Dose-dependant acute or subacute disease caused by Burkholderia pseudomallei strain NCTC 13392 in a BALB/c aerosol model of infection.

AIMS: The goal of this study was to examine, for the first time, the virulence and pathogenicity of aerosolized Burkholderia pseudomallei, strain NCTC 13392, in BALB/c mice in order to develop an animal model for testing novel medical countermeasures (MCMs) for the treatment of human acute and subacute (a disease state between acute and chronic) melioidosis. METHODS AND RESULTS: BALB/c mice were exposed to varying doses of aerosolized bacteria. Acute disease was seen in animals exposed to a very-high dose (≥103  CFU per animal) and death occurred 3-4 days postchallenge (pc). Bacteria were detected in the lungs, liver, kidney and spleen. In contrast, animals exposed to a low dose (<10 CFU per animal) survived to the end of the study (day 30 pc) but developed weight loss, a bacterial tissue burden and increasing clinical signs of infection from day 20 pc onwards, mimicking a subacute form of the disease. Pathological changes in the tissues mirrored these findings. CONCLUSIONS: This proof of concept study has shown that B. pseudomallei strain NCTC 13392 is virulent and pathogenic in BALB/c mice, when delivered by aerosol. By varying the doses of aerosolized bacteria it was possible to mimic characteristics of both human acute and subacute melioidosis, at the same time, within the same study. SIGNIFICANCE AND IMPACT OF THE STUDY: Burkholderia pseudomallei, the aetiological agent of melioidosis, causes a serious and often fatal disease in humans and animals. Novel MCMs are urgently needed for both public health and biodefense purposes. The present model provides a useful tool for the assessment and evaluation of new MCMs (e.g. therapeutics and vaccines) and offers the potential for testing new treatments for both subacute to chronic and acute melioidosis prior to human clinical trials.

Functional consequences of I56ii Dlx enhancer deletion in the developing mouse forebrain.

Dlx homeobox genes encode a group of transcription factors that play an essential role during developmental processes including maintaining the differentiation, proliferation and migration of GABAergic interneurons. The Dlx1/2 and Dlx5/6 genes are expressed in the forebrain and are arranged in convergently transcribed bigene clusters, with I12a/I12b and I56i/I56ii cis-regulatory elements (CREs) located in the intergenic region of each cluster respectively. We have characterized the phenotypic consequences of deleting I56ii on forebrain development and spatial patterning of corridor cells that are involved in guiding thalamocortical projections. Here we report that deletion of I56ii impairs expression of Dlx genes and that of potential targets including Gad2 as well as striatal markers Islet1, Meis2, and Ebf1. In addition, I56ii deletion reduces both the binding of DLX2 in the Dlx5/Dlx6 intergenic region and the presence of H3K9Ac at the Dlx5/Dlx6 locus, consistent with the reduced expression of these genes. Deletion of I56ii reduces the expression of the ISLET1 and CTIP2 in the striatum and disrupts the number of parvalbumin and calretinin expressing cells in the adult somatosensory cortex of the ΔI56ii mice. These data suggest an important regulatory role for I56ii in the developing forebrain by means of a potential regulatory mechanism which may regulate the expression of Dlx genes, notably Dlx6 as well as the spatial patterning of the ventral telencephalon, including possibly corridor cells.

Sequence of pathogenic events in cynomolgus macaques infected with aerosolized monkeypox virus.

