Detection of circulating tumor cells in patients with gastrointestinal tract cancer using RT-PCR and its clinical implications.

To investigate the relationship between the presence of circulating tumor cells in different stages of gastrointestinal tract cancer and the subsequent relapse or distant metastasis, circulating levels of CEA mRNA was serially examined at an interval of 10.6+/-4.5 or 13.7+/-3.0 months in gastric or colorectal cancer patients, respectively. CEA mRNA was measured by means of RT-PCR amplification as an indicator for micrometastatic malignant cells. Seven of twenty-nine respectable gastric cancer patients (24.1%) [EGC: 2/9 (22.2%), AGC IIIa: 1/5 (20.0%), AGC IIIb: 4/15 (26.6%)] were positive for CEA mRNA on the initial test and 10 of 29 patients (34.4%) [EGC: 2/ 9 (22.2%), AGC IIIa: 1/5 (20.0%), AGC IIIb: 7/15 (46.7%)] were positive on a follow-up test. Only in AGC IIIb, the positive rate for CEA mRNA increased about twice and 6 of 7 positive cases (85.7%) relapsed within 2.6+/-2.4 months after the follow-up test. In colorectal cancer, 4 of 19 patients (21.1%) [B2: 1/6 (16.7%), C2: 3/13 (23.0%)] were positive on the initial test and 10 of 19 patients (52.6%) [B2: 4/6 (66.7%), C2: 6/13 (46.2%)] were positive on a follow-up test showing an increase in positive rates during a follow-up, however, no significant correlation between CEA mRNA positivity and subsequent relapse was demonstrated. These results suggest that an early tumor cell dissemination may occur in gastrointestinal tract cancer without subsequent relapse, however, the serial regular examination of CEA mRNA level may contribute to predicting a subsequent relapse in AGC IIIb in gastric cancer.

Identification of the minimal essential RNA sequences responsible for site-specific targeting of the Leishmania RNA virus 1-4 capsid endoribonuclease.

The Leishmania RNA virus 1-4 capsid protein possesses an endoribonuclease activity responsible for single-site-specific cleavage within the 450-nucleotide 5' untranslated region of its own viral RNA transcript. To characterize the minimal essential RNA determinants required for site-specific cleavage, mutated RNA transcripts were examined for susceptibility to cleavage by the virus capsid protein in an in vitro assay. Deletion analyses revealed that all determinants necessary for accurate cleavage are encoded in viral nucleotides 249 to 342. Nuclease mapping and site-specific mutagenesis of the minimal RNA sequence defined a stem-loop structure that is located 40 nucleotides upstream from the cleavage site (nucleotide 320) and that is essential for accurate RNA cleavage. Abrogation of cleavage by disruption of base pairing within the stem-loop was reversed through the introduction of complementary nucleotide substitutions that reestablished the structure. We also provide evidence that divalent cations, essential components of the cleavage reaction, stabilized the stem-loop structure in solution. That capsid-specific antiserum eliminated specific RNA cleavage provides further evidence that the virus capsid gene encodes the essential endoribonuclease activity.

Host cell proteins bind specifically to the capsid-cleaved 5' end of Leishmaniavirus RNA.

Leishmaniavirus (LRV) is a double-stranded RNA (dsRNA) virus that persistently infects some strains of the protozoan parasite, Leishmania. LRV generates a short transcript, corresponding to the 5' end of the positive-sense RNA (320 nt), via a cleavage event mediated by the viral capsid protein on the full-length positive sense RNA transcript. To address the possibility that the RNA cleavage represents a regulatory mechanism for maintaining persistent infection, the interactions between Leishmania cytoplasmic proteins and in vitro synthesized viral transcripts were studied. In gel mobility shift experiments, three specific RNA/protein complexes were formed between cellular proteins and the cleaved viral transcript, and three major proteins were labeled by UV cross-linking. No protein binding activity was observed for either the short (320 nt) or full-length RNA transcripts. However, the two cleavage reaction products were able to form stable RNA/RNA complexes. We present a model in which the virus is targeting its own transcript for cleavage to promote binding of host factors to cryptic domains inaccessible in the full-length transcript.

Specific in vitro cleavage of a Leishmania virus capsid-RNA-dependent RNA polymerase polyprotein by a host cysteine-like protease.

