Radiosensitivity of peripheral blood lymphocytes in autoimmune disease

The proliferation of peripheral blood lymphocytes, cultured with Con A, can be inhibited by ionizing radiation. Lymphocytes from patients with conditions associated with autoimmunity, such as rheumatoid arthritis, systemic lupus erythematosus and polymyositis, are more radiosensitive than those from healthy volunteers or patients with conditions not associated with autoimmunity. The nuclear material isolated from the lymphocytes of patients with autoimmune diseases is, on average, lighter in density than the nuclear material from most healthy controls. This difference in density is not related to increased sensitivity to ionizing radiation but the degree of post-irradiation change in density (lightening) is proportional to the initial density, i.e. more dense nuclear material always shows a greater upward shift after radiation. The recovery of preirradiation density of nuclear material, 1 h after radiation exposure, taken as an indication of DNA repair, correlates with the radiosensitivity of lymphocyte proliferation (Con A response); failure to return to pre-irradiation density being associated with increased sensitivity of proliferative response. These results require extension but, taken with previously reported studies of the effects of DNA methylating agents, support the idea that DNA damage and its defective repair could be important in the aetio-pathogenesis of autoimmune disease.     

Functional gene analysis suggests different acetogen populations in the bovine rumen and tammar wallaby forestomach

Reductive acetogenesis via the acetyl coenzyme A (acetyl-CoA) pathway is an alternative hydrogen sink to methanogenesis in the rumen. Functional gene-based analysis is the ideal approach for investigating organisms capable of this metabolism (acetogens). However, existing tools targeting the formyltetrahydrofolate synthetase gene (fhs) are compromised by lack of specificity due to the involvement of formyltetrahydrofolate synthetase (FTHFS) in other pathways. Acetyl-CoA synthase (ACS) is unique to the acetyl-CoA pathway and, in the present study, acetyl-CoA synthase genes (acsB) were recovered from a range of acetogens to facilitate the design of acsB-specific PCR primers. fhs and acsB libraries were used to examine acetogen diversity in the bovine rumen and forestomach of the tammar wallaby (Macropus eugenii), a native Australian marsupial demonstrating foregut fermentation analogous to rumen fermentation but resulting in lower methane emissions. Novel, deduced amino acid sequences of acsB and fhs affiliated with the Lachnospiraceae in both ecosystems and the Ruminococcaeae/Blautia group in the rumen. FTHFS sequences that probably originated from nonacetogens were identified by low "homoacetogen similarity" scores based on analysis of FTHFS residues, and comprised a large proportion of FTHFS sequences from the tammar wallaby forestomach. A diversity of FTHFS and ACS sequences in both ecosystems clustered between the Lachnospiraceae and Clostridiaceae acetogens but without close sequences from cultured isolates. These sequences probably originated from novel acetogens. The community structures of the acsB and fhs libraries from the rumen and the tammar wallaby forestomach were different (LIBSHUFF, P < 0.001), and these differences may have significance for overall hydrogenotrophy in both ecosystems.     

Quantitation and diversity analysis of ruminal methanogenic populations in response to the antimethanogenic compound bromochloromethane

Methyl coenzyme-M reductase A (mcrA) clone libraries were generated from microbial DNA extracted from the rumen of cattle fed a roughage diet with and without supplementation of the antimethanogenic compound bromochloromethane. Bromochloromethane reduced total methane emissions by c. 30%, with a resultant increase in propionate and branched chain fatty acids. The mcrA clone libraries revealed that Methanobrevibacter spp. were the dominant species identified. A decrease in the incidence of Methanobrevibacter spp. from the clone library generated from bromochloromethane treatment was observed. In addition, a more diverse methanogenic population with representatives from Methanococcales, Methanomicrobiales and Methanosacinales orders was observed for the bromochloromethane library. Sequence data generated from these libraries aided in the design of an mcrA-targeted quantitative PCR (qPCR) assay. The reduction in methane production by bromochloromethane was associated with an average decrease of 34% in the number of methanogenic Archaea when monitored with this qPCR assay. Dissociation curve analysis of mcrA amplicons showed a clear difference in melting temperatures for Methanobrevibacter spp. (80-82 degrees C) and all other methanongens (84-86 degrees C). A decrease in the intensity of the Methanobrevibacter spp. specific peak and an increase for the other peak in the bromochloromethane-treated animals corresponded with the changes within the clone libraries.     

Effect of sulfur supplements on cellulolytic rumen micro-organisms and microbial protein synthesis in cattle fed a high fibre diet

AIM: To examine the effect of sulfur-containing compounds on the growth of anaerobic rumen fungi and the fibrolytic rumen bacteria Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes in pure culture and within the cattle rumen. METHODS AND RESULTS: The effect of two reduced sulfur compounds, 3-mercaptopropionic acid (MPA) or 3-mercapto-1-propanesulfonic acid as the sole S source on growth of pure fibroyltic fungal and bacterial cultures showed that these compounds were capable of sustaining growth. An in vivo trial was then conducted to determine the effect of sulfur supplements (MPA and sodium sulfate) on microbial population dynamics in cattle fed the roughage Dichanthium aristatum. Real-time PCR showed significant increases in fibrolytic bacterial and fungal populations when cattle were supplemented with these compounds. Sulfate supplementation leads to an increase in dry matter intake without a change in whole tract dry matter digestibility. CONCLUSIONS: Supplementation of low S-containing diets with either sodium sulfate or MPA stimulates microbial growth with an increase in rumen microbial protein supply to the animal. SIGNIFICANCE AND IMPACT OF THE STUDY: Through the use of real-time PCR monitoring, a better understanding of the effect of S supplementation on discrete microbial populations within the rumen is provided.     

Protein methyltransferase activities in commercial in vitro translation systems

Protein arginine methylation is a well-known post-translational modification that has been shown to occur in rabbit reticulocyte in vitro translation lysates (RRL); however, it is not known whether this is a general feature of in vitro-produced proteins from other eukaryotic cell-free translation systems, particularly insect-derived lysates (ICL). Because methylation can affect protein localization, RNA binding and protein-protein interactions this may be of great importance as in vitro-produced proteins are often used in assays of protein function. Here, I report the presence of base-stable and base-labile methyltransferase activities in RRL, ICL and wheat germ in vitro extracts (WGE). Indeed, the presence of CARM1 in RRL and ICL and a class II protein arginine methyltransferase activity (PRMT5/7) is documented in all three systems. Additionally, the lysine methyltransferase that modifies eukaryotic elongation factor 1A (eEF-1A) was detected in ICL and WGE. Importantly, using a defined set of substrates under identical conditions I show that all three in vitro systems contain different complements of the various methyltransferases. These data suggest that three systems can be used in a complementary fashion to investigate the effect(s) of post-translational modification on protein function.