UNLABELLED: To evaluate new vaccines when human efficacy studies are not possible, the FDA's "Animal Rule" requires well-characterized models of infection. Thus, in the present study, the early pathogenic events of monkeypox infection in nonhuman primates, a surrogate for variola virus infection, were characterized. Cynomolgus macaques were exposed to aerosolized monkeypox virus (10(5) PFU). Clinical observations, viral loads, immune responses, and pathological changes were examined on days 2, 4, 6, 8, 10, and 12 postchallenge. Viral DNA (vDNA) was detected in the lungs on day 2 postchallenge, and viral antigen was detected, by immunostaining, in the epithelium of bronchi, bronchioles, and alveolar walls. Lesions comprised rare foci of dysplastic and sloughed cells in respiratory bronchioles. By day 4, vDNA was detected in the throat, tonsil, and spleen, and monkeypox antigen was detected in the lung, hilar and submandibular lymph nodes, spleen, and colon. Lung lesions comprised focal epithelial necrosis and inflammation. Body temperature peaked on day 6, pox lesions appeared on the skin, and lesions, with positive immunostaining, were present in the lung, tonsil, spleen, lymph nodes, and colon. By day 8, vDNA was present in 9/13 tissues. Blood concentrations of interleukin 1ra (IL-1ra), IL-6, and gamma interferon (IFN-γ) increased markedly. By day 10, circulating IgG antibody concentrations increased, and on day 12, animals showed early signs of recovery. These results define early events occurring in an inhalational macaque monkeypox infection model, supporting its use as a surrogate model for human smallpox. IMPORTANCE: Bioterrorism poses a major threat to public health, as the deliberate release of infectious agents, such smallpox or a related virus, monkeypox, would have catastrophic consequences. The development and testing of new medical countermeasures, e.g., vaccines, are thus priorities; however, tests for efficacy in humans cannot be performed because it would be unethical and field trials are not feasible. To overcome this, the FDA may grant marketing approval of a new product based upon the "Animal Rule," in which interventions are tested for efficacy in well-characterized animal models. Monkeypox virus infection of nonhuman primates (NHPs) presents a potential surrogate disease model for smallpox. Previously, the later stages of monkeypox infection were defined, but the early course of infection remains unstudied. Here, the early pathogenic events of inhalational monkeypox infection in NHPs were characterized, and the results support the use of this surrogate model for testing human smallpox interventions.

Efficacy of liposome-encapsulated ciprofloxacin in a murine model of Q fever.

Encapsulation of antibiotics may improve treatment of intracellular infections by prolonging antibiotic release and improving antibiotic uptake into cells. In this study, liposome-encapsulated ciprofloxacin for inhalation (CFI) was evaluated as a postexposure therapeutic for the treatment of Coxiella burnetii, the causative agent of Q fever. Intranasal treatment of male A/Jola (A/J) mice with CFI (50 mg/kg of body weight) once daily for 7 days protected mice against weight loss and clinical signs following an aerosol challenge with C. burnetii. In comparison, mice treated twice daily with oral ciprofloxacin or doxycycline (50 mg/kg) or phosphate-buffered saline (PBS) lost 15 to 20% body weight and exhibited ruffled fur, arched backs, and dehydration. Mice were culled at day 14 postchallenge. The weights and bacterial burdens of organs were determined. Mice treated with CFI exhibited reduced splenomegaly and reduced bacterial numbers in the lungs and spleen compared to mice treated with oral ciprofloxacin or doxycycline. When a single dose of CFI was administered, it provided better protection against body weight loss than 7 days of treatment with oral doxycycline, the current antibiotic of choice to treat Q fever. These data suggest that CFI has potential as a superior antibiotic to treat Q fever.

Placement of coiled catheters into the paravertebral space.

There are conflicting results with regard to the use of catheter-based techniques for continuous paravertebral block. Local anaesthetic spread within the paravertebral space is limited and the clinical effect is often variable. Discrepancies between needle tip position and final catheter position can also be problematic. The aim of this proof-of-concept study was to assess the reliability of placing a newly developed coiled catheter in human cadavers. Sixty Tuohy needles and coiled catheters were placed under ultrasound guidance, three on each side of the thoracic vertebral column in 10 human cadavers. Computed tomography was used to assess needle tip and catheter tip locations. No catheter was misplaced into the epidural, pleural or prevertebral spaces. The mean (SD) distance between catheter tips and needle tips was 8.2 (4.9) mm. The median (IQR [range]) caudo-cephalad spread of contrast dye injectate through a subset of 20 catheters was 4 (4-5[3-8]) thoracic segments. All catheters were removed without incident. Precise paravertebral catheter placement can be achieved using ultrasound-guided placement of a coiled catheter.

Survival of Mycobacterium tuberculosis during experimental aerosolization and implications for aerosol challenge models.

