The effects of polymorphisms in the DGAT1, leptin and growth hormone receptor gene loci on body energy, blood metabolic and reproductive traits of Holstein cows.

The objective of this study was to examine the impact of polymorphisms in the acyl-CoA:diacylglycerol acyltransferase (DGAT1), leptin and growth hormone receptor genes on body energy (body condition score, total body energy content and cumulative effective energy balance) and blood metabolic traits (levels of beta-hydroxybutyrate, glucose and non-esterified fatty acids), measured once before the first calving and then repeatedly throughout first lactation in 497 Holstein cows. The influence of the same polymorphisms on cow reproductive performance and health during the first and second lactations was also assessed. Several reproductive traits were considered including interval, conception and insemination traits, as well as incidence of metritis and reproductive problems. Genotyping was performed using PCR-RFLP (DGAT1, leptin) or allele-specific PCR (growth hormone receptor). For each locus, the effect of allele substitution on body energy and blood metabolic traits was estimated using random regression models. The same effect on reproductive traits was assessed with single-trait mixed linear models. Significant (P<0.05) effects on specific reproductive traits were observed. DGAT1 and growth hormone receptor alleles responsible for significant increases in milk production were found to have an adverse effect on reproduction, while the leptin allele responsible for significant increase in milk production was linked to marginally increased metritis frequency. Furthermore, the three studied loci were also found to significantly (P<0.05) affect certain body energy and blood metabolic traits. Several associations are published for the first time, but these should be confirmed by other investigators before the polymorphisms are used in gene-assisted selection.

Mycotic encephalitis and nephritis in a dog due to infection with Cladosporium cladosporioides.

The dematiaceous fungus Cladosporium cladosporioides is a widely distributed saprophyte that is reported to occasionally infect the lung, skin, eye and brain of humans. This report describes a German shepherd dog with granulomatous encephalitis and nephritis due to C. cladosporioides infection. Although the fungal organisms appeared non-pigmented in haematoxylin and eosin stained sections, they were readily identified with histochemical stains. Semi-nested polymerase chain reaction using universal fungal primers amplified fungal DNA from fixed tissue that had identity to that of C. cladosporioides on sequencing.

Primary gastric choriocarcinoma in a dog.

Primary gastric choriocarcinoma (PGC) is a rare neoplasm to date only reported in humans. This report describes a canine gastric tumour with microscopical, histochemical and immunohistochemical features of PGC. The tumour diffusely infiltrated the submucosa and muscularis propria of the pylorus and anterior duodenum, and metastasized to the gastric lymph node. Immunohistochemically, the neoplastic cells displayed aberrant expression of beta-catenin and E-cadherin, but normal expression of the adenomatous polyposis coli (APC) protein. Expression of the oncogenes c-myc and Ras was also increased. These observations suggest that this canine PGC had synchronous activation of both the Wnt/beta-catenin and Ras signalling pathways of carcinogenesis.

A deletion in the R region of long terminal repeats in small ruminant lentiviruses is associated with decreased pathology in the lung.

A particular variant of the maedi visna virus (MVV) that although present in blood causes no clinical signs in infected sheep has been described. This variant carries a 13-14 nucleotide deletion in the R region of the proviral long terminal repeats. The hypothesis that this specific deletion may be associated with low pathogenicity has been investigated by comparing the distribution of proviral sequences, the histopathological lesions and the expression of viral proteins in the brain, lungs and udders of sheep naturally infected with viral strains carrying the deletion. Provirus could be demonstrated in most of the tissues examined from sheep infected with either type of virus, and the tissue-derived virus carried the typical deletion in the study flock animals. Histopathological analysis revealed that the lungs were significantly less affected in the animals infected with virus carrying the deletion. Concomitantly, viral expression was significantly reduced in the lungs of these animals. The findings suggest that the reduced pathogenicity of MVV with the specific deletion in the R region is not due to a restriction in the availability of specific tissues to infection, but is associated with a reduced capacity for viral expression in the lungs.

Detection of maedi-visna virus in the liver and heart of naturally infected sheep.

Maedi-visna virus (MVV) in sheep, which infects mainly cells of the monocyte/macrophage lineage, produces changes in the lung, mammary gland, brain and joints. In this study, however, the liver and heart of six naturally infected sheep were examined for the presence of the virus. MVV proviral DNA was demonstrated by polymerase chain reaction (PCR) analysis, and immunohistochemical examination revealed viral antigens in the cytoplasm of hepatocytes and cardiac myocytes. Although histopathological examination showed mild to moderate, chronic lymphocytic cholangiohepatitis and myocarditis and the presence of small lymphoid aggregates, the typical maedi lymphoproliferative lesions (lymphoid follicle-like structures of considerable size with germinal centres) were not seen in the liver and heart. These novel findings suggest that, although the macrophage is the main cell for productive viral replication, the liver and heart represent additional MVV targets.