Antibodies raised against baculovirus-expressed RNA-dependent RNA polymerase (RDRP) recognized a 95-kDa antigen and two smaller proteins in sucrose-purified Leishmania virus particles isolated from infected parasites. The 95-kDa antigen is similar in size to one predicted by translation of the RDRP open reading frame (ORF) alone. In an effort to reconcile in vitro observations of translational frameshifting on Leishmania RNA virus 1-4 transcripts, we have developed an in vitro cleavage assay system to explore the possibility that the fusion polyprotein is proteolytically processed. We show that coincubation a synthetic Cap-Pol fusion protein with lysates from Leishmania parasites yields major cleavage products similar in size to those encoded by the individual capsid and RDRP genes as well as the antigens detected in vivo. The major 82- and 95-kDa major cleavage products are specifically immunoprecipitated by capsid- or polymerase-specific antibodies, respectively, showing that cleavage occurs at or near the junction of the two functional domains. Protease inhibitor studies suggest that a cysteine-like protease is responsible for cleavage in the in vitro assay system developed here. From these results, we suggest that failure to detect a capsid-polymerase fusion protein produced by translational frameshifting in vivo may be due to specific proteolytic processing.

Hygromycin B resistance mediates elimination of Leishmania virus from persistently infected parasites.

A series of pX63-HYG derivatives encoding Leishmania RNA virus 1-4 (LRV1-4) sequences were electroporated into cells of Leishmania strain M4147, a virus-infected strain of L. guyanensis. After 6 weeks of drug selection (hygromycin B), transfected parasites lacked detectable quantities of viral genomic double-stranded RNA, viral capsid protein, and RNA-dependent RNA polymerase (RDRP) activity. Evidence of viral infection was not recovered upon removal of the drug. While viral RNA transcripts were produced from electroporated expression vectors, as determined by reverse transcription-PCR, viral antigens were not detected, suggesting that the antiviral effects of hygromycin B are mediated through translation inhibition. A short-term selection study suggests that the LRV1-4 elimination may not only be a function of hygromycin B as a protein synthesis inhibitor but also possibly related to the mechanism of hygromycin B resistance in Leishmania strains.

Dihydroxyacetone synthase from a methanol-utilizing carboxydobacterium, Acinetobacter sp. strain JC1 DSM 3803.

Acinetobacter sp. strain JC1 DSM 3803, a carboxydobacterium, grown on methanol was found to show dihydroxyacetone synthase, dihydroxyacetone kinase, and ribulose 1,5-bisphosphate carboxylase, but no hydroxypyruvate reductase and very low hexulose 6-phosphate synthase, activities. The dihydroxyacetone synthase was found to be expressed earlier than the ribulose 1,5-bisphosphate carboxylase. The dihydroxyacetone synthase was purified 19-fold in eight steps to homogeneity, with a yield of 9%. The final specific activity of the purified enzyme was 1.12 micromol of NADH oxidized per min per mg of protein. The molecular weight of the native enzyme was determined to be 140,000. Sodium dodecyl sulfate-gel electrophoresis revealed a subunit of molecular weight 73,000. The optimum temperature and pH were 30 degrees C and 7.0, respectively. The enzyme was inactivated very rapidly at 70 degrees C. The enzyme required Mg2+ and thiamine pyrophosphate for maximal activity. Xylulose 5-phosphate was found to be the best substrate when formaldehyde was used as a glycoaldehyde acceptor. Erythrose 4-phosphate, glycolaldehyde, and formaldehyde were found to act as excellent substrates when xylulose 5-phosphate was used as a glycoaldehyde donor. The Kms for formaldehyde and xylulose 5-phosphate were 1.86 mM and 33.3 microM, respectively. The enzyme produced dihydroxyacetone from formaldehyde and xylulose 5-phosphate. The enzyme was found to be expressed only in cells grown on methanol and shared no immunological properties with the yeast dihydroxyacetone synthase.

Cleavage site mapping and substrate-specificity of Leishmaniavirus 2-1 capsid endoribonuclease activity.

The Leishmaniavirus capsid protein possesses an RNA endoribonuclease activity that cleaves viral positive-sense RNA at a specific, single site within the 5' untranslated region. The site of cleavage in LRV1-4 RNA was previously mapped to nucleotide 320 of the LRV1-4 genome. Here we show that an LRV2-1-derived substrate RNA transcript is also cleaved at a single site in an in vitro cleavage assay with LRV2-1 virions. Precise RNA cleavage site mapping in this divergent Old World virus, LRV2-1, confirms that cleavage is occurring within a region of homology to the LRV1 isolates. Substrate RNA transcripts possessing viral sequences from LRV1-4 or LRV2-1 genomes were assayed for susceptibility to cleavage by the cognate and noncognate capsid endoribonucleases to determine the level of substrate specificity.

Purification and some properties of ribulose 1,5-bisphosphate carboxylases/oxygenases from Acinetobacter sp. strain JC1 and Hydrogenophaga pseudoflava.

