Potential biomarker for early risk assessment of prostate cancer.

BACKGROUND: Catechol estrogen quinones (CEQ) derived from 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2) react with DNA to form depurinating--N7Gua and--N3Ade adducts. This damage leads to mutations that can initiate breast and prostate cancer. To determine whether this damage occurs in humans, urine samples from men with prostate cancer and benign urological conditions, and healthy controls were analyzed. The objective was determining whether any of the cancer patients had formed the depurinating 4-OHE1(E2)-1-N3Ade adducts. METHODS: The adducts were extracted from samples by using affinity columns equipped with a monoclonal antibody developed for detecting 4-OHE1(E2)-1-N3Ade adducts. Eluted extracts were separated by capillary electrophoresis with field-amplified sample stacking and/or ultraperformance liquid chromatography. Absorption/luminescence spectroscopies and mass spectrometry were used to identify the adducts. RESULTS: 4-OHE1-1-N3Ade was detected at higher levels in samples from subjects with prostate cancer (n = 7) and benign urological conditions (n = 4) compared to healthy males (n = 5). CONCLUSION: This is the first demonstration that CEQ-derived DNA adducts are present in urine samples from subjects with prostate cancer.

Development of monoclonal antibodies to 4-hydroxyestrogen-2-N-acetylcysteine conjugates: immunoaffinity and spectroscopic studies.

Catechol estrogen quinones (CEQ) derived from oxidation of the catechol estrogens 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2) can conjugate with glutathione (GSH), a reaction that prevents damage to DNA and can provide biomarkers of exposure to CEQs. Monoclonal antibodies (MAb) to 4-OHE1(E2)-2-N-acetylcysteine [4-OHE1(E2)-2-NAcCys] were developed and characterized by immunological and spectroscopic studies. The NAcCys conjugate is the hydrolytic product of the corresponding conjugate with GSH, followed by N-acetylation of cysteine. MAbs were produced by immunizing mice with 4-OHE1(E2)-2-NAcCys attached to an appropriate linker that was conjugated to keyhole limpet hemocyanin (KLH). Hybridoma cell lines were screened using 4-OHE1(E2)-2-NAcCys conjugated to ovalbumin (OA). There is no immunological cross-reactivity between KLH and OA. Hence, positive hybridoma cell lines secreting antibody against 4-OHE1(E2)-2-NAcCys could be rapidly identified using OA-4-OHE1(E2)-2-NAcCys. An affinity column was developed and used to purify MAb against 4-OHE1(E2)-2-NAcCys. The purified MAb was immobilized on an agarose bead column. This column was used to capture and preconcentrate the hapten of interest out of urine samples. A number of structurally related standards were used to estimate the selectivity and specificity of the chosen MAb. Capillary electrophoresis (CE) with field-amplified sample stacking in absorbance detection mode and laser-induced low temperature luminescence measurements were used to identify and quantitate the 4-OHE1(E2)-2-NAcCys conjugates and related compounds released from the affinity column. Femtomole detection limits have been demonstrated. Future prospects in clinical diagnostics for testing human exposure to CEQ by urine analysis are briefly addressed.

Integration of the PiGMaP and USDA maps for porcine chromosome 14.

In order to align two previously published genetic linkage maps, a set of four of the United States Department of Agriculture (USDA) microsatellite linkage markers was mapped in the International Pig Gene Mapping Project (PiGMaP) reference families. Two-point linkage analysis was used between these USDA markers and the set of genes and markers previously mapped on the PiGMaP chromosome 14 map. Markers with threshold lod scores of three or greater were used for multipoint map construction. The USDA and PigGMaP linkage maps of chromosome 14 were aligned using the four USDA microsatellite markers along with three markers that are common to both maps. The PiGMaP genetic linkage map order for chromosome 14 was confirmed and the map was expanded to 193 cM with addition of the new markers.

Transformation of Mollicutes with single-stranded Tn4001 DNA.

Acholeplasma oculi ISM1499 and Mycoplasma gallisepticum were transformed with single-stranded and double-stranded plasmids containing Tn4001. The transposon mobilized to the chromosome using both single-stranded and double-stranded DNA at the same frequency. M. gallisepticum transformed at a 2 log lower frequency than did A. oculi ISM1499. Restriction enzyme digestion of single-stranded DNA indicated homologous base pairing in the inverted repeat regions, which could account for the transpositional activity of single-stranded DNA.

