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.

Novel applications of polymerase chain reaction to urinary nucleic acid analysis.

DNA fragments from cells that have died throughout the body not only appear in the bloodstream but also cross the kidney barrier into the urine. The relatively low molecular weight (150-200 bp) of this Transrenal DNA should be considered when deciding on methods of isolation and analysis. In particular, if polymerase chain reaction (PCR) is used for amplification and detection of specific sequences, then the reduction of amplicon size will significantly enhance sensitivity. Detection of DNA mutations is also made more difficult by the presence of a large excess of a wild-type allele. Using K-RAS mutations as an example, two ways around this problem--enriched PCR and stencil-aided mutation analysis-are described, based on selective pre-PCR elimination of wild-type sequences.

Transrenal DNA testing: progress and perspectives.

Transrenal DNA (Tr-DNA) is a recently discovered class of extracellular urinary DNA that originates from cells dying throughout the body. Postapoptotic DNA is known to appear in the circulating plasma, but it is now recognized that a portion of these fragments cross the kidney barrier and appear in urine in the form of 150-200-bp fragments. Tr-DNA containing fetal sequences has been isolated from the urine of pregnant women, tumor-specific mutations have been detected in Tr-DNA from patients with colon and pancreatic tumors, and donor DNA has been found in Tr-DNA isolated from recipient urine. Furthermore, proviral HIV DNA, bacterial and parasite DNA sequences have been detected in Tr-DNA from infected patients. Potential applications of Tr-DNA-based tests cover a very broad area of molecular diagnostics and genetic testing, including prenatal detection of inherited diseases, tumor diagnostics and therapeutic monitoring and detection of infectious agents. The Tr-DNA test is expected to have utility in treatment monitoring, transplantation monitoring, drug development and broad public health screening, where a noninvasive, common-platform diagnostic technology has particular value. This review describes some of the highlights of Tr-DNA technology applications, advantages over existing technologies and potential problems anticipated in test development.

Transrenal DNA as a diagnostic tool: important technical notes.

A small portion of DNA from apoptotic cells escapes complete degradation, appears in blood as oligonucleosomal-size fragments, is excreted in the urine, and can be used for diagnostic purposes. More detailed study revealed that transrenal DNA (Tr-DNA) belongs to a relatively low molecular-weight (150-250 bp) fraction, thereby requiring more careful attention to methods employed for purification and analysis. For example, here it is demonstrated that the QIAamp blood kit purifies primarily high molecular-weight DNA from serum, whereas the Guanidine/Promega Wizard Resin (GITC/WR) method purifies primarily low molecular-weight DNA. As a result, sensitivity in detection of K-RAS mutations in serum of patients with colorectal tumors is significantly higher with DNA isolated with the GITC/WR method than with the QIAamp kit. Amplicon size is also extremely important in analysis of Tr-DNA, because the shorter the amplicon, the higher is the sensitivity of biomarker detection in Tr-DNA. One hundred fifty-seven and 87 bp amplicons were employed for detection of mutant K-RAS in DNA isolated from 0.1 mL of urine obtained from 15 patients with pancreatic cancer. Mutant K-RAS was found in Tr-DNA of 3 and 10 patients with the long and short amplicons, respectively. The sensitivity and specificity of detection of mutant sequences are reduced in the presence of high excess of a respective wild-type allele, but they can be significantly increased through application of enriched polymerase chain reaction (PCR), peptide nucleic acid (PNA) clamped PCR, and/or stencil-aided mutation analysis (SAMA), based on selective pre-PCR elimination of wild-type sequences.

Apoptosis in the myocardium: much is still expected.

Apoptosis continues to be a controversial concept and subject of debate among scientists regarding its value as the basis for new therapeutic strategies. Today, it is widely accepted that the death of cardiac myocytes under a variety of conditions appears to be apoptotic based on a variety of criteria. However, the significance of these observations and how the insights into apoptotic molecular pathways may provide novel therapeutic targets remains to be determined. It is important to reconsider the pertinent underlying mechanisms of apoptosis regulation, and how these molecular pathways may be viewed in the functioning, intact heart. This knowledge can be applied in pursuit of practical goals in a search for new ways to prevent myocardial damage following such injuries as ischaemia/reperfusion or exposure to cardiotoxic drugs. Although recent literature contains reports of positive findings, there has not yet been a rigorous application of the model of apoptosis in the myocardium, and the potential for development of new therapeutic strategies is not yet understood.

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.

Biochemical and immunologic properties of the nonstructural proteins of the hepatitis C virus: implications for development of antiviral agents and vaccines.