Ribulose 1,5-bisphosphate carboxylases/oxygenases (RuBisCOs) of two carboxydobacteria, Acinetobacter sp. strain JC1 and Hydrogenophaga pseudoflava, grown on carbon monoxide were purified and partially characterized. RuBisCO of Acinetobacter sp. JC1 was purified 5-fold in eight steps to homogeneity, with a yield of 1.6%. The final specific activity of the purified enzyme was 39.5 nmol CO2 incorporated per min per mg protein. The molecular weight of the native enzyme was determined to be 520,000. Sodium dodecyl sulfate-gel electrophoresis revealed two nonidentical subunits of molecular weights 53,500 and 15,000. The Km and Vmax for CO2 were 36.7 microM and 296.1 nmol per min per mg protein, respectively, and those for ribulose 1,5-bisphosphate were 3.7 microM and 770 nmol per min per mg protein, respectively. The enzyme of H. pseudoflava was purified 55-fold in eight steps to homogeneity, with a yield of 3.6%. The final specific activity was 304.3 nmol CO2 incorporated per min per mg protein. The molecular weight of the enzyme was estimated to be 505,000. The enzyme was found to have two kinds of nonidentical subunits of molecular weights 51,500 and 14,000. The Km and Vmax for CO2 were found to be 16.4 microM and 777.8 nmol per min per mg protein, respectively, and those for ribulose 1,5-bisphosphate were 0.1 microM and 436.2 nmol per min per mg protein, respectively. The N-terminal amino acid sequences of the large and small subunits of Acinetobacter sp. JC1 enzyme were Ala-Asp-Arg-Trp-Asn-Ala-Gly-Val-IIe-Pro-Tyr-Ala-Glu-Met-Gly and Met-Arg-Ile-Thr-Glu-Gly-Thr-Phe-Ser-Tyr-Leu-Pro-Asp-Phe-Thr, respectively. The sequences of the H. pseudoflava enzyme were Ala-Thr-Lys-Thr-Tyr-Asu-Ala-Gly-Val-Lys-Glu-Tyr-Trp-Ser-Thr and Met-Ser-Met-Gln-Asp-Tyr-His-Ser-Arg-Leu-Ser-Asp-Pro-Ala-Ile, respectively. The peptide map of RuBisCO from Acinetobacter sp. JC1 grown on carbon monoxide was different from that of the bacterium grown on methanol. The two RuBisCOs, however, were found to be identical in N-terminal residue and antigenic property. The RuBisCO of Acinetobacter sp. JC1 was found to share no immunological properties with those of H. pseudoflava, Oligotropha carboxidovorans and Pseudomonas carboxydohydrogena.

The complete sequence of Leishmania RNA virus LRV2-1, a virus of an Old World parasite strain.

A complete cDNA sequence is reported for LRV2-1, the first Leishmania RNA virus known to infect an Old World parasite, Leishmania major. Sequence analyses show that LRV2-1 differs significantly from members of the LRV1 genus which infect New World parasites. The data support a view that transmission of LRV is strictly vertical and suggest that LRV predate the divergence of Old and New World parasites. As a consequence of this divergence, conserved features can be identified for the first time in Leishmaniavirus proteins. A finding that the virus capsid and polymerase genes do not overlap is unique among the known Totiviridae and infers that a gag-pol fusion protein cannot be produced simply via tRNA slippage in LRV2-1.

Purification and some properties of carbon monoxide dehydrogenase from Acinetobacter sp. strain JC1 DSM 3803.

A brown carbon monoxide dehydrogenase from CO-autotrophically grown cells of Acinetobacter sp. strain JC1, which is unstable outside the cells, was purified 80-fold in seven steps to better than 95% homogeneity, with a yield of 44% in the presence of the stabilizing agents iodoacetamide (1 mM) and ammonium sulfate (100 mM). The final specific activity was 474 mumol of acceptor reduced per min per mg of protein as determined by an assay based on the CO-dependent reduction of thionin. Methyl viologen, NAD(P), flavin mononucleotide, flavin adenine dinucleotide, and ferricyanide were not reduced by the enzyme, but methylene blue, thionin, and dichlorophenolindophenol were reduced. The molecular weight of the native enzyme was determined to be 380,000. Sodium dodecyl sulfate-gel electrophoresis revealed at least three nonidentical subunits of molecular weights 16,000 (alpha), 34,000 (beta), and 85,000 (gamma). The purified enzyme contained particulate hydrogenase-like activity. Selenium did not stimulate carbon monoxide dehydrogenase activity. The isoelectic point of the native enzyme was found to be 5.8; the Km of CO was 150 microM. The enzyme was rapidly inactivated by methanol. One mole of native enzyme was found to contain 2 mol of each of flavin adenine dinucleotide and molybdenum and 8 mol each of nonheme iron and labile sulfide, which indicated that the enzyme was a molybdenum-containing iron-sulfur flavoprotein. The ratio of densities of each subunit after electrophoresis (alpha:beta:gamma = 1:2:6) and the number of each cofactor in the native enzyme suggest a alpha 2 beta 2 gamma 2 structure of the enzyme. The carbon monoxide dehydrogenase of Acinetobacter sp. strain JC1 was found to have no immunological relationship with enzymes of Pseudomonas carboxydohydrogena and Pseudomonas carboxydovorans.