Transformation of Mycoplasma gallisepticum with Tn916, Tn4001, and integrative plasmid vectors.

Mycoplasma gallisepticum causes respiratory disease in avian species, but little is known about its mechanism(s) of pathogenesis. These studies were undertaken in order to develop genetic systems for analysis of potential virulence factors. M. gallisepticum was transformed with plasmids containing one of the gram-positive transposons Tn916 or Tn4001, which inserted randomly into the mycoplasmal chromosome. Plasmids containing cloned chromosomal DNA were also constructed and tested for integration into regions of DNA homology derived either from chromosomal fragments or from the gentamicin resistance marker from Tn4001. These studies demonstrate that M. gallisepticum is amenable to transformation with both transposons and integrative vectors.

Comparison of a commercial DNA probe test and three cultivation procedures for detection of Mycobacterium paratuberculosis in bovine feces.

Diagnosis of paratuberculosis using the IDEXX DNA probe test and 3 methods for cultivation of Mycobacterium paratuberculosis from fecal specimens were compared. Twenty-one of 170 fecal specimens were DNA probe test positive, whereas 35 specimens were positive by 1 or more of the cultivation methods evaluated. Four specimens were DNA probe test positive but were negative by fecal culture. The probe test detected M. paratuberculosis DNA in 62.9% of the specimens positive by a sedimentation culture method, in 56.6% of those positive by a centrifugation culture method, and in 65.4% of the specimens positive by the Cornell culture method. Specificity of the DNA probe test was approximately 97% relative to all culture methods. Generally, the probe test detected M. paratuberculosis DNA in fecal specimens from animals shedding at least 10(4) M. paratuberculosis colony forming units per gram of feces. Although the probe test did not detect all of the cattle shedding M. paratuberculosis, it was possible to identify cattle shedding the greatest number of organisms in 3 days compared with a minimum of 6 weeks required for positive culture results. The centrifugation method resulted in the most isolations of M. paratuberculosis after 12 weeks of incubation. However, contamination also was greatest when the centrifugation method was used. Contamination was best controlled using the Cornell method. The sedimentation method was the least time consuming and yielded results similar to those of the other 2 methods.

Identification of restriction fragment length polymorphisms in DNA from Mycobacterium paratuberculosis.

DNAs from 34 mycobactin-dependent isolates of Mycobacterium paratuberculosis and 1 isolate of M. paratuberculosis 18 were digested with four restriction endonucleases. Southern hybridization experiments were performed with a 32P-labeled oligonucleotide DNA probe derived from the sequence of IS900, an insertion sequence present in 15 to 20 copies per M. paratuberculosis chromosome. The probe hybridized with DNA from each of the mycobactin-dependent isolates, and restriction fragment length polymorphisms were detected among the isolates with each restriction endonuclease used. Restriction fragment length polymorphism analysis may permit identification of various strains of M. paratuberculosis, which has not been possible with other techniques.

Analysis of restriction endonuclease fragment patterns of DNA from Mycobacterium paratuberculosis.

The DNA from 31 isolates and a reference strain of Mycobacterium paratuberculosis was digested individually with restriction endonucleases BstE II and Pst I. DNA fragments were separated by gel electrophoresis and analyzed. The isolates were from 23 American states, Argentina and Nova Scotia. Twenty-seven were isolated from cattle, two from goats and two from sheep. With the exception of one isolate from cattle, all had restriction endonuclease fragment patterns identical to the fragment patterns for the reference strain, M. paratuberculosis ATCC 19698T. These results confirm other reports and indicate that organisms identified as typical M. paratuberculosis isolates are genetically very similar. It may be possible to use restriction endonuclease analysis to differentiate isolates of M. paratuberculosis from other slowly growing mycobacteria. The genetic similarity also indicates that it may be possible to develop a diagnostic probe that is specific for M. paratuberculosis.

