Expression of murine sarcoma virus genes in uninfected rat cells subjected to anaerobic sress.

Exposure of uninfected rat cells in tissue culture to anaerobic culture conditions induces transcription of RNA corresponding to the two principal constituents of rat-derived type-C sarcoma virus genomes: (i) those specific rat cell sequences present in the Kirsten and Harvey murine sarcoma virus genomes, and (ii) an endogenous type-C rat leukemia virus.

A cancer-associated lactate dehydrogenase is expressed in normal retina.

An unusual isozyme of lactate dehydrogenase, lactate dehydrogenase k, is found in high concentrations in cultured cells transformed by the Kirsten murine sarcoma virus and in many human cancer tissues. In experiments described here high levels of a lactate dehydrogenase k activity were detected in extracts of normal rodent retina. This activity had the same key properties as the human tumor isozyme, namely, a highly cathodic electrophoretic mobility and inhibition of enzymatic activity by oxygen and 5',5'-dipurinenucleoside tetraphosphates. Expression of this activity in the retina may be related to the high aerobic glycolysis characteristic of the retina, a metabolic feature shared with many tumors.

Real-time polymerase chain reaction (PCR) assays for the specific detection and quantification of seven Eimeria species that cause coccidiosis in chickens.

Coccidiosis of chickens is an economically important disease caused by infection with species of Eimeria. The oocysts of some of the seven recognized species are difficult to distinguish morphologically and for this reason diagnostic laboratories are increasingly utilizing DNA-based technologies for the specific identification of Eimeria. The real-time PCR provides both sensitivity and speed for the analysis of DNA samples, and the approach has the capability of quantifying DNA. Together with a protocol for the extraction of DNA directly from faecal samples, real-time PCR assays have been established for the detection and quantification of seven species of Eimeria that infect chickens in Australia. The assays target one genetic marker, the second internal transcribed spacer of nuclear ribosomal DNA (ITS-2), use TaqMan MGB technology with species-specific probes, and can be multiplexed in pairs such that the seven species of Eimeria can be screened in four reaction tubes. A test screen of commercial flocks identified more Eimeria-infected chickens than were detected by coproscopic examination for oocysts. These molecular assays can also be used for the quality control of mixed-species vaccines. The ability to multiplex the assays makes them particularly practical for screening samples from chickens with mixed-species infections where the relative abundance of each Eimeria species present is required.

The distribution of Fasciola hepatica in Queensland, Australia, and the potential impact of introduced snail intermediate hosts.

A survey was conducted to establish the distribution of the liver fluke, Fasciola hepatica, in the state of Queensland, Australia, and to evaluate the impact of the introduced snail intermediate hosts, Pseudosuccinia columella and Austropeplea viridis. Serum samples from a total of 5103 homebred cattle in 142 beef herds distributed throughout the state and 523 pooled milk samples from dairy herds from the state's major dairying regions were tested for antibodies to F. hepatica by ELISA. Snails were collected on infected properties around the limits of the F. hepatica distribution. F. hepatica infection was detected in 44 dairy herds and two beef herds. The distribution of infected herds indicates that F. hepatica is established only in southeast Queensland. The distribution there was patchy but the parasite was more widespread than suggested by an earlier survey. The predominant intermediate host species found along the northern limit of the distribution was P. columella. We conclude that the introduction of P. columella and A. viridis has not yet had a major impact on the distribution of F. hepatica in Queensland. However, the presence of P. columella, which is much more adaptable to tropical habitats than the native intermediate host, Austropeplea tomentosa, at the northern limit of the F. hepatica distribution suggests that there is potential for the parasite to expand its range.

p53 tumor suppressor gene status and the degree of genomic instability in sporadic colorectal cancers.