The reliability of isokinetic and isometric leg strength measures among individuals with symptoms of mild osteoarthritis.

AIM: The aim of the study is to evaluate the test-retest reliability of measures of isokinetic and isometric leg strength and joint function among individuals exhibiting symptoms of mild osteoarthritis. Reliable procedures are needed to assess the effectiveness of an intervention on osteoarthritic symptoms. METHODS: Test-retest reliability of two leg strength protocols was assessed using the intraclass correlation coefficient (R). Testing was completed on two occasions separated by 7 days. Eighteen subjects (9 male and 9 female; 54.1+/-11 years) completed an isokinetic testing trial, which consisted of a set of 5 maximal repetitions of the quadriceps and hamstrings at 60 deg/s followed by a set of 15 maximal contractions at 180 deg/s with a 2-min rest between sets and an isometric testing trial, which consist of 3 maximal contractions of the quadriceps for 6 s with a 30-s rest between contractions at 30, 45, and 80 degrees of knee flexion for a total of 9 isometric contractions. A 90-s rest occurred between angles. RESULTS: Most of the isokinetic variables showed moderate to high intraclass reliability (ICC). Two of the calculated isokinetic variables (work fatigue at 180 degrees /s for extension and for flexion) showed low intraclass reliability (ICC=0.78, resp. ICC=0.6). All calculated ICC values of the isometric variables were moderate to high. CONCLUSIONS: Test-retest reliability of isokinetic and isometric leg strength was high, allowing the intervention protocol to monitor changes in leg strength and joint function among those exhibiting symptoms of mild osteoarthritis.

Detection of maedi-visna virus in the kidneys of naturally infected sheep.

Infections with maedi-visna virus (MVV) cause progressive inflammation in different organs, mainly the lung, mammary gland, brain and joints. The aim of the present study was to investigate whether the kidney represents a viral target in natural MVV infection. For this, kidney samples from 13 sheep naturally infected with MVV were examined by histology, polymerase chain reaction (PCR), and immunohistochemistry. The kidneys of nine animals showed membranoproliferative glomerulonephritis and interstitial nephritis. The inflammatory infiltrate consisted of lymphocytes, plasma cells and macrophages. Interestingly, lymphoid follicles resembling those known to occur in other MVV-infected tissues were observed. Lung tissue from the same animals had typical MVV lesions, such as lymphofollicular hyperplasia and interstitial pneumonia. Maedi-visna proviral DNA sequences were detected in renal and lung tissue samples from these nine sheep by PCR, and the specificity of the amplified products was further verified by DNA sequencing. Moreover, MVV-specific immunohistochemistry revealed viral antigen in affected kidneys and lungs. These results suggest that the kidney may be a common target in natural MVV infection, and raise the issue of the role of this organ in the disease.

A novel deletion in the LTR region of a Greek small ruminant lentivirus may be associated with low pathogenicity.

Greek small ruminant lentivirus (SRLV) strains remain relatively uncharacterized at the molecular level, despite the fact that lentiviral diseases of small ruminants are known to be widespread in the country. In the present study, we investigated the sequence diversity of the LTR region in Greek SRLV strains from sheep with and without disease symptoms, since sequence differences within this genomic area have been shown to lead to SRLVs with distinct replication rates. The AP-4 and AML (vis) motifs and the TATA-box were highly conserved among Greek strains, whereas the two AP-1 sites exhibited some substitutions. Pairwise comparisons with reference strains revealed that Greek LTR sequences were closer to the ovine strains (25.7% average divergence) rather than the caprine strain CAEV (59.1% average divergence). The most striking difference observed between the two groups of animals was a 13-14 nucleotide deletion in the strains obtained from the asymptomatic sheep. The deletion was located within the R region of LTR, which was also found to be much less homologous (39.6% average divergence) than the U3 and U5. Taken together, our data suggest that the R region of LTR may be involved in virus transcriptional activation. Furthermore, a specific deletion within this region may, at least in part, be associated with low pathogenicity of some SRLV strains.

First partial characterisation of small ruminant lentiviruses from Greece.