AIMS: We undertook a series of experiments to investigate factors that contribute to variation in Mycobacterium tuberculosis viability and infectivity, during experimental aerosolization, with an aim to optimize a strategy to enable a more reproducible delivered dose within animal models of tuberculosis. METHODS AND RESULTS: The viability and infectivity of the challenge suspension was determined in relation to aerosolization time, concentration, method of preparation and M. tuberculosis strain. Challenge stocks generated from frozen aliquots of M. tuberculosis were shown to undergo a 1 log(10) CFU ml(-1) decrease in viability during the first 10 min of aerosolization. This correlated with a decrease in surface lung lesions developing in guinea pigs challenged during this time. The phenomenon of decreased viability in vitro was not observed when using freshly grown, nonfrozen cells of M. tuberculosis. The viability of aerosolized bacilli at the point of inhalation relative to the point of aerosolization always remained constant. CONCLUSION: Based on these findings, we have developed an improved strategy by which to reproducibly deliver aerosol infection doses to individually challenged animals and separately challenged groups of animals. SIGNIFICANCE AND IMPACT OF THE STUDY: Study of the aerobiological characteristics of micro-organisms is a critical step in the validation of methodology for aerosol infection animal models, particularly where large numbers of animals and nonhuman primates are used.

Chemiluminescence of phagocytic cells caused by N-formylmethionyl peptides.

N-formylmethionyl (F-Met) peptides, when added alone to macrophages or polymorphonuclear leukocytes (PMN), were found to induce a chemiluminescent response of shorter duration than that produced by the commonly employed particulate stimulant, zymosan. The cellular nature of F-Met peptide-induced chemiluminescence was indicated by its dependence on cell concentration, and by its inhibition by cell disruption, heat inactivation, or previous maximal stimulation by the peptides. Comparison of PMN and macrophages from different species showed that the maximal chemiluminescent response seen in the dose-response curve of F-Met- Phe was different in different cell types. Chemiluminescence reached highest values in human PMN, it was intermediate in guinea pig macrophages and PMN, and in rabbit PMN; but it was nonexistent in rabbit alveolar macrophages and very low in rabbit peritoneal macrophages. A definite relationship was observed between peptide structure and chemiluminescent activity. Met-Phe, F- Met and Phe were inactive even at millimolar concentrations, while F-Met-Phe caused chemiluminescence at micromolar concentrations. Four active peptides were tested in guinea pig, rabbit, and human PMN, and in guinea pig alveolar and peritoneal macrophages. The relative activity of these peptides was the same in all cells studied, e.g. F-Met-Leu-Phe >> F-Met-Phe > F-Met-Val > F- Met-Ala. The values of ED50 for each peptide were also comparable to previously reported ED50 values of these peptides in inducing lysosomal enzyme release. These results were seen both in the presence and absence ofthe chemiluminescent oxidant indicator, luminol. Low concentrations of superoxide dismutase (10 mug/ml) completely inhibited chemiluminescence caused by the F-Met peptides, suggesting the involvement of 0(2)(-) or O(2)(-)-derived compounds in this response. Sodium azide, an inhibitor of peroxidase reactions, had either no effect or a slight inhibitory effect on chemiluminescence. However, when the extracellular release of lysosomal enzymes was induced by cytochalasin B, an azide- inhibitable enhancement of chemiluminescence was seen in PMN, but not in macrophages. This effect appears to be correlated with the presence of granule-associated myeloperoxidase. Although azide-inhibitable peroxidases could be a potential source of light, they did not appear to be a significant contributor in these experiments. Based on these results and on those of previous investigators, we postulate that the F-Met-peptides stimulate 0(2)(-) production in addition to stimulating lysosomal enzyme release and chemotaxis. The similar structure- activity relationship which appears to exist for these processes may indicate that they are all initiated by a single receptor mechanism. Since F-Met peptides are formed in bacteria it is likely that their actions represent an important physiologic response.

Recurrent posterior shoulder instability.

Recurrent posterior shoulder instability is an uncommon condition. It is often unrecognized, leading to incorrect diagnoses, delays in diagnosis, and even missed diagnoses. Posterior instability encompasses a wide spectrum of pathology, ranging from unidirectional posterior subluxation to multidirectional instability to locked posterior dislocations. Nonsurgical treatment of posterior shoulder instability is successful in most cases; however, surgical intervention is indicated when conservative treatment fails. For optimal results, the surgeon must accurately define the pattern of instability and address all soft-tissue and bony injuries present at the time of surgery. Arthroscopic treatment of posterior shoulder instability has increased application, and a variety of techniques has been described to manage posterior glenohumeral instability related to posterior capsulolabral injury.

RNA type C virus antigens in hamster cells transformed by carcinogenic DNA viruses and chemicals.