BACKGROUND: Genomic instability reflects the propensity and the susceptibility of the genome to acquire multiple alterations and, in turn, is believed to be a driving force behind multistep carcinogenesis. Although the molecular basis of genomic instability in sporadic colorectal cancers remains largely a mystery, mutation of the p53 tumor suppressor gene (also known as TP53) has been proposed to play an integral role in this process. However, a dilemma exists in that p53 mutation appears to be a late event in the progression of sporadic colorectal tumors, whereas genomic instability, serving as a facilitator of tumor progression, is envisioned as occurring early in this process. PURPOSE: We evaluated the relationship between p53 mutation and the major form of genomic instability in sporadic colorectal tumors, namely, that involving DNA breakage, which leads to chromosomal translocations, insertions, deletions, and gene amplification. METHODS: Fifty-eight sporadic colorectal tumors that had been previously evaluated for genomic instability were analyzed for p53 mutations. These tumors were from consecutively diagnosed patients. Genomic instability was quantified by use of inter-simple sequence repeat polymerase chain reaction analysis that employed (CA)8RG and (CA)8RY primers (R = purine [A or G]; Y = pyrimidine [C or T]); a genomic instability index (a measure of the number of alterations in tumor DNA in comparison with normal DNA, expressed as a percent) was calculated for each tumor. Mutation of the p53 gene in exons 5-9 was determined by use of single-strand conformational polymorphism-polymerase chain reaction analysis and DNA sequencing. Chi-squared analysis was used to determine the statistical significance of differences between groups of tumors. Reported P values are two-sided. RESULTS: p53 mutations were identified in 29 (50%) of the 58 tumors. The median genomic instability index value was 3.3%. Nineteen (65.5%) of the 29 tumors with p53 mutations had genomic instability indices that were less than the median value (range, 0%-2.6%); the remaining 10 (34.5%) tumors had genomic instability indices that were greater than the median (range, 3.9%-13.0%). Eleven (37.9%) of the 29 tumors with wild-type p53 genes had genomic instability indices that were less than the median value (range, 0%-2.6%), whereas the remaining 18 tumors had genomic instability indices above the median (range, 3.9%-11.7%). There was a statistically significant association between a lesser degree of genomic instability and the presence of p53 mutations (P = .032). CONCLUSIONS AND IMPLICATIONS: Tumors with no or minimal evidence of genomic instability are more likely to harbor p53 mutations than tumors with evidence of substantial genomic instability. p53 mutations play an important role in the development of cancers but do not appear to initiate or promote genomic instability in sporadic colorectal tumors.

Selection and characterisation of two attenuated vaccine lines of Eimeria tenella in Australia.

OBJECTIVE: To attenuate two strains of Eimeria tenella by selecting for precocious development and evaluate the strains in characterisation trials and by field evaluation, to choose one precocious line for incorporation into an Australian live coccidiosis vaccine for poultry. DESIGN: Two strains from non-commercial flocks were passaged through chickens while selecting for precocious development. Each strain was characterised for drug sensitivity, pathogenicity, protection against homologous and heterologous challenge, and oocyst output in replicated experiments in which the experimental unit was a cage of three birds. Oocyst output and/or body weight gain data collected over a 10 to 12 day period following final inoculation were measured. Feed conversion ratios were also calculated where possible. RESULTS: Fifteen passages resulted in prepatent periods reduced by 24 h for the Redlands strain (from 144 h to 120 h) and 23 h for the Darryl strain (from 139 h to 116 h). Characterisation trials demonstrated that each precocious line was significantly less pathogenic than its parent strain and each effectively induced immunity that protected chickens against challenge with both the parent strain and other virulent field strains. Both lines had oocyst outputs that, although significantly reduced relative to the parent strains, remained sufficiently high for commercial vaccine production, and both showed susceptibility to coccidiostats. CONCLUSION: Two attenuated lines have been produced that exhibit the appropriate characteristics for use in an Australian live coccidiosis vaccine.

Fluctuation of a cancer-associated lactate dehydrogenase during S phase of the cell cycle in HeLa cells.

An unusual isozyme of lactate dehydrogenase, originally detected in Kirsten sarcoma virus-infected cells and later shown to be induced in normal mammalian cells by anaerobic shock, has also been reported at elevated levels in several human carcinomas. This enzyme is subject to inhibition by guanosine triphosphate and by the dinucleosides 5',5"'-diadenosine tetraphosphate and 5',5"'-diguanosine tetraphosphate (4). Fluctuation of the activity of this enzyme in soluble extracts of synchronized HeLa cells suggests the enzyme may be linked to DNA synthesis. The lactate dehydrogenase K activity increased in early S phase and then decreased to nearly undetectable levels during the period of most active DNA synthesis. This was observed in cells synchronized by thymidine excess or by aphidicolin, an inhibitor of DNA polymerase alpha.

Expression of an unusual isozyme of lactate dehydrogenase in the serum of cancer patients and comparison with carcinoembryonic antigen.