Small ruminant lentivirus (SRLV) infections are widespread in Greece, but SRLVs have never been isolated and characterized. In this study, we present the sequence of a 574-nucleotide (191-amino acid) region of the gag gene of SRLV strains from four sheep and one goat from a single geographic area of Greece. All five sequences appeared to be closely related at both nucleotide (2.1-14.2% variation) and deduced amino acid (1.6-4.2% variation) level. Greek SRLV strains were closer to ovine prototypic strains (average divergence 16.8%) than to the caprine strain CAEV-Co (21% divergence). By amino acid composition, the Greek SRLVs were on the average more than twice as distant from CAEV-Co as from other ovine strains. Phylogenetic analysis suggested that Greek strains segregate into a unique group, separate from, but related to, other ovine prototype sequences.

Quantitative RT-PCR luminometric hybridization assay with an RNA internal standard for cytokeratin-19 mRNA in peripheral blood of patients with breast cancer.

OBJECTIVES: To develop a highly sensitive quantitative RT-PCR hybridization assay for the determination of CK-19 mRNA in peripheral blood of patients with breast cancer. PATIENTS AND METHODS: Quantification of CK-19 mRNA was based on the coamplification of CK-19 mRNA with a recombinant CK-19 RNA internal standard (CK-19 RNA-IS) through RT-PCR. The biotinylated amplification products were immobilized on steptavidin coated wells, hybridized with digoxigenin labeled probes and determined through an antidigoxigenin antibody conjugated to alkaline phosphatase by luminometric detection. The developed luminometric hybridization assay was validated with samples containing total RNA of known amounts from CK-19 expressing cells (MCF-7) in the presence of 1 microg total RNA isolated from peripheral blood mononuclear cells (PBMC) of healthy controls and a constant amount of CK-19 RNA-IS. The method was applied for the quantitative determination of CK-19 mRNA in the peripheral blood of 26 healthy volunteers, 14 patients with stage IV breast cancer and 37 patients with stage I/II breast cancer before chemotherapy. RESULTS: Luminescence ratios for CK-19 mRNA and CK-19 RNA-IS were linearly related to the number of MCF-7 cells within the range of 1 to 2000 cells. The overall reproducibility of the assay (between-run) varied between 8.9% and 13.4%. The method can clearly detect CK-19 mRNA from 1 MCF-7 cell in the presence of 10(6) normal PBMC and is highly specific as none of the 26 healthy controls tested had detectable CK-19 mRNA levels, while 10 out of 14 (71.4%) and 9 out of 37 (24.3%) patients with stage IV and stage I/II breast cancer, respectively, were tested positive. CONCLUSION: The developed quantitative RT-PCR hybridization assay for CK-19 is reproducible, highly sensitive and specific, and can be used for a large-scale prospective evaluation of clinical samples.

Prevalence of genetic mutations that predispose to thrombophilia in a Greek Cypriot population.

Several hereditary disorders, particularly those affecting the physiological anticoagulation systems, have been well established as risk factors for venous thromboembolism. In the present study, we investigated the prevalence of the following thrombogenic mutations in a Greek-Cypriot population: the G1691 factor V Leiden mutation, the G20210A mutation in the prothrombin gene, and the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR). All three variants have been documented to be significant risk factors for various cardiovascular conditions. Ninety unrelated subjects were screened. For the Leiden mutation, 11 subjects (12.2%) were heterozygous and one (1.1%) was homozygous. Seven subjects (7.8%) were heterozygous for the G20210A variant in prothrombin; no homozygotes were identified. The C677T mutation in MTHFR was found in 40 individuals in the heterozygous state (44.4%), and in 16 individuals in the homozygous state (17.8%). These data demonstrate that Greek-Cypriots have an increased frequency of thrombogenic mutations, and suggest that screening for these mutations should be seriously considered, especially when surgery or pregnancy is planned. This is the first study for the frequency of mutations in risk factors that predispose to thrombophilia on the island of Cyprus.

p53 gene mutation, tumor p53 protein overexpression, and serum p53 autoantibody generation in patients with breast cancer.