The passive hemagglutination inhibition technique was used to test serologically for the presence of Syrian hamster type C virus antigen(s) (SHCVA) in a wide variety of normal and transformed hamster cells and tissues. SHCVA could not be detected in normal tissues or nonneoplastic tissues of tumor-bearing Syrian hamsters. Normal hamster embryo cells or cells transformed in vitro by simian adenovirus, by chemical alone, or doubly transformed by simian adenovirus and chemical did not contain SHCVA; however, SHCVA was found in a majority of tumors resulting from transplantation of these in vitro-transformed cells. No consistent pattern was observed in the capacity of individual transformed cell lines to produce SHCVA-positive or -negative tumors. When cells of a given transformed line were inoculated at 4 sites on each of 8 hamsters, SHCVA-positive tumors were found not to be randomly distributed but rather to be clustered on a few animals. SHCVA could be detected in only a few primary tumors induced by inoculation of carcinogenic DNA viruses; however, both the incidence and titer of SHCVA were significantly increased in a variety of transplanted tumors. These data suggest that SHCVA may be introduced into transplanted, transformed hamster cell tumors during passage in the host animal. Alternatively, in vivo conditions may allow expression of viral antigens not found under in vitro conditions; however, if this is true, only certain animals appear to be capable of activating SHCVA.

Chemical enhancement of viral transformation in Syrian hamster embryo cells by gaseous and volatile chlorinated methanes and ethanes.

Methods were developed for exposing cells in vitro to gases or vapors of volatilized organic liquids. Compounds were selected for their industrial importance, environmental impact, and suspected role in the etiology of some human cancers. Exposure chambers were designed for easy insertion of dishes of cultured cells and were equipped with inlet and outlet ports for introduction and purging of test gases. A gas delivery system utilizing a mass flow meter was used for the quantitative distribution of test gases into exposure chambers. For volatile compounds, appropriate volumes of cold (4 degrees) liquids in glass Petri dishes were quickly placed into chambers, the system sealed, and the compounds rapidly volatilized at 37 degrees. For exposure, the cells and chambers were placed in an incubator and rocked at a constant rate so that a portion of the cells was always in direct contact with the test gases or vapors. Known sample volumes were removed after various treatment times and test gas concentrations determined by standard gas chromatographic techniques. After exposure, the cells were removed and assayed for viability and increased sensitivity to viral transformation. Under these experimental conditions, the volatile liquids 1,1,1-trichloroethane, dichloromethane, chloroform, 1,2-dichloroethane, and 1,1-dichloroethane significantly enhanced transformation of Syrian hamster embryo cells by SA7 adenovirus, while acetone exerted no effect. The gases chloromethane and vinyl chloride were also active in this test system, while bromomethane, methane, and ethane were inactive. Incorporation of some of these compounds into liquid cell culture medium for cell treatment was either unsuccessful or produced only a weak enhancement response. Methodology is now available to evaluate volatile and gaseous carcinogens or mutagens and can be used to identify their mechanisms of action and the relative hazards of these agents to human health.

Relationships between benzo(a)pyrene-DNA adduct levels and genotoxic effects in mammalian cells.

The effectiveness of benzo(a)pyrene [B(a)P]-DNA binding as an internal dosimeter was evaluated. Data were obtained from concurrent studies, measuring B(a)P induced genotoxic effects and DNA adducts in several short-term bioassay systems: cytotoxicity, gene mutation, and sister chromatid exchange in Chinese hamster V79 cells; cytotoxicity, gene mutation, and chromosome aberrations in mouse lymphoma L5178Y TK+/-; cytotoxicity and enhanced virus transformation in Syrian hamster embryo cells; and cytotoxicity and morphological transformation in C3H10T1/2CL8 mouse embryo fibroblasts. Both total B(a)P-DNA binding and specific B(a)P-DNA adducts were measured. N2-(10 beta-[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]yl)deoxyguanosine [BPDE I-dGuo] was one of the major adducts identified in all bioassay systems. DNA binding and genotoxic responses varied significantly between bioassays. Each genetic end point was induced with a differing efficiency on a per adduct basis. However, the relationships between frequency of genetic effect or morphological transformation and B(a)P-DNA binding or BPDE I-dGuo were linear within a given assay. In order to compare biological end points of diverse frequencies in diverse biological systems, a doubling adduct level, expressed as the number of BPDE I-dGuo adducts per unit of DNA required to double the induced frequency of biological response, was applied to the data.

Tumorigenesis and genotoxicity of ethyl carbamate and vinyl carbamate in rodent cells.