Two studies reported here demonstrate a statistically significant association between metastatic cancer and the appearance of the k isozyme of lactate dehydrogenase in serum of affected patients. The first study included 190 coded samples from three types of cancer patients and matched controls; the second included 155 preoperative and 200 postoperative colorectal cancer patients. In the second, plasma carcinoembryonic antigen was compared with serum k isozyme of lactate dehydrogenase as an indicator of the presence of metastatic cancer. This comparison showed that both markers were independently useful for assessing patient status and predicted that a combination of the two should be a better discriminator for the presence of metastases than either marker alone.

Anoxia-inducible endonuclease activity as a potential basis of the genomic instability of cancer cells.

Normal rat fibroblasts exhibit a staged response to anoxia which in several respects parallels processes activated in malignant tumor cells. We describe here a new element of the anoxic response, the induction by anoxia of a sequestered endonuclease activity. Such activity is elevated approximately 3-fold within anoxic fibroblasts and during Hirt DNA isolation is able to digest chromatin to produce a nucleosomal ladder. However, DNA is not measurably affected within intact cells, and cells retain complete viability as the endonuclease is induced. The anoxia-inducible endonuclease acts without specificity for DNA sequence. Trace leakage of this endonuclease into the nucleus has obvious potential to underlie the known propensity of anoxic cells to undergo amplification and may be associated with the break-related genomic instability of cancer cells.

An unusual oxygen-sensitive lactate dehydrogenase isozyme associated with Kirsten murine sarcoma virus in human serum.

An unusual isozyme of lactate dehydrogenase, lactate dehydrogenase associated with Kirsten murine sarcoma virus (LDHk) was found in the sera of many patients with malignant tumors, while the sera of healthy persons had little or no such activity. This isozyme was detectable only when assayed in a nitrogen atmosphere, and its activity showed little or no relationship to the total lactate dehydrogenase activity as measured by a standard clinical assay. The activity of serum LDHk appeared to be correlated with the presence of known metastases. Increased serum LDHk appeared in a wide variety of patients with cancer, although it appeared to be more common in certain types of cancer. Increased serum LDHk activity was also found in the sera of some patients with nonmalignant disease. The activity of serum LDHk may be useful to monitor recurrence or response to therapy in certain types of cancer.

An anoxia inducible endonuclease and enhanced DNA breakage as contributors to genomic instability in cancer.

Fischer rat embryo fibroblasts subjected to temporary anoxia followed by an aerobic recovery period show genomic instability in the form of highly elevated CAD gene amplification rates. As revealed by flow cytometric analysis this is associated with DNA breakage in vivo, followed by repair during the recovery period. Such genomic instability parallels expression of a M(r) 29,000/31,000 endonuclease; this enzyme requires no added divalent metal ion and has a pH optimum of about 6.5. The same endonuclease was found to be expressed within healing wounds and in four of ten human colorectal cancers but was not seen in eight normal colorectal tissue samples. Our results indicate that DNA breakage resulting from endogenous endonuclease activity can have a substantial effect in modulating genomic instability.

Intrachromosomal genomic instability in human sporadic colorectal cancer measured by genome-wide allelotyping and inter-(simple sequence repeat) PCR.

We have used genome-wide allelotyping with 348 polymorphic autosomal markers spaced, on average, 10 cM apart to quantitate the extent of intrachromosomal instability in 59 human sporadic colorectal carcinomas. We have compared instability measured by this method with that measured by inter-(simple sequence repeat) PCR and microsatellite instability assays. Instability quantitated by fractional allelic loss rates was found to be independent of that detected by microsatellite instability analyses but was weakly associated with that measured by inter-(simple sequence repeat) PCR. A set of seven loci were identified that were most strongly associated with elevated rates of fractional allelic loss and/or inter-(simple sequence repeat) PCR instability; these seven loci were on chromosomes 3, 8, 11, 13, 14, 18, and 20. A lesser association was seen with two loci flanking p53 on chromosome 17. Coordinate loss patterns for these loci suggest that at least two separate sets of cooperating loci exist for intrachromosomal genomic instability in human colorectal cancer.

Retrotransposon-like VL30 elements are efficiently induced in anoxic rat fibroblasts.

