with loss of Na+,K+-ATPase activity in rat hepatic allograft rejection.

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.

Reduction of non-steroidal anti-inflammatory drug induced gastric injury and leucocyte endothelial adhesion by octreotide.

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.

The effect of verapamil on mitochondrial calcium content in normoxic, hypoxic and reoxygenated rat liver.

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.

A comparison of hemolysis rates using intravenous catheters versus venipuncture tubes for obtaining blood samples.

OBJECTIVE: The primary purpose of this study was to compare the rate of hemolysis in blood samples obtained by an i.v. catheter versus the rate in samples obtained by venipuncture (Vacutainer tubes and needles; Becton Dickinson Vacutainer Systems, Franklin Lakes, N.J.). Subsequently, variance in i.v. catheter diameter was reviewed to determine its influence on hemolysis rate of i.v. catheter aspirate. DESIGN: A randomized, prospective study was used to evaluate hemolysis differences between the two blood sampling methods. A descriptive, retrospective review of study data was used to evaluate the importance of the variable i.v. catheter diameter. METHODS: The study group consisted of patients who came to the emergency department and required both an i.v. infusion and blood sampling for determination of electrolyte levels and complete blood cell count. Pediatric patients (younger than 16 years) were excluded. The ED patients who qualified for the study were randomly assigned to either group A or B. The blood samples for patients in the A group were obtained through the i.v. catheter at the time of insertion. The i.v. catheters ranged in size from 24 gauge to 14 gauge. Patients in the B group also had insertion of an i.v. line, but their blood samples were obtained by Vacutainer venipuncture at a separate site. The Vacutainer needle was standardized at 21 gauge. All blood samples were collected by one of seven experienced ED nurses. The nurse who collected the blood sample for an study patient was responsible for result follow-up. RESULTS: A total of 165 patients participated in the study; 87 patients were assigned to the A (i.v.) group, and 78 patients participated in the B (venipuncture) group. In group A a total of 12 of 87 (13.7%) blood samples hemolyzed. Hemolysis occurred in 3 of 78 (3.8%) of group B samples. These findings were statistically significant (p < 0.05). When we examined the variable i.v. catheter diameter, we noted a lower incidence of hemolysis with larger catheter diameters: 24 gauge (100%), 22 gauge (25%), 20 gauge (15%), 18 gauge (10%), 16 gauge (0%), 14 gauge (0%). This findings was statistically significant (p < 0.05). CONCLUSIONS: Hemolysis of blood samples obtained by an i.v. catheter was significantly higher than when blood was obtained through Vacutainer venipuncture. There is an inverse correlation between i.v. catheter diameter and the rate of hemolysis.

DNA fragmentation in mouse organs during endotoxic shock.

The systemic inflammatory response syndrome has still an unpredictable outcome, and patients often die of multiple organ failure despite circulatory stabilization therapy. The still incompletely understood pathophysiological mechanisms include organ damage due to direct toxic actions of cytokines elicited by overactivation of the host response. To study this process of organ failure in experimental septic shock, we injected mice with a lethal dose of endotoxin and examined apoptotic and necrotic tissue damage biochemically, histologically, and ultrastructurally. Endotoxin administration caused oligonucleosomal as well as random DNA fragmentation in liver, lung, kidney, and intestine. In the liver, DNA fragmentation was not restricted to hepatocytes but also occurred in nonparenchymal cells. The DNA fragmentation was mediated by tumor necrosis factor and attenuated by endogenous nitric oxide release. Unlike the situation in D-galactosamine-sensitized mice, in which injection or release of tumor necrosis factor causes massive hepatocyte apoptosis, liver failure due to high doses of endotoxin was characterized by single-cell necrosis, a low incidence of apoptosis, and simultaneous damage to nonparenchymal cells. We conclude that, even though endotoxin causes cytokine-mediated DNA fragmentation in several organs including the liver, hepatocyte apoptosis itself seems to be a minor phenomenon in high-dose endotoxic shock in mice.

Promoters controlling expression of the alternative nitrogenase and the molybdenum uptake system in Rhodobacter capsulatus are activated by NtrC, independent of sigma54, and repressed by molybdenum.

The alternative nitrogenase of Rhodobacter capsulatus is expressed only under conditions of nitrogen and molybdenum depletion. The analysis of anfA-lacZ fusions demonstrated that this dual control occurred at the level of transcription of anfA, which encodes a transcriptional activator specific for the alternative nitrogenase. The anfA promoter was found to be activated under nitrogen-limiting conditions by NtrC in a sigma54-independent manner. In addition, anfA transcription was repressed by traces of molybdenum. This molybdenum-dependent repression of anfA was released in R. capsulatus mutants carrying either lesions in the high-affinity molybdenum uptake system (modABCD) or a double deletion of mopA and mopB, two genes encoding molybdenum-pterin-binding proteins. The expression of the molybdenum transport system itself was shown to be negatively regulated by molybdenum and, unexpectedly, to be also regulated by NtrC. This finding is in line with the presence of two tandemly arranged DNA motifs located in front of the R. capsulatus mopA-modABCD operon, which are homologous to R. capsulatus NtrC binding sites. Mapping of the transcriptional initiation sites of mopA and anfA revealed promoter sequences exhibiting significant homology to each other but no homology to known prokaryotic promoters. In addition, a conserved DNA sequence of dyad symmetry overlapping the transcriptional initiation sites of mopA and anfA was found. Deletions within this element resulted in molybdenum-independent expression of anfA, indicating that this DNA sequence may be the target of MopA/MopB-mediated repression.