Vinyl carbamate (VC) is a suspect metabolic intermediate in ethyl carbamate (EC) carcinogenesis. In the present studies, EC and VC were evaluated for their relative abilities to induce adenomas and sister chromatid exchanges (SCEs) in lung cells of A/J, C3HeB/FeJ, and C57BL/6J strain mice. For both end points, animals were administered a single i.p. injection of the test chemical. Percentage of mice with adenomas and number of adenomas per mouse were compared among the three strains 24 weeks following exposure to EC or VC. Although the relative order of strain sensitivity was the same for both chemicals: A/J greater than C57BL/6J greater than C3HeB/FeJ, VC was much more potent than EC. For SCE analysis of primary lung cells cultured from treated animals, EC and VC showed potency differences similar to those observed for tumorigenesis. All three mouse strains revealed significant dose-dependent increases in SCE frequency. However, there was no strain specificity for this effect. SCE persistence over time was also compared in treated A/J and C57BL/6J mice. Although EC- and VC-induced SCE frequencies declined over a 2-week observation period, again, there was no strain specificity for this effect. VC was also tested for enhancement of SA7 virus transformation of Syrian hamster embryo cells. Significant concentration-dependent increases in cell transformation frequency were observed.

A highly conserved enhancer in the Dlx5/Dlx6 intergenic region is the site of cross-regulatory interactions between Dlx genes in the embryonic forebrain.

Four Dlx homeobox genes, Dlx1, Dlx2, Dlx5, and Dlx6 are expressed in the same primordia of the mouse forebrain with temporally overlapping patterns. The four genes are organized as two tail-to-tail pairs, Dlx1/Dlx2 and Dlx5/Dlx6, a genomic arrangement conserved in distantly related vertebrates like zebrafish. The Dlx5/Dlx6 intergenic region contains two sequences of a few hundred base pairs, remarkably well conserved between mouse and zebrafish. Reporter transgenes containing these two sequences are expressed in the forebrain of transgenic mice and zebrafish with patterns highly similar to endogenous Dlx5 and Dlx6 expression. The activity of the transgene is drastically reduced in mouse mutants lacking both Dlx1 and Dlx2, consistent with the decrease in endogenous Dlx5 and Dlx6 expression. These results suggest that cross-regulation by Dlx proteins, mediated by the intergenic sequences, is essential for Dlx5 and Dlx6 expression in the forebrain. This hypothesis is supported by cotransfection and DNA-protein binding experiments. We propose that the Dlx genes are part of a highly conserved developmental pathway that regulates forebrain development.

Enhancement of choline uptake by glycine in hamster heart.

Choline uptake by the isolated hamster heart has been shown to be inhibited by exogenous ethanolamine. In this study, the effect of glycine on choline uptake was investigated. At 0.01-1.0 mM glycine in the perfusate, an enhancement of choline uptake (30%) by the isolated heart was observed. Despite the higher choline uptake, the presence of glycine did not affect the rate of phosphatidylcholine biosynthesis. At higher glycine concentration (50 mM), the enhancement of choline uptake was abolished. Exogenous choline had no effect on the uptake of glycine. We postulate that choline and glycine are transported by separate mechanisms, and that glycine may play a regulatory role in the control of choline uptake by the hamster heart.

Cardiolipin is synthesized from exogenous phosphatidylglycerol in rat heart.

De novo biosynthesis of phosphatidylglycerol and cardiolipin in the isolated intact rat heart was shown to occur from newly synthesized phosphatidic acid via the formation of cytidine-5'-diphosphate-1,2-diacylglycerol (Hatch, G.M. (1994) Biochem. J. 297, 201-208). The biosynthesis of new cardiolipin was investigated in isolated rat hearts perfused with exogenous phosphatidylglycerol. Phosphatidylglycerol was rapidly (within 5 min) incorporated into the heart when hearts were perfused with either phosphatidyl-[14C]glycerol or NBD-phosphatidylglycerol. In hearts perfused with phosphatidyl-[14C]glycerol for up to 30 min the amount of radioactivity observed in phosphatidylglycerol was maximum by 5 min of perfusion and remained constant throughout the perfusion period. In the presence of 1-50 microM phosphatidylglycerol, the amount of radioactive phosphatidylglycerol incorporated into the heart was not affected. There was a time-dependent accumulation of radioactivity incorporated into cardiolipin. In addition, radioactivity was incorporated with time into lysophosphatidylglycerol. No significant amount of radioactivity was associated with other phospholipids involved in the biosynthesis of cardiolipin, including phosphatidic acid and cytidine-5'-diphosphate-1,2-diacylglycerol, precursors of cardiolipin biosynthesis via the CDP-DG pathway. We postulate that cardiac cardiolipin may be synthesized from exogenous phosphatidylglycerol independent of phosphatidylglycerol synthesized within the heart.

