Mycobacterium tuberculosis DNA detection in soluble fraction of urine from pulmonary tuberculosis patients.

SETTING: A tertiary care and research institution in Italy. BACKGROUND: Small DNA fragments from cells dying throughout the body have been detected in urine (transrenal DNA [Tr-DNA]). OBJECTIVE: To test the hypothesis that Mycobacterium tuberculosis Tr-DNA could be detected in the urine of pulmonary tuberculosis (TB) patients. DESIGN: We studied 43 patients with culture-confirmed pulmonary TB with no evidence of extra-pulmonary involvement, 10 patients with pulmonary diseases other than TB and 13 healthy controls. DNA was extracted from urine and analysed by semi-nested polymerase chain reaction (PCR). RESULTS: M. tuberculosis-specific sequences were found in the urine of 34 of 43 (79%) TB patients studied, whereas all controls were negative. The transrenal nature of M. tuberculosis DNA was demonstrated by two lines of evidence: first, separate analysis of supernatants and sediments from eight of the study patients found seven positive supernatants but only two matched positive sediments. Second, M. tuberculosis-specific sequences were amplified by semi-nested PCR with primers designed for short but not large amplicons. CONCLUSION: Small M. tuberculosis DNA fragments may be detected in the urine of a significant proportion of patients with pulmonary TB. If these observations are confirmed by larger studies, Tr-DNA technology could represent a new approach for detecting pulmonary M. tuberculosis infection.

Circulating nucleic acids and apoptosis.

It is well documented that plasma contains DNA from tissues throughout the body, including developing fetuses, and tumors. A portion of this DNA crosses the kidney barrier and appears in urine (i.e., transrenal DNA). However, molecular, cellular, and physiological mechanisms of the circulating DNA phenomenon and renal clearance are in an early phase of investigation. Here, we discuss possible forms of circulating DNA, factors affecting representation of different tissues and genomic sequences in plasma DNA, possible mechanisms of renal DNA clearance, and technical problems encountered in DNA isolation from urine. We suggest that apoptotic cells are an important source of DNA in both plasma and urine. Further analysis of the data has led us to propose that a significant portion of circulating DNA can be represented in apoptotic bodies.

Apoptosis induction and cell cycle perturbation in established cell lines by peroxysomicine A1 (T-514).

Peroxysomicine A1, a novel potential anticancer compound induced cell death in established cell lines and in a primary culture of rat neonatal cardiomyocytes. Non-transformed cells are less sensitive to the compound than transformed cell lines. Fluorescent microscopy of dying cells stained with DNA-specific dyes revealed chromatin condensation and nuclear fragmentation as well as membrane blebbing characteristic of apoptosis. Flow cytometry of cells treated with peroxysomicine A1, demonstrated appearance of cells containing less than 2C DNA, that indicated degradation of nuclear DNA, another hallmark of apoptotic cell death. Z-VAD, a nonspecific caspase inhibitor, prevented DNA fragmentation but not cell death registered by permeabilization of cell outer membrane. Peroxysomicine A1 also inhibited proliferation of various cell lines. Flow cytometry analysis showed significant accumulation of dividing cells in G2/M phases of cell cycle indicating, most likely delay in G2. These results provide initial insight into the mechanisms of action of peroxysomicine A1 and suggest that peroxysomicine A1 is a potent inhibitor of cell proliferation and inducer of apoptosis and may be a useful antineoplastic chemotherapeutic agent.

Apoptosis and the heart: a brief review.

Cardiomyopathies are observed with increasing frequency in association with AIDS and HIV infection. Although indirect evidence exists suggesting an association between apoptosis regulation and HIV infection, there is yet no direct evidence that HIV-associated cardiomyopathies involve increased level of apoptosis in the heart. However, since it is now known that apoptosis plays a significant role in heart injury associated with other conditions such as ischemia/reperfusion and heart failure, there is a possibility that dysregulation of apoptosis plays a similarly important role in HIV-associate cardiomyopathies. Here we will briefly review the evidence that apoptotic death of cardiomyocytes occurs and what novel therapeutic strategies may be suggested.

Antiapoptotic activity of the Bowman-Birk inhibitor can be attributed to copurified phospholipids.