Infection with the hepatitis C virus (HCV) is the major cause of non-A, non-B hepatitis worldwide. The viral genome, a positive-sense, single-stranded, 9.6-kb long RNA molecule, is translated into a single polyprotein of about 3,000 amino acids. The viral polyprotein is proteoytically processed to yield all the mature viral gene products. The genomic order of HCV has been determined to be C-->E1-->E2-->p7-->NS2-->NS3-->NS4A-->NS4B-->NS5A++ +-->NS5B. C, E1, and E2 are the virion structural proteins. Whereas the function of p7 is currently unknown, NS2 to NS5B are thought to be the nonstructural proteins. Generation of the mature nonstructural proteins relies on the activity of viral proteinases. Cleavage at the NS2-NS3 junction is accomplished by a metal-dependent autocatalytic proteinase encoded within NS2 and the N-terminus of NS3. The remaining downstream cleavages are effected by a serine proteinase contained also within the N-terminal region of NS3. NS3, in addition, contains an RNA helicase domain at its C-terminus. NS3 forms a heterodimeric complex with NS4A. The latter is a membrane protein that acts as a cofactor of the proteinase. Although no function has yet been attributed to NS4B, NS5A has been recently suggested to be involved in mediating the resistance of the HCV to the action of interferon. Finally, the NS5B protein has been shown to be the viral RNA-dependent RNA polymerase. This article reviews the current understanding of the structure and the function of the various HCV nonstructural proteins with particular emphasis on their potential as targets for the development of novel antiviral agents and vaccines.

Biochemical characterization of a hepatitis C virus RNA-dependent RNA polymerase mutant lacking the C-terminal hydrophobic sequence.

The RNA-dependent RNA polymerase activity of hepatitis C virus is carried out by the NS5B protein. The full-length protein was previously purified as a non-fusion protein from insect cells infected with a recombinant baculovirus. The characterization is now described of a C-terminal hydrophobic domain deletion mutant of NS5B purified from E. coli. In addition to increased solubility, deletion of this sequence also positively affected the polymerase enzymatic activity. The efficiency of nucleotide polymerization of both the full-length and the C-terminal truncated enzymes were compared on homopolymeric template-primer couples as well as on RNA templates with heteropolymeric sequences. The largest difference in the polymerase activity was observed on the latter. On all the templates, the increased activity could be ascribed, at least in part, to enhanced template turnover of the deletion mutant with respect to the full-length enzyme. The elongation rates of the two enzyme forms were compared under single processive cycle conditions. Under these conditions, both the full-length and the deletion mutant were able to incorporate about 700 nt/min.

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.

Identification of a novel sequence at the 3' end of the GB virus B genome.

GB virus B (GBV-B) is a virus of the family Flaviviridae that infects small primates (Saguinus sp. [tamarins]) and shows similarities to hepatitis C virus (HCV) in genome organization, protein function, tissue tropism, and pathogenicity. This suggests the possibility of using tamarins infected by GBV-B or GBV-B/HCV chimeric viruses as a surrogate animal model of HCV infection. To achieve the construction of such chimeric viruses, it is essential to produce a complete and infectious GBV-B genomic RNA. We have identified a novel sequence at the 3' end of the GBV-B genome and show that it can be arranged in a secondary structure resembling that of the 3' end of the HCV genome, which is known to be essential for infectivity.

Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus.

We report the crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus, a major human pathogen, to 2.8-A resolution. This enzyme is a key target for developing specific antiviral therapy. The structure of the catalytic domain contains 531 residues folded in the characteristic fingers, palm, and thumb subdomains. The fingers subdomain contains a region, the "fingertips," that shares the same fold with reverse transcriptases. Superposition to the available structures of the latter shows that residues from the palm and fingertips are structurally equivalent. In addition, it shows that the hepatitis C virus polymerase was crystallized in a closed fingers conformation, similar to HIV-1 reverse transcriptase in ternary complex with DNA and dTTP [Huang H., Chopra, R., Verdine, G. L. & Harrison, S. C. (1998) Science 282, 1669-1675]. This superposition reveals the majority of the amino acid residues of the hepatitis C virus enzyme that are likely to be implicated in binding to the replicating RNA molecule and to the incoming NTP. It also suggests a rearrangement of the thumb domain as well as a possible concerted movement of thumb and fingertips during translocation of the RNA template-primer in successive polymerization rounds.

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.

Multiple enzymatic activities associated with recombinant NS3 protein of hepatitis C virus.