VL30 elements are a multigene family within the class of retroviruses and retrotransposons. We have characterized the response of normal rat fibroblasts to anoxia, in which endogenous VL30 element expression is strongly induced. Optimal induction up to 500-fold occurs under complete anoxia, although a lesser response is seen under atmospheres up to 2% oxygen. Phorbol esters and diacylglycerol, which induce mouse VL30 RNA approximately eightfold, show no effect on the rat VL30 system. The hypoxic conditions optimal for rat VL30 induction represent a mild cellular stress, with no reduction in cell viability during the induction period. Although the precise physiological role of this fibroblast response to temporary anoxia is unknown, it may occur during wound healing. The induction of VL30 by anoxia provides a unique model system wherein a member of the mammalian retrovirus/retrotransposon family is highly responsive to a common physiological signal.

Evaluation of a commercially available enzyme-linked immunosorbent assay for detecting antibodies to Fasciola hepatica and Fasciola gigantica in cattle, sheep and buffaloes in Australia.

A commercially available ELISA for detecting antibodies to liver fluke was evaluated for use in Australia. Milk and serum samples from cattle and sheep in which infection with Fasciola hepatica was confirmed by detection of eggs in faeces were used to estimate sensitivity. Similar samples collected from cattle and sheep outside the F. hepatica-endemic area were used to estimate specificity. The ELISA was also evaluated for detecting antibodies to F. hepatica in milk from sheep and antibodies to Fasciola gigantica in sera from cattle and buffaloes, but with small numbers of samples. In cattle, the sensitivity and specificity of the ELISA were 98.2% and 98.3% using serum and 97.7% and 99.3% using milk. In infected herds, 41.4% and 41.5% of animals were positive in the serum and milk ELISAs, respectively, whereas F. hepatica eggs were found in faecal samples from 26.5% of animals. In sheep, the sensitivity of the ELISA was 96.9% and the specificity was 99.4%. In infected flocks, 60.2% of animals were positive in the serum ELISA and F. hepatica eggs were found in faecal samples 52.2% of animals. There was perfect agreement in the ELISA between paired serum and milk samples collected from ewes. The assay detected antibodies in sera from cattle and buffaloes with natural and experimental F. gigantica infections. In the experimentally infected animals, antibodies were detected 2 weeks post-infection. We conclude that the ELISA will be a valuable tool for diagnosing F. hepatica infections in cattle and sheep. The assay may also be useful for diagnosing F. gigantica infections but further studies are required to establish sensitivity and specificity.

Germline and somatic mutation analyses in the DNA mismatch repair gene MLH3: Evidence for somatic mutation in colorectal cancers.

DNA mismatch repair is of considerable scientific and medical importance because of its essential role in maintaining genomic integrity, and its association with hereditary non-polyposis colon cancer (HNPCC). Germline mutations in five mismatch repair genes (MLH1, MSH2, PMS1, PMS2, and MSH6) have been associated with HNPCC susceptibility. Our laboratory recently identified MLH3, a novel DNA mismatch repair gene. We screened the MLH3 coding sequence in 60 probands with increased genetic risk factors for colorectal cancer susceptibility and no mutations in the other candidate genes. No definite MLH3 germline mutations were found. We subsequently screened 36 colon tumors, and discovered an appreciable frequency of somatic MLH3 coding mutations in MSI-H tumors (25%). In four of six tumors, evidence of biallelic inactivation was noted. Furthermore, MLH3 nonsense mutations were identified in two of 12 microsatellite stable (MSS) tumors with 14q24 loss of heterozygosity. While our analyses do not exclude the existence of germline MLH3 mutations in patients with increased genetic risk factors for colorectal cancer susceptibility, they suggest such mutations are uncommon in this patient population. The finding of an appreciable frequency of somatic MLH3 mutations is consistent with a possible role for this gene in the progression of colorectal cancer tumorigenesis. Hum Mutat 17:389-396, 2001. Published 2001 Wiley-Liss, Inc.

Influence of age on accumulation of alpha-actin in satellite-cell-derived myotubes in vitro.