Previous studies have shown that extracts from soy possess potent antiapoptotic activity in in vitro and in vivo models. We recently reported that this antiapoptotic activity can be attributed to the presence of specific phospholipids. In this study, a conventional preparation of the soy-derived Bowman-Birk inhibitor (BBI) was tested for antiapoptotic activity in a C3H/10T1/2 cell serum deprivation assay. The BBI preparation was separated into lipid- or protein-containing fractions by organic extraction. The lipid fraction contained only antiapoptotic activity; the protein fraction contained only enzyme inhibition activity. We therefore conclude that the antiapoptotic activity of the BBI preparation is due to specific phospholipids that copurify with BBI. These phospholipids retain their antiapoptotic activity after autoclave treatment, whereas autoclave treatment of the protein fraction results in a loss of its enzyme inhibition activity.

Soy-derived antiapoptotic fractions protect gastrointestinal epithelium from damage caused by methotrexate treatment in the rat.

The ability of a previously described soy-derived antiapoptotic fraction (SDAAF), a soy water extract (Lexirin), and raw soy flour to inhibit methotrexate (MTX)-induced gastrointestinal damage was evaluated by histological examination of duodenal/jejunal sections from MTX-treated rats. Male Sprague-Dawley rats were fed diets containing casein as a sole protein source or diets supplemented with fractions isolated from soy (SDAAF or Lexirin) before and after MTX treatment. The soy fractions were also shown to inhibit serum deprivation-induced programmed cell death (apoptosis) in mouse embryonic C3H10T1/2 cells. Protein sequence (Lexirin) and enzyme activity (Lexirin and SDAAF) were also analyzed. Rats that received SDAAF- and Lexirin-supplemented diets had significantly reduced necrotic and apoptotic damage in the duodenal mucosa, as demonstrated by difference in villi height, mitotic activity, epithelial cell height, and inflammatory response.

Aging and apoptosis control.

The phenomenon of aging is distinct from processes associated with advanced age known to increase risk of diseases, such as cancer. Furthermore, the process of aging is not necessarily related to phenomena such as in vitro replicative senescence; however, any unifying hypothesis of aging must account for all age-dependent phenomena, including senescence. It is proposed that apoptosis forms the ultimate protective process for preservation of phenotypic fidelity in multicellular organisms since it is the process by which the organism detects damage and replaces the defective cell. Time-dependent degeneration of apoptosis control is the rate-limiting step in the process of aging.

Edg-2/Vzg-1 couples to the yeast pheromone response pathway selectively in response to lysophosphatidic acid.

We have functionally expressed the human cDNA encoding the putative lysophosphatidic acid (LPA) receptor Edg-2 (Vzg-1) in Saccharomyces cerevisiae in an attempt to determine the agonist specificity of this G-protein-coupled receptor. LPA activated the pheromone response pathway in S. cerevisiae expressing Edg-2 in a time- and dose-dependent manner as determined by induction of a pheromone-responsive FUS1::lacZ reporter gene. LPA-mediated activation of the pheromone response pathway was dependent on mutational inactivation of the SST2 gene, the GTPase-activating protein for the yeast G alpha protein (the GPA1 gene product). This indicates that, in sst2 delta yeast cells, Edg-2 can efficiently couple to the yeast heterotrimeric G-protein in response to LPA and activate the yeast mitogen-activated protein kinase pathway. The Edg-2 receptor showed a high degree of specificity for LPA; other lyso-glycerophospholipids, sphingosine 1-phosphate, and diacyl-glycerophospholipids did not activate FUS1::lacZ. LPA analogs including a cyclic phosphoester form and ether-linked forms of LPA activated FUS1::lacZ, although fatty acid chains of 6 and 10 carbons did not activate FUS1::lacZ, suggesting a role for the side chain in ligand binding or receptor activation. These results indicate that Edg-2 encodes a highly specific LPA receptor.

Ceramide is involved in triggering of cardiomyocyte apoptosis induced by ischemia and reperfusion.