OBJECTIVES: Autoantibodies against the p53 tumor suppressor protein have been detected in the serum of a proportion of patients with various cancers. The generation of such antibodies has been proposed to be due to either tumor p53 protein accumulation or to the type of p53 gene mutation. These hypotheses are examined in the present study. DESIGN AND METHODS: Using immunofluorometric assays, we studied 195 patients with primary breast cancer for the presence of p53 antibodies in serum and p53 protein accumulation in the corresponding tumor. Seventeen patients (9%) were p53 antibody-positive and 77 (40%) overexpressed p53. Ten of the 17 p53 antibody-positive patients had tumor p53 accumulation and 7 were negative for p53. Statistical analysis revealed a weak association between the presence of p53 antibodies and p53 protein accumulation (p = 0.05). Direct DNA sequencing of exons 1-11 of the p53 gene was performed for 16 p53 antibody-positive and 16 p53 antibody-negative patients. RESULTS: Five of the seropositive and eight of the seronegative patients had a p53 gene mutation. Four of the five mutations in the p53 antibody-positive patients affected a Tyr residue, whereas none of the gene abnormalities in the seronegative patients had such an effect. CONCLUSIONS: We conclude that p53 antibodies tend to develop in patients with tumor p53 accumulation, but p53 accumulation is neither sufficient nor necessary for the generation of the immune response. Further, p53 antibody-positive patients do not have higher frequency of p53 gene mutations than p53 antibody-negative patients, but the former patient group is associated with a Tyr substitution in the protein product.

Characterization of the BRCA1-like immunoreactivity of human seminal plasma.

OBJECTIVES: The subcellular localization of the breast cancer susceptibility gene product BRCA1 has been controversial. Discrepant results have been reported during the past 3 years, partially because of the unavailability of highly specific reagents for BRCA1 protein. Our objective was to characterize the BRCA1-like immunoreactivity that is detected in human seminal plasma by using monoclonal and polyclonal antibodies that are supposedly specific for BRCA1 protein. METHODS: We used immunologic, chromatographic, and protein sequencing techniques to detect the immunoreactivity of BRCA1 in seminal plasma and to purify and partially identify the immunoreactive species. RESULTS: We present data indicating that two BRCA1 antibodies, SG-11 and D-20, which were thought to be free of cross-reactivities, strongly interact with proteins present in human seminal plasma. This cross-reactivity is detectable even at seminal plasma dilutions as high as 10(6)-fold, and it is effectively blocked by peptides that capture the binding site of either SG-11 or D-20 antibodies. Purification and characterization of the immunoreactive compound revealed that this consists of a macromolecular complex that contains semenogelins. The D-20 polyclonal antibody was found to cross-react with purified semenogelins I and II; the SG-11 monoclonal antibody appeared to recognize a component of the macromolecular complex that was not semenogelin. CONCLUSIONS: Our data demonstrate that the BRCA1 antibodies SG-11 and D-20 strongly interact with seminal plasma proteins and are not highly specific for BRCA1 protein. It is thus suggested that BRCA1 antibodies should be used with caution until reagents free of interference are developed and evaluated. In light of the very high cross-reactivity of the two antibodies with seminal plasma proteins, we recommend that new BRCA1 antibodies should be examined for cross-reactivity with seminal plasma proteins to verify specificity.

Immunofluorometric assay of p53 protein versus sequencing of p53 exons 5 to 9 for the detection of p53 abnormalities in ovarian carcinoma.

p53 alteration, detected as mutation of the p53 gene or as accumulation of mutant p53 protein, is a common feature of most malignancies, including ovarian carcinoma, and may identify patients with unfavorable prognosis and resistance to chemotherapy. Tumor tissues from 55 patients with well or poorly differentiated (grades 1 or 3) primary epithelial ovarian carcinoma were assessed both for p53 protein overexpression by a sensitive time-resolved immunofluorometric assay employing DO-1 and CM-1 antibodies, and for genetic p53 abnormalities by direct sequencing of PCR-amplified exons 5 to 9. Sixteen p53 mutations (29%), including 3 deletions causing frameshifts as well as one nonsense and 12 missense point mutations were found in all exons except exon 9. Overexpression of p53 protein, defined as a concentration exceeding the 75th percentile, was found in 15 cases (27%), 10 of which had missense mutations (P < 0.01). Tumors with nonsense and frameshift mutations were p53-negative by immunoassay. Both p53 mutation (P = 0.04) and p53 protein accumulation (P < 0.01) were associated with stage III-IV disease, while p53 mutation was more closely related to grade 3 lesions (P = 0.04) and serous histotype (P = 0.01). These results indicate that p53 protein accumulation correlates well with missense point mutation in carcinoma of the ovary and, together with other evidence that p53 abnormality may be prognostic of outcome in this disease, suggest that the immunoassay of p53 protein may have clinical value.

Identification of deletions and insertions in the p53 gene using multiplex PCR and high-resolution fragment analysis: application to breast and ovarian tumors.