The amino-terminal signal peptide on the porcine transmissible gastroenteritis coronavirus matrix protein is not an absolute requirement for membrane translocation and glycosylation.

cDNA clones mapping within the first 2601 bases of the 3' end of the porcine transmissible gastroenteritis corona-virus (TGEV) genome were sequenced by the method of Maxam and Gilbert and an open reading frame yielding a protein having properties of the matrix (M or E1) protein was identified. It is positioned at the 5' side of the nucleocapsid (N) gene from which it is separated by an intergenic stretch of 12 bases. The deduced M protein comprises 262 amino acids, has a molecular weight of 29,544, is moderately hydrophobic, and has a net charge of +7 at neutral pH. Thirty-four percent of its amino acid sequence is homologous with the M protein of the bovine coronavirus (BCV), 32% with that of the mouse hepatitis coronavirus (MHV), and 19% with that of the avian infectious bronchitis coronavirus (IBV). Judging from alignment with the BCV, MHV, and IBV M proteins, the amino terminus of the TGEV M protein extends 54 amino acids from the virion envelope which compares with only 28 for BCV, 26 for MHV, and 21 for IBV. Eleven of the sixteen amino-terminal amino acids are hydrophobic and the positions of charged amino acids around this sequence suggest that the first 16 amino acids comprise a potentially cleavable signal peptide for membrane insertion. A similar sequence is not found in the M proteins of BCV, MHV, or IBV. When mRNA from infected cells, or RNA prepared by in vitro transcription of the reconstructed M gene, was translated in vitro in the presence of microsomes, the M protein became translocated and glycosylated. When a protein without the amino-terminal signal peptide was made by translating a truncated version of the M gene transcript, some translocation and glycosylation also occurred suggesting that the amino-terminal signal peptide on the TGEV M protein is not an absolute requirement for membrane translocation. Interestingly, the amino-terminal peptide did not appear to be cleaved during in vitro translation in the presence of microsomes suggesting that a step in virion assembly may be required for proper exposure of the cleavage site to the signal peptidase.

Neutralization of porcine transmissible gastroenteritis virus by complement-dependent monoclonal antibodies.

Monoclonal antibodies (MAB) to each of the 3 major structural proteins of transmissible gastroenteritis virus (TGEV) of swine were compared, using their virus-neutralizing (VN) activity in the presence or absence of guinea pig, rabbit, or swine complement. The MAB against the peplomer protein had similar VN titers for TGEV in the presence or absence of complement from any source. The MAB against the matrix protein had VN activity for TGEV only in the presence of complement, whereas MAB specific for the nucleocapsid protein did not possess VN activity in the presence or absence of complement. Serum from sows containing antibodies against TGEV peplomer and matrix proteins had slightly higher VN titers in the presence of complement than in the absence of complement. High concentrations of complement from swine serum (128 U) had little effect on the infectivity of TGEV for swine testes cells, whereas 32 U of complement from rabbit serum and 64 U of complement from guinea pig serum were able to neutralize virulent and attenuated TGEV in the absence of known antibodies for TGEV. Complement (less than or equal to 8 U) from any source did not decrease the infectivity of TGEV by greater than 0.5 log10 units.

Diagnosis of porcine and bovine enteric coronavirus infections using cloned cDNA probes.

Molecular clones representing the first 2,000 bases from the 3' end of the porcine transmissible gastroenteritis coronavirus genome and the first 2,160 bases from the 3' end of the bovine enteric coronavirus genome were used in dot blot hybridization assays to detect viral RNA from cell culture and from fecal specimens. In each case, the cloned DNA represents approximately 10% of the genome. The cloned sequence for each virus encompasses the 3' noncoding region, the nucleocapsid protein gene, and a large portion of the matrix protein gene. 32P-labeled cDNA probes prepared from these clones detected as little as 25 pg of RNA from the parental virus but did not detect RNA from the nonparental virus even when amounts of up to 10 ng per dot were used. This specificity reflects the antigenic diversity between these two coronaviruses. The hybridization assay could also detect coronaviruses antigenically closely related to the parental virus but not coronaviruses belonging to an antigenically unrelated subgroup. Dot blot hybridization for transmissible gastroenteritis coronavirus diagnosis was compared with the routine procedures of virus isolation and electron microscopy as a diagnostic test.