Thyroxine stimulates the acylation of lysophosphatidylethanolamine in rat heart.

The acylation of cardiac lysophosphatidylethanolamine (LPE) was examined in rats treated with thyroid hormone. Rats were treated for five consecutive days with thyroxine (250 microg/kg) and controls were treated with saline. On the sixth day after an overnight fast, the hearts were removed and perfused in the Langendorff mode with 0.1 mM [1-14C]oleic acid. Radioactivity incorporated into phosphatidylethanolamine (PE) was increased 1.5-fold (P < 0.025) compared to controls. Radioactivity incorporated into phosphatidylcholine was not effected. The pool size of phosphatidylethanolamine and de novo biosynthesis of this phospholipid from [3H(G)]serine or [1,2-14C]ethanolamine were unaltered by thyroxine treatment. Treatment of rats with thyroxine resulted in a 1.5-fold (P < 0.025) increase in the relative percent of oleic acid in cardiac phosphatidylethanolamine. Thyroxine treatment resulted in a 1.8-fold (P < 0.025) increase in cardiac microsomal acyl-coenzyme A:1-acyl glycerophosphorylethanolamine acyltransferase activity compared to controls whereas, phospholipase A, acyl-coenzyme A hydrolase and fatty acyl-coenzyme A synthase activities were unaltered. The results demonstrate that the reacylation of cardiac LPE is regulated by thyroid hormone.

The protein phosphatase inhibitor, okadaic acid, inhibits phosphatidylcholine biosynthesis in isolated rat hepatocytes.

There is evidence that phosphatidylcholine (PC) biosynthesis in hepatocytes is regulated by a phosphorylation-dephosphorylation mechanism. The phosphatases involved have not been identified. We, therefore, investigated the effect of okadaic acid, a potent protein phosphatase inhibitor, on PC biosynthesis via the CDP-choline pathway in suspension cultures of isolated rat hepatocytes. Okadaic acid caused a 15% decrease (P less than 0.05) in [Me-3H]choline uptake in continuous-pulse labeling experiments. After 120 min of treatment, the labeling of PC was decreased 46% (P less than 0.05) with a corresponding 20% increase (P less than 0.05) in labeling of phosphocholine. Cells were pulsed with [Me-3H]choline for 30 min and subsequently chased for up to 120 min with choline in the absence or presence of okadaic acid. The labeling of phosphocholine was increased 86% (P less than 0.05) and labeling of PC decreased 29% (P less than 0.05) by 120 min of chase in okadaic acid-treated hepatocytes. The decrease of label in PC was quantitatively accounted for in the phosphocholine fraction. Incubation of hepatocytes with both okadaic acid and CPT-cAMP did not produce an additive inhibition in labeling of PC. Choline kinase and cholinephosphotransferase activities were unaltered by treatment with okadaic acid. Hepatocytes were incubated with digitonin to cause release of cytosolic components. Cell ghost membrane cytidylyltransferase (CT) activity was decreased 37% (P less than 0.005) with a concomitant 33% increase (P less than 0.05) in released cytosolic cytidylyltransferase activity in okadaic acid-treated hepatocytes. We postulate that CT activity and PC biosynthesis are regulated by protein phosphatase activity in isolated rat hepatocytes.

The acylation of lysophosphatidylglycerol in rat heart: evidence for both in vitro and in vivo activities.

