I .A technique for the separation and colorimetric estimation of z,4-diaminopimelic acid (DAP) using automated ion-exchange chromatography coupled with an acid ninhydrin detection system is described.2. Only traces of DAP were found in rumen protozoa and no DAP was detected in rumen fluid prepared by ultracentrifugation or dialysis.3. The concentration of DAP in rumen bacteria from sheep on a constant feeding regimen, and the ratio of nitrogen to DAP for these bacteria were found to be constant over a 3-month period.4. The method has proved suitable for the estimation of bacterial N in the duodenal digesta of ruminants.5. The contribution of bacterial N to the total N leaving the abomasum of a lactating cow fitted with a permanent re-entrant cannula in the duodenum was found to be 5 0 % .In ruminants a substantial proportion of dietary protein is fermented in the rumen, and the degradation products are utilized for microbial protein synthesis. The protein which becomes available for digestion and absorption beyond the rumen includes unchanged feed protein and microbial protein. ) and may be used to estimate the bacterial contribution to ruminal and duodenal nitrogenous constituents. This paper describes. an improved method for estimating DAP using ion-exchange chromatography and an acid ninhydrin reagent based on the procedure of el-Shazly & Hungate (1966). The method has been used to assess the daily contribution of bacterial N to the total N passing into the duodenum of a lactating dairy cow. K. HUTTON, F. J. BAILEY AND E. F. ANNISON M A T E R I A L S A N D M E T H O D S Experimental animalsA mature Clun Forest wether with a rumen fistula, given I kg hay and IOO g rolled oats once daily, was used as a source of rumen bacteria when samples were required to assess the variation in N : DAP ratio over a 3-month period for an animal on a constant feeding regimen.A 4-year-old lactating Ayrshire cow in the 8th month of lactation and yielding 8.3 kg of milk daily was used for the estimation of bacterial N entering the duodenum. This animal had a rumen fistula and a re-entrant cannula in the proximal duodenum about 7 cm from the pyloric sphincter. The maintenance requirement of the cow was supplied by barley straw and a protein balancer offered at 11.00 and 17.00 hours. Production concentrates (400 g/kg milk) were given at 7.00 and 16.00 hours. The total intake of nutrients was slightly in excess of the minimum requirements recommended by the Agricultural Research Council (1965). Paper impregnated with chromic oxide (72.2 g chromic oxide/zoo g paper) was added to the rumen of the cow once daily as a marker in the measurement of digesta flow. Collection and preparation of digestaRumen contents were strained through four layers of surgical gauze, and about 400 ml of strained liquor were centrifuged at IOO g for I min to remove protozoa and dense food particles, and to yield a bacteria-rich supernatant fluid. This bacterial fraction was centrifuged at 22 ooo g for 10 min to yield clarified rumen liquor (CRL) and a precipi...

Classical immunoanalytical techniques are based on native antigens and their precipitation with specific antibodies: they are not very appropriate for the investigation of processed denatured mixtures. This report describes an immunoassay based on the more recent but well-established enzyme-linked immunosorbent assay (ELISA) procedure, which in this case is specific for soya proteins that have been solubilised in urea and then 'renatured' by removing or diluting the denaturant. Levels of soya protein 100 g-I total protein (nominally 100%) were determined with 34 commercial soya products, using a standard reference antigen. Normal expected levels were observed with flours (average 107 %) and isolates (108 %), while results with concentrates (82 " , ) and texturates (79 %) were somewhat lower. Some specialised products gave little or no response, presumably because they are hydrolysed. Meat, milk, egg, wheat and field bean proteins displayed negligible interference. These preliminary results suggest that the ELISA procedure will provide a convenient general method for the qualitative characterisation and quantitative estimation of individual proteins in food products even after severe processing; it seems more attractive than many other methods reported. Imrnunoassay is capable of large numbers of inexpensive but sophisticated determinations of specific food components. The food analyst should regard ELISA (for the determination in situ of specific antigens in mixtures) as a powerful technique complementary to classical chromatographic and electrophoretic methods which depend on separation before determination.

Analysis of trypsin-treated extracts from cooked meat products can be used to assess their soya protein content. The technique is based on fractionation of positively charged peptides using an automatic cation exchange amino acid analyser system. A peptide, unique to digests of soya protein, is used for quantitation purposes. Assessment of the quantitative performance is not yet complete, but results so far indicate that the lower limit of detection will be 5-10 g soya protein per 100 g total protein. The accuracy is expected to be f 5 %.

Dimethylarginine dimethylaminohydrolases (DDAHs) catalyze the hydrolysis of methylarginines to yield L-citrulline and methylamines as products. DDAHs and their central roles in methylarginine metabolism have been characterized for eukaryotic cells. While DDAHs are known to exist in some bacteria, including Streptomyces coelicolor and Pseudomonas aeruginosa, the physiological importance and genetic regulation of bacterial DDAHs remain poorly understood. To provide some insight into bacterial methylarginine metabolism, this study focused on identifying the key elements or factors regulating DDAH expression in P. aeruginosa PAO1. First, results revealed that P. aeruginosa can utilize N G ,N Gdimethyl-L-arginine (ADMA) as a sole source of nitrogen but not carbon. Second, expression of the ddaH gene was observed to be induced in the presence of methylarginines, including N G -monomethyl-L-arginine (L-NMMA) and ADMA. Third, induction of the ddaH gene was shown to be achieved through a mechanism consisting of the putative enhancer-binding protein PA1196 and the alternative sigma factor RpoN. Both PA1196 and RpoN were essential for the expression of the ddaH gene in response to methylarginines. On the basis of the results of this study, PA1196 was given the name DdaR, for dimethylarginine dimethylaminohydrolase regulator. Interestingly, DdaR and its target ddaH gene are conserved only among P. aeruginosa strains, suggesting that this particular Pseudomonas species has evolved to utilize methylarginines from its environment.IMPORTANCE Methylated arginine residues are common constituents of eukaryotic proteins. During proteolysis, methylarginines are released in their free forms and become accessible nutrients for bacteria to utilize as growth substrates. In order to have a clearer and better understanding of this process, we explored methylarginine utilization in the metabolically versatile bacterium Pseudomonas aeruginosa PAO1. Our results show that the transcriptional regulator DdaR (PA1196) and the sigma factor RpoN positively regulate expression of dimethylarginine dimethylaminohydrolases (DDAHs) in response to exogenous methylarginines. DDAH is the central enzyme of methylarginine degradation, and its transcriptional regulation by DdaR-RpoN is expected to be conserved among P. aeruginosa strains.