Nucleotide sequence of the porcine transmissible gastroenteritis coronavirus matrix protein gene.

cDNA clones mapping within the first 2601 bases of the 3' end of the TGEV genome were sequenced completely or in part by the method of Maxam and Gilbert and open reading frames were examined. One reading frame yielding a protein having properties of the matrix (M) protein was identified. It is positioned at the immediate 5' side of the nucleocapsid (N) gene but is separated by an intergenic region of 12 bases. The deduced M protein is comprised of 262 amino acids, has a molecular weight of 29,544, is moderately hydrophobic, and has an amino acid sequence homology of approximately 36% with the mouse hepatitis coronavirus, 37% with the bovine enteric coronavirus, and 28% with the avian infectious bronchitis virus. Judging from an alignment with MHV and IBV proteins, the amino terminus of the TGEV M protein extends 54 amino acids from the virion envelope which compares with 26 for MHV and 21 for IBV.

Complement-dependent neutralization of transmissible gastroenteritis virus by monoclonal antibodies.

Monoclonal antibodies (MAb) to each of the 3 major structural proteins of transmissible gastroenteritis virus (TGEV) of swine were compared as to their virus neutralizing activity in the presence or absence of guinea pig complement. MAbs to the peplomer protein had neutralizing activity for TGEV with or without complement and the titers were similar in either case. MAbs to the matrix protein had neutralizing activity for TGEV only in the presence of complement. Antibodies to the nucleocapsid protein were without neutralizing activity with or without complement. High concentrations of guinea pig complement, but not swine complement, had neutralizing activity for TGEV even in the absence of any known TGEV antibodies.

Sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene.

The 3' end of the 20-kb genome of the Purdue strain of porcine transmissible gastroenteritis coronavirus (TGEV) was copied into cDNA after priming with oligo(dT) and the double-stranded product was cloned into the PstI site of the pUC9 vector. One clone of 2.0-kb contained part of the poly(A) tail and was sequenced in its entirety using the chemical method of Maxam and Gilbert. Another clone of 0.7 kb also contained part of the poly(A) tail and was sequenced in part to confirm the primary structure of the most 3' end of the genome. Two potential, nonoverlapping genes were identified within the 3'-terminal 1663-base sequence from an examination of open reading frames. The first gene encodes a 382-amino acid protein of 43,426 mol wt, that is the apparent nucleocapsid protein on the basis of size, chemical properties, and amino acid sequence homology with other coronavirus nucleocapsid proteins. It is flanked on its 5' side by at least part of the matrix protein gene. The second encodes a hypothetical 78-amino acid protein of 9101 mol wt that is hydrophobic at both ends. A 3'-proximal noncoding sequence of 276 bases was also determined and a conserved stretch of 9 nucleotides near the poly(A) tail was found to be common among TGEV, the mouse hepatitis coronavirus, and the avian infectious bronchitis coronavirus.

Carbon dioxide metabolism by Capnocytophaga ochracea: identification, characterization, and regulation of a phosphoenolpyruvate carboxykinase.

Cell suspensions of Capnocytophaga ochracea incorporated [14C]NaHCO3 into a major four-carbon fermentation product, succinate, and cell-free extracts from this organism contained high levels of phosphoenolpyruvate carboxykinase (PEPCK). PEPCK is the major, if not the only, CO2(HCO-3)-fixing enzyme in C. ochracea since cell-free extracts were devoid of pyruvate-dependent and other phosphoenolpyruvate (PEP)-dependent CO2(HCO-3)-fixing enzymes. The reaction products of the enzyme, which was partially purified by diethylaminoethylcellulose column chromatography, were identified as adenosine 5'-triphosphate (ATP) and oxalacetate. The enzyme showed maximum activity when manganese (Mn2+) was the divalent cation in the incubation mixture, and it had an absolute requirement for the nucleoside 5-'diphosphate adenosine 5'-diphosphate (ADP). PEPCK showed a sigmoidal kinetic response to the Mn2+ concentration and homotropic interactions in its kinetic responses to each of its three substrates PEP, ADP, and CO2(HCO-3). The (S)0.5v values for Mn2+, PEP, ADP, and CO2(CHO-3) were approximately 0.08, 0.3, 0.1, and 10 mM, respectively, and Hill coefficients for these same ligands were 2.60, 1.7, 1.9, and 3.0, respectively. In addition, C. ochracea PEPCK is under metabolic control by the nucleoside -5'-triphosphate ATP, and it also showed a sigmoidal kinetic response to this allosteric effector. The Hill coefficient for ATP was 2.70.