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) contains at least two domains associated with multiple enzymatic activities; a serine protease activity resides in the N-terminal one-third of the protein, whereas RNA helicase activity and RNA-stimulated nucleoside triphosphatase activity are associated with the C-terminal portion. To study the possible mutual influence of these enzymatic activities, a full-length NS3 polypeptide of 67 kDa was expressed as a nonfusion protein in Escherichia coli, purified to homogeneity, and shown to retain all three enzymatic activities. The protease activity of the full-length NS3 was strongly dependent on the activation by a synthetic peptide spanning the central hydrophobic core of the NS4A cofactor. Once complexed with the NS4A-derived peptide, the full-length NS3 protein and the isolated N-terminal protease domain cleaved synthetic peptide substrates with comparable efficiency. We show that, as in the case of the isolated protease domain, the protease activity of full-length NS3 undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B and NS5A-NS5B. We have also characterized and quantified the NS3 ATPase, RNA helicase, and RNA-binding activities under optimized reaction conditions. Compared with the isolated N-terminal and C-terminal domains, recombinant full-length NS3 did not show significant differences in the three enzymatic activities analyzed in independent in vitro assays. We have further explored the possible interdependence of the NS3 N-terminal and C-terminal domains by analyzing the effect of polynucleotides on the modulation of all NS3 enzymatic functions. Our results demonstrated that the observed inhibition of the NS3 proteolytic activity by single-stranded RNA is mediated by direct interaction with the protease domain rather than with the helicase RNA-binding domain.

Complex of NS3 protease and NS4A peptide of BK strain hepatitis C virus: a 2.2 A resolution structure in a hexagonal crystal form.

The crystal structure of the NS3 protease of the hepatitis C virus (BK strain) has been determined in the space group P6(3)22 to a resolution of 2.2 A. This protease is bound with a 14-mer peptide representing the central region of the NS4A protein. There are two molecules of the NS3(1-180)-NS4A(21'-34') complex per asymmetric unit. Each displays a familiar chymotrypsin-like fold that includes two beta-barrel domains and four short alpha-helices. The catalytic triad (Ser-139, His-57, and Asp-81) is located in the crevice between the beta-barrel domains. The NS4A peptide forms an almost completely enclosed peptide surface association with the protease. In contrast to the reported H strain complex of NS3 protease-NS4A peptide in a trigonal crystal form (Kim JL et al., 1996, Cell 87:343-355), the N-terminus of the NS3 protease is well-ordered in both molecules in the asymmetric unit of our hexagonal crystal form. The folding of the N-terminal region of the NS3 protease is due to the formation of a three-helix bundle as a result of crystal packing. When compared with the unbound structure (Love RA et al., 1996, Cell 87:331-342), the binding of the NS4A peptide leads to the ordering of the N-terminal 28 residues of the NS3 protease into a beta-strand and an alpha-helix and also causes local rearrangements important for a catalytically favorable conformation at the active site. Our analysis provides experimental support for the proposal that binding of an NS4A-mimicking peptide, which increases catalytic rates, is necessary but not sufficient for formation of a well-ordered, compact and, hence, highly active protease molecule.

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.

Rational design and functional expression of a constitutively active single-chain NS4A-NS3 proteinase.

BACKGROUND: The proteinase domain of the hepatitis C virus NS3 protein is involved in the maturation of the viral polyprotein. A central hydrophobic domain of the NS4A protein is required as a cofactor for its proteolytic activity. The three-dimensional structure of the proteinase domain alone and complexed with an NS4A-derived peptide has been solved recently and revealed that the N terminus of the proteinase is in near proximity to the C terminus of the cofactor. To study the molecular basis of the enzyme activation by its cofactor and to overcome the difficulties of structural and functional investigation associated with a two-species complex, we rationally designed a link to bridge the two molecules in order to have a single polypeptide construct. RESULTS: The engineered construct led to the production of a stable, monomeric protein with proteolytic activity that is independent from the addition of a synthetic peptide representing the cofactor domain of the NS4A protein. The protein is active on both protein and synthetic peptide substrates. Spectroscopic and kinetic analysis of the recombinant NS4A-NS3 single-chain proteinase demonstrated features superimposable with the isolated NS3 proteinase domain complexed with the NS4A cofactor. CONCLUSIONS: We designed a very tight connection between the NS3 and NS4A polypeptide chains with the rationale that this would allow a more stable structure to be formed. The engineered single-chain enzyme was indistinguishable from the NS3 proteinase complexed with its NS4A cofactor in all enzymatic and physico-chemical properties investigated.

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.