We have developed a simple and highly efficient method to detect deletions and insertions in the p53 gene. All 11 exons of the p53 gene were amplified along with a control sequence in four multiplex PCR reactions in the presence of fluorescein-labeled primers. The PCR products were resolved on an automated sequencing gel and the DNA fragments were detected by fluorescence. Using this method, we screened 7 DNA specimens from ovarian tumors, 19 from breast tumors, and 26 from normal breast tissues. No abnormality was found in any of the DNA samples extracted from the normal tissues. A 19 base pair deletion in exon 5 of the p53 gene was detected in one ovarian tumor. Insertions were identified in two breast and two ovarian tumors. The insertions were identical in 3 of these tumors and consisted of a 16 bp repeat within intron 3 of the p53 gene. It appears that the insertion within intron 3 may represent a hot spot for duplication of the normal sequence at that site.

Rapid sequencing of the p53 gene with a new automated DNA sequencer.

p53 is the most commonly mutated gene in human cancers. Approximately 90% of the p53 gene mutations are localized between domains encoding exons 5 to 8. Sequencing methods currently available are tedious and time-consuming and are not suitable for routine laboratory testing. In an effort to identify a simple and rapid sequencing method, we analyzed 16 preselected breast tumors and 18 preselected ovarian tumors, using a newly developed automated DNA sequencer. p53 gene mutations had been previously identified in these tumors, using a conventional automated sequencing procedure. Exons 5 to 8 were amplified by PCR, and the PCR products were subsequently subjected to cycle sequencing with the Sanger chain termination method, using Cy5.5-labeled primers. The sequencing mixture was then resolved on a newly developed automated DNA sequencer that can sequence approximately 300 bases of DNA in 30 min. Of these 16 breast tumors, two had mutations in exon 5, four in exon 6, three in exon 7, and three in exon 8. Of the 18 ovarian tumors, two had mutations in exon 5, five in exon 6, two in exon 7, and three in exon 8. In all cases, we identified the same mutations by both the new and the conventional sequencing procedures. Most mutations affected an arginine codon. These data demonstrate that the new method has the capability to provide accurate sequencing information in a fraction of the time and labor in comparison with current automated sequencing techniques. When such procedures are used, DNA sequencing may become a routine tool for identifying clinically important mutations for diagnosis and prognosis of patients with genetic, malignant, infectious, and other diseases.

Exon 5 of the p53 gene is a target for deletions in ovarian cancer.

Missense point mutations, leading to inactivation of the p53 tumor suppressor gene product, are currently the most frequent alterations in human cancer. Little, however, is known about small intragenic deletions or insertions occurring in this locus of chromosome 17. We have analyzed 56 primary ovarian tumors for the presence of such abnormalities. The analysis was based on multiplex PCR amplification of exons 1 through 11 of the p53 gene and fragment analysis of the generated PCR products. Mutations were detected in 14% (8 of 56) of the tumors. Deletions were much more prevalent than insertions (seven vs one). Six of the deletions and the insertion affected exon 5, and the other deletion was in exon 7. Two deletions and the insertion did not disrupt the reading frame; the protein product was expressed in the tumor at high concentrations in all three cases. The other five deletions generated a frameshift, which is predicted to result in the production of a truncated protein product. In the case of the deletions, a 2-5-bp repeat was present close to the detected deletion, whereas the insertion duplicated the sequence immediately upstream of the insertion site. Overall our findings indicate that small intragenic p53 deletions/insertions are not rare events in ovarian cancer, and that p53 exon 5 is the target in the vast majority (88%) of the cases.

Frequency of expression of prostate-specific antigen mRNA in lung tumors.

The presence of prostate-specific antigen (PSA) protein and messenger RNA (mRNA) was studied in 52 primary lung tumor tissues. The PSA protein was detected more frequently and at higher levels in lung tumor extracts from men. The levels of PSA protein in tumor extracts correlated with preoperative and postoperative serum PSA levels, suggesting a possible contamination of the tumor extracts with PSA from residual blood in the tumor vasculature. The PSA mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot hybridization in 24 (68%) of 35 tumors from men, in 9 (53%) of 17 tumors from women, and in 5 (71%) of 7 adjacent normal lung tissue specimens. The levels of PSA protein did not associate with patient age, the tumor stage, grade, or histologic type, or the nodal status. Similarly, PSA mRNA was not associated with any clinicopathologic variables, but squamous cell carcinomas, especially in men, were more frequently positive. A by-product of the RT-PCR procedure was cloned and sequenced and found to be a 450-base pair sequence not previously deposited in the data bank. We conclude that PSA mRNA and protein frequently can be detected in lung tumors and normal tissues from men and women but at levels much lower than those seen in breast carcinomas in women. The significance of the new 450-base pair sequence remains to be determined.