Involvement of ceramide signaling in the initiation of apoptosis induction in myocardial cells by in vitro and in vivo ischemia and reperfusion was analyzed. Synthetic cell permeable C2-ceramide induced apoptotic death of rat neonatal cardiomyocytes in vitro. In vitro ischemia (oxygen/serum/glucose deprivation) led to a progressive accumulation of ceramide in cardiomyocytes. After 16 hours of simulated in vitro reperfusion (readdition of oxygen, serum and glucose), the level of ceramide in surviving cells was found to have returned to baseline, whereas, levels in nonadherent dead cells remained high. In the rat heart left coronary artery occlusion model, ischemia with the subsequent reperfusion, but not ischemia alone, induced apoptosis in myocardial cells as demonstrated by DNA electrophoresis and measurement of soluble chromatin degradation products. The content of ceramide in ischemic area was elevated to 155% baseline levels at 30 minutes, and to 330% after 210 minutes of ischemia. Ischemia (30 minutes) followed by reperfusion (180 minutes) increased the ceramide level to 250% in the ischemic area. The combination of results obtained in both in vitro and animal models demonstrate for the first time that ceramide signaling can be involved in ischemia/reperfusion death of myocardial cells.

Interferon-gamma modulates a p53-independent apoptotic pathway and apoptosis-related gene expression.

Interferon (IFN)-gamma increases the sensitivity of tumor cell lines, many of which are p53 mutants, to tumor necrosis factor-alpha-mediated and anti-Fas antibody-mediated cell death. To better understand the mechanism of IFN-gamma action in modulating the cell death response independently of p53 function, we analyzed the death of the human colon adenocarcinoma cell line, HT-29, following treatment with IFN-gamma and various cytotoxic agents. Here we show that IFN-gamma modulates cell death by sensitizing the cells to killing by numerous pro-apoptotic stimuli but not pro-necrotic stimuli. Furthermore, we show that select genes from several important apoptosis-related gene families are induced by IFN-gamma, including the apoptosis-signaling receptors CD95 (Fas/APO-1) and TNFR 1 and interleukin-1beta-converting enzyme (Ice) family members Ice, CPP32 (Yama, apopain), ICErel-II (TX, Ich-2), Mch-3 (ICE-LAP3, CMH-1), Mch-4, and Mch-5 (MACH, FLICE). Of the bcl-2 family members, IFN-gamma directly induced bak but notably not bax, which is activated by p53. The IFN-responsive transcriptional activator interferon regulatory factor-1 was also strongly induced and translocated into the nucleus following IFN-gamma treatment. We propose that IFN-gamma modulates a p53-independent apoptotic pathway by both directly and indirectly inducing select apoptosis-related genes.

Antiapoptotic compound to enhance hypothermic liver preservation.

BACKGROUND: Apoptosis (programmed cell death) occurs as a consequence of global organ ischemia during isolation and storage prior to transplantation. If apoptosis is inhibited during ischemia, organ preservation should be improved, and the length of time for permissible storage may be increased. The objective of this study was to test the effect of a newly developed antiapoptotic compound, LXR-015, during extended hypothermic liver preservation. METHODS: Three groups of 12 rats each were studied. In the normal group, liver function was studied immediately after harvesting. In the study group, harvested livers were flushed with Euro-Collins solution (30 ml/kg body weight) containing LXR-015 at a concentration equivalent to 9 mg/kg animal body weight (300 microg/ml). The livers were then stored at 4 degrees C for 24 hr before liver function was studied. In the control group, harvested livers were flushed with Euro-Collins solution without LXR-015 and then stored at 4 degrees C for 24 hr before liver function was studied. RESULTS: Portal venous flow was higher (P<0.05) in the normal and study groups compared with the control group. Portal venous resistance was lower (P<0.05) in the normal and study groups compared with the control group. Liver tissue oxygen consumption in the study group was significantly higher than in both the normal and control groups (P<0.05). Liver enzyme production (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine kinase) was higher in the control group than in either the study or normal group (P<0.05). Bile production in both the normal and study groups was higher than in the control group (P<0.05). The liver tissue wet to dry weight ratio in both the normal and study groups was lower than in the control group (P<0.05). Histopathology studies revealed fewer apoptotic bodies (P<0.05) in both the normal (1.70+/-0.15 per high-power field) and study groups (2.08+/-0.10 per high-power field) than in the control group (7.92+/-.33 per high-power field). CONCLUSIONS: Adding an antiapoptotic compound, LXR-015, to Euro-Collins solution significantly improves hypothermic preservation of the rat liver compared with Euro-Collins solution alone.