The reacylation of lysophospholipids back to their parent molecules is important for attaining the appropriate fatty acyl composition in many phospholipids and for preventing the accumulation of arrhythmia generating lysophospholipids in the heart. In this study, we report the presence of an active acyltransferase activity for lysophosphatidylglycerol reacylation to phosphatidylglycerol in rat heart membrane preparations. The activity of acyl-Coenzyme A:1-acylglycerophosphorylglycerol acyltransferase in rat heart subcellular fractions was in the order of microsomal > mitochondrial > cytosol. The activity in the membrane fractions were characterized and found to have a pH optimum in the alkaline range. However, significant enzyme activity was observed at physiological pH. With oleoyl-Coenzyme A as substrate, the microsomal activity had a preference for lysophosphatidylglycerol substrates in the order of myristoyl > palmitoyl > oleoyl > stearoyl. The apparent K(m) values for 1-palmitoylglycerophosphorylglycerol and oleoyl-Coenzyme A were 9.4 and 7.1 microM, respectively. In contrast, the mitochondrial activity had a preference for lysophosphatidylglycerol substrates in the order of oleoyl > myristoyl = stearoyl = palmitoyl. The apparent K(m) values for 1-oleoylglycerophosphorylglycerol and oleoyl-Coenzyme A were 17.8 and 18.0 microM, respectively. Both membrane activities were heat labile as pre-incubation at 55 degrees C for 1 min completely abolished the activity. However, pre-incubation at 50 degrees C resulted in different profiles of inactivation in both microsomal and mitochondrial fractions. Both membrane activities were inhibited by high concentrations of lysophosphatidylglycerol and affected to a similar extent by various detergents. To demonstrate whether reacylation of lysophosphatidylglycerol to phosphatidylglycerol occurred in vivo, isolated rat hearts were perfused for 60 min in the Langendorff mode with 0.1 microM 1-palmitoylglycerophosphoryl[14C]glycerol bound to albumin. 1-Palmitoylglycerophosphoryl[14C]glycerol was readily taken up by the isolated perfused rat heart and significant synthesis of phosphatidyl[14C]glycerol was observed. The findings indicate the presence of an acyl-Coenzyme A:1-acylglycerophosphorylglycerol acyltransferase activity in the rat heart subcellular membranes which is capable of catalyzing lysophosphatidylglycerol acylation to phosphatidylglycerol in vitro and in vivo.

Effect of NaF and okadaic acid on the subcellular distribution of CTP: phosphocholine cytidylyltransferase activity in rat liver.

The effect of preincubation of rat liver post-mitochondrial supernatant with NaF and okadaic acid on the subcellular distribution of CTP: phosphocholine cytidylyltransferase activity was investigated. NaF (20 mM) inhibited the time-dependent activation of cytidylyltransferase activity in post-mitochondrial supernatant. Subcellular fractionation of the post-mitochondrial supernatant revealed that cytidylyltransferase activity in the microsomal fraction was decreased and activity in the cytosolic fraction increased with time of preincubation with NaF compared to controls. Okadaic acid is a specific and potent inhibitor of type 1 and 2A phosphoprotein phosphatases. Preincubation of cytosol with 5 microM okadaic acid inhibited the time-dependent activation of cytosolic cytidylyltransferase activity. Preincubation of post-mitochondrial supernatants with 5 microM okadaic acid inhibited the time-dependent activation of cytidylyltransferase activity by 13% at 45 min and 16% at 60 min of preincubation compared to controls. Microsomal cytidylyltransferase activity was decreased 27% at 45 min and 31% at 60 min with a corresponding retention of cytosolic cytidylyltransferase activity of 21% at 45 min and 37% at 60 min of preincubation with okadaic acid compared to controls. We postulate that the activity of the type 1 and/or type 2A phosphoprotein phosphatases affect the subcellular distribution of CTP: phosphocholine cytidylyltransferase activity in rat liver.

Induction of free radicals in hepatocytes, mitochondria and microsomes of rats by ochratoxin A and its analogs.

Oxidative damage may be one of the manifestations of cellular damage in the toxicity of ochratoxin A (OA). OA; its three natural analogs, OB, OC and O alpha; and three synthetic analogs, the ethyl amide of OA (OE-OA), O-methylated OA (OM-OA), and the lactone-opened OA (OP-OA) were used to study free radical generation in hepatocytes, mitochondria and microsomes from rats. Electron paramagnetic resonance spectroscopy (EPR) using alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN) as a spin trapping agent showed an enhanced free radical generation due to the addition of NADPH to the microsomes. An EPR signal was not observed in the mitochondria and hepatocyte samples when they were treated with a variety of agents. Addition of OM-OA together with NADPH and Fe3+ to the microsomes resulted in a strong EPR signal compared with the other analogs, whereas the signal could be quenched by the addition of catalase. OM-OA does not have a dissociable phenolate group and does not chelate Fe3+. The spin adduct hyperfine splitting constants indicated the presence of alpha-hydroxyethyl radicals resulting from generated hydroxyl radicals, which were trapped by 4-POBN. The results also suggested that the production of hydroxyl radicals by OA does not require a dissociable phenolate group or the prior formation of an OA-Fe complex.