. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 444 D. MUTTON AND V. J. SMITH : V*- 1a 1b. Figure 1. Amoehocyles oi Actinui cqinna isolated from the mesenteric filaments alter 45-min incuba- tion on glass coverslips. (a) A spread hyaline (phagocytic) type cell containing numerous small, usually nonrefractile, inclusions. Scale bar = 5 ^m. (h) An intact granular cell containing many large, highly re- tractile granules. Scale bar = 5 ^m. was stored on ice until use. Protein in the ALS was deter- mined by the method of Bradford (1976) with bovine serum albumin as standa
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 444 D. MUTTON AND V. J. SMITH : V*- 1a 1b. Figure 1. Amoehocyles oi Actinui cqinna isolated from the mesenteric filaments alter 45-min incuba- tion on glass coverslips. (a) A spread hyaline (phagocytic) type cell containing numerous small, usually nonrefractile, inclusions. Scale bar = 5 ^m. (h) An intact granular cell containing many large, highly re- tractile granules. Scale bar = 5 ^m. was stored on ice until use. Protein in the ALS was deter- mined by the method of Bradford (1976) with bovine serum albumin as standard and routinely adjusted by di- lution with OS to 2 mg ml~' before use. Aliquots (80 ^1) of the ALS were then dispensed in quadruplicate wells of a 96-well (flat-bottomed) microtiter plate and mixed with 10 jul of MB and 10 n\ of P. immobilis (stock con- centration 2 X 105 mP1). For controls, 80 ^1 of OS was substituted for the ALS. The absorbance of each of the control and experimental wells was read at 570 nm after incubation at 20°C for 0, 3, 6, 12, and 24 h. Because the bactericidal assays with whole amoebo- cytes indicated that the response involves lysis (see be- low), we undertook to ascertain whether the antibacte- rial properties of A. ecniina ALS are due at least in part to lysozyme activity. A modification of the procedure described in Findlay and Smith (1995) was used. Briefly, ALS samples were prepared as above but in salt- supplemented phosphate-buffered saline (ssPBS; 20mA/ Na2HPO4-2H:O; 45mA/ KH2PO4: M NaCl; pH ). They were serially diluted to protein concentrations of 1500, 1000, 500, or 200 /*g ml~', and 100 i/l of each dilution was mixed with 900 n\ of Micro- coccus luteus cell walls (Sigma) previously suspended in ssPBS to give an absorbance of at 570 nm. An extra tube containing PBS in place of ALS was set up as the negative control. To provide both positive controls and a standard curve by which any lysozyme-like activity in A. equina ALS coul
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Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