A soy-derived antiapoptotic fraction decreases methotrexate toxicity in the gastrointestinal tract of the rat.

The ability of a soy-derived antiapoptotic fraction to inhibit methotrexate-induced gastrointestinal toxicity was examined. Male Sprague-Dawley rats treated with methotrexate were fed diets containing casein as a sole protein source or diets supplemented with a protein-phospholipid fraction isolated from soy flour. This soy fraction has also been shown to inhibit serum deprivation-induced programmed cell death (apoptosis) in the mouse embryonic C3H10T1/2 cell. Rats that received high doses of the soy-derived antiapoptotic fraction-supplemented diets experienced significantly less weight loss and diarrhea and better maintained their pretreatment appetite.

SARPs: a family of secreted apoptosis-related proteins.

Quiescent mouse embryonic C3H/10T1/2 cells are more resistant to different proapoptotic stimuli than are these cells in the exponential phase of growth. However, the exponentially growing 10T1/2 cells are resistant to inhibitors of RNA or protein synthesis, whereas quiescent cells die upon these treatments. Conditioned medium from quiescent 10T1/2 cells possesses anti-apoptotic activity, suggesting the presence of protein(s) that function as an inhibitor of the apoptotic program. Using differential display technique, we identified and cloned a cDNA designated sarp1 (secreted apoptosis-related protein) that is expressed in quiescent but not in exponentially growing 10T1/2 cells. Hybridization studies with sarp1 revealed two additional family members. Cloning and sequencing of sarp2 and sarp3 revealed 38% and 40% sequence identity to sarp1, respectively. Human breast adenocarcinoma MCF7 cells stably transfected with sarp1 or infected with SARP1-expressing adenovirus became more resistant, whereas cells transfected with sarp2 displayed increased sensitivity to different proapoptotic stimuli. Expression of sarp family members is tissue specific. sarp mRNAs encode secreted proteins that possess a cysteine-rich domain (CRD) homologous to the CRD of frizzled proteins but lack putative membrane-spanning segments. Expression of SARPs modifies the intracellular levels of beta-catenin, suggesting that SARPs interfere with the Wnt-frizzled proteins signaling pathway.

Prevention of rat neonatal cardiomyocyte apoptosis induced by simulated in vitro ischemia and reperfusion.

Apoptosis, or programmed cell death, is an active metabolic response to physiological signals or exposure to cytotoxic agents. Recent evidence has shown that the cell death response can be modified by agents presumed to be unrelated to the initial signal, but capable of interfering with the molecular mechanisms of the apoptotic pathway progression. Here we show the results of investigations on the use of a phospholipid-based pharmaceutical preparation for suppression of myocardial damage. First, we show that serum or serum/glucose deprivation, in vitro ischemia with subsequent simulated reperfusion, inhibition of protein synthesis, and treatment with ceramide, staurosporine, adriamycin, cis-platinum and menadione induce apoptotic death in a primary culture of rat neonatal cardiomyocytes. Then we demonstrate that a mixture of specific phospholipids, which has been originally purified from soy flour on the basis of its anti-apoptotic activity, prevents cardiomyocyte death induced by serum or serum/glucose deprivation, by ischemia with subsequent simulated reperfusion, and by ceramide, but not by other cytotoxic treatments. This suggests that ceramide, a lipid secondary messenger which triggers apoptosis induced by some cytotoxic agents, may be involved in the process of signaling ischemia/reperfusion induced apoptotic death of cardiomyocytes. These results further demonstrate that an active pharmaceutical preparation for the suppression of cardiomyocyte death can be formulated based upon a novel strategy of apoptosis modification.

Molecular cloning of a high-affinity receptor for the growth factor-like lipid mediator lysophosphatidic acid from Xenopus oocytes.