Because muscle satellite cells have been implicated in the process of muscle growth and mass regulation, as well as regeneration, alterations in the capacity of satellite cells to differentiate and accumulate muscle specific proteins during aging could play a role in the process of senile muscle atrophy. Skeletal muscle satellite cells were cultured from male rats from the following four age groups: neonatal rats (less than 5 days of age), growing rats (1--3 months of age, adult rats (9--12 months of age) and old rats (more than 24 months of age). A series of experiments was conducted in which cultures were harvested at daily intervals following fusion, and the amount of alpha-actin per myotube nucleus was determined. Analysis of maximum actin accumulation in myotubes from each experiment within each age group revealed no significant differences among cells derived from growing, adult or old rats; however, myotubes differentiating from neonatal muscle cells were able to accumulate more than three times as much alpha-actin per myotube nuclei as cells from the other three age groups. This result may reflect fundamental differences between authentic satellite cells and myogenic cells of prenatal origin. Aside from differences between neonatal cells and satellite cells, satellite cells from old muscle do not appear to have a diminished capacity to accumulate muscle-specific proteins following differentiation into muscle fibers.

Species differentiation in the Didymozoidae (Digenea): restriction fragment length differences in internal transcribed spacer and 5.8S ribosomal DNA.

Genetic differences among six didymozoid species distinguished on morphological criteria (Didymozoon species A, B, E, F and G, and Neometadidymozoon helicis) were sought by restriction fragment length analysis of internal transcribed spacer (ITS) and 5.8S ribosomal DNA. DNA fragments comprising ITS1, 5.8S and ITS2 were amplified by polymerase chain reaction for each of the six species. Digestion with the restriction enzyme (RE) Rsa I yielded four to six fragments, approximately 65-775 base pairs long. Digestion with another RE, Sau 3AI, yielded five to eight fragments, approximately 25-775 base pairs long. No intraspecific variation was detected but there were many interspecific differences. All six species distinguished by morphology are genetically distinct. Thus, the reliability of the morphological characters originally used to separate the species was confirmed.

Ecology and diversity of digenean trematodes of reef and inshore fishes of Queensland.

coral reefs harbour an extraordinary, concentrated diversity of life. What are the implications of this for parasites? After the corals themselves, the most striking component of coral reefs is the fishes. Individual coral reefs may harbour as many as a thousand species of fishes. Like most fishes, those of coral reefs bear remarkable loads of parasites. Records of digenean trematodes from 214 species of fishes from the Great Barrier Reef and 103 species of fishes from inshore Australian waters are compared to examine the ecological expression of parasite diversity on coral reefs. Coral reef fish had an overall prevalence of infection of 70% compared with 48% for their inshore counterparts and averaged 2.61 species of digenean per host species as compared with only 1.41 for the inshore group. A total of 236 species of Digenea has been collected from reef fishes. Most of the digeneans are concentrated in just a few families. Host-specificity of digeneans of reef fishes is variable but, on average, each species infects 2.37 host species. Characteristics of the fauna studied so far are used to predict that the 1300 fishes of the Australian Great Barrier Reef are likely to harbour some 2270 species of Digenea.

A DNA-based demonstration of a three-host life-cycle for the Bivesiculidae (Platyhelminthes: Digenea)

Immature bivesiculid trematodes collected from the intestine of Thlalassoma lunare (Labridae) are shown to be morphologically consistent with adults of Bivesicula claviformis from Epinephelus fasciatus (Serranidae). In addition, the immature bivesiculids have the same sequence for the second internal transcribed spacer of the ribosomal DNA. Comparison with three other species of Bivesiculidae showed differences of between 23% and 30%. These results show that bivesiculids may have three-host life-cycles in addition to the two-host life-cycles that have been demonstrated previously. The three-host life-cycle enables bivesiculids to infect large carnivorous fishes.

Single-strand restriction fragment length polymorphism analysis of the second internal transcribed spacer (ribosomal DNA) for six species of Eimeria from chickens in Australia.

Species of Eimeria from chickens from Australia were characterised using a polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) approach. The ribosomal DNA region spanning the second internal transcribed spacer (ITS-2) was amplified from genomic DNA by PCR, digested separately with three restriction endonucleases (CfoI, Sau3AI and TaqI) and the fragments separated by denaturing gel electrophoresis. The PCR products amplified from the six species varied from approximately 70 to 620 bp on agarose gels, with differences in size and number of bands among species, but no apparent variation within a species. The PCR-RFLP analysis of ITS-2 amplicons on denaturing gels gave characteristic profiles for individual species (except for minor variation in profiles within some species). The results indicate that ITS-2 contains useful genetic markers for the identification of six Eimeria species occurring in Australia.