Lysophosphatidic acid (1-acyl-2-lyso-snglycero-3-phosphate, LPA) is a multifunctional lipid mediator found in a variety of organisms that span the phylogenetic tree from humans to plants. Although its physiological function is not clearly understood, LPA is a potent regulator of mammalian cell proliferation; it is one of the major mitogens found in blood serum. In Xenopus laevis oocytes, LPA elicits oscillatory Cl- currents. This current, like other effects of LPA, is consistent with a plasma membrane receptor-mediated activation of G protein-linked signal transduction pathways. Herein we report the identification of a complementary DNA from Xenopus that encodes a functional high-affinity LPA receptor. The predicted structure of this protein of 372 amino acids contains features common to members of the seven transmembrane receptor superfamily with a predicted extracellular amino and intracellular carboxyl terminus. An antisense oligonucleotide derived from the first 5-11 predicted amino acids, selectively inhibited the expression of the endogenous high-affinity LPA receptors in Xenopus oocytes, whereas the same oligonucleotide did not affect the low-affinity LPA receptor. Expression of the full-length cRNA in oocytes led to an increase in maximal Cl- current due to increased expression of the high-affinity LPA receptor, but activation of the low-affinity receptor was, again, unaffected. Oocytes expressing cRNA prepared from this clone showed no response to other lipid mediators including prostaglandins, leukotrienes, sphingosine 1-phosphate, sphingosylphosphorylcholine, and platelet-activating factor, suggesting that the receptor is highly selective for LPA.

Post-ischemic apoptotic death of rat neonatal cardiomyocytes.

Despite the clinical importance of cardiomyocyte death following ischemia and reperfusion, little is known about the nature of the process. In primary rat neonatal cardiomyocyte cultures, cell death was induced by ischemia (deprivation of oxygen, serum and glucose) and reperfusion. We report here that ischemia induced primarily necrosis, whereas subsequent reperfusion induced apoptosis. Apoptosis of rat neonatal cardiomyocytes could not be prevented by protein synthesis inhibitors, suggesting that molecular components of the apoptotic pathway pre-exist in these cells. IGFs and calpain inhibitors had no effect on necrotic death during ischemia, but they significantly reduced apoptotic death during reperfusion. These results support the concept that inhibition of post-ischemic apoptotic death in the myocardium may provide a valuable new therapeutic strategy for the treatment of acute myocardial ischemia.

Modulation of apoptosis by the widely distributed Bcl-2 homologue Bak.

Members of the Bcl-2 family of proteins are characterized by their ability to modulate cell death. Bcl-2 and some of its homologues inhibit apoptosis, whereas other family members, such as Bax, will accelerate apoptosis under certain conditions. Here we describe the identification and characterization of a complementary DNA that encodes a previously unknown Bcl-2 homologue designated Bak. Like Bax, the bak gene product primarily enhances apoptotic cell death following an appropriate stimulus. Unlike Bax, however, Bak can inhibit cell death in an Epstein-Barr-virus-transformed cell line. The widespread tissue distribution of Bak messenger RNA, including those containing long-lived, terminally differentiated cell types, suggests that cell-death-inducing activity is broadly distributed, and that tissue-specific modulation of apoptosis is controlled primarily by regulation of molecules that inhibit apoptosis.

Apoptosis and its role in human disease.

In a landmark paper published over two decades ago, Kerr et al. proposed the term apoptosis "for a hitherto little recognized mechanism of controlled cell deletion, which appears to play a complementary but opposite role to mitosis in the regulation of animal cell populations". In the ensuing years, this natural cell death process was studied at the basic science level, primarily with a view to understanding its roles in cancer and in the development and maintenance of the immune system. More recently, however, evidence has suggested a role for the failure of normal apoptosis control in many of the major diseases of the industrialized world. Though complex, apoptosis appears amenable to therapeutic intervention. The range of modern pharmaceutical strategies available to treat such disregulated gene-directed processes offers promise for advances in the control of cancer, immune system and neurodegenerative disorders, heart disease, and perhaps even the aging process itself.

Apoptosis in C3H/10T1/2 mouse embryonic cells: evidence for internucleosomal DNA modification in the absence of double-strand cleavage.

Apoptosis in embryonic C3H/10T1/2 (clone 8) cells is marked by specific changes in morphology and DNA fragmentation that differ from those found in apoptotic thymocytes. These results demonstrate that ultrastructural changes within the nucleus associated with endonucleolytic degradation are linked with structural degradation at higher levels of chromatin organization. Strand modifications within the internucleosomal linker region are shown to involve alkaline-sensitive sites that appear to be sensitive to S1 endonuclease. Our results suggest that apoptosis is not dependent upon internucleosomal cleavage and may reveal the penultimate step and the nature of the metabolic cascade that leads to cell death.