Detection of serum-dependent cytotoxic activity of Campylobacter jejuni and its characteristics.
We previously set forth appropriate three assay systems using Chinese hamster ovary cells to detect the Campylobacter jejuni cytotoxin. Although we could not reach a conclusion because the cytotoxins shown in this study were not purified, at least three different cytotoxins were detected in these assay systems. The first cytotoxin in the presence of fetal calf serum (FCS) was heat-labile and the molecular weight (Mw) was estimated at 50-100 k by ultrafiltration. The second cytotoxin detected in the presence of newborn calf serum (NCS) was heat-stable and Mw was estimated at 0.5-3.0 k. The third cytotoxin detected in serum-free culture (SFC) assay was heat-labile and non dialyzable. However, Mw was not estimated since the low Mw and heat-stable cytotoxin was also detected in this assay. The cytotoxic activity detected in FCS and NCS assays, but not that detected in SFC assay, was completely abolished by treatment with a reducing agent. In contrast, the cytotoxicity detected in both FCS and NCS assays was not inactivated by such an enzyme as trypsin, lipase, neuraminidase, and beta-galactosidase. When the filtrate was heated at 100 degrees C to inactivate the heat-labile cytotoxin, the cytotoxic activity was detected in the NCS assay but not in FCS assay. However, when NCS was added to this heated filtrate, the cytotoxicity was restored in FCS assay. Furthermore, when normal rabbit serum (NRS) was added, no cytotoxicity was restored. The cytotoxic activity in SFC assay was completely inactivated with FCS or NRS. These findings suggest that the cytotoxic activity is dependent on serum added to tissue culture medium and that the substance amplifying and/or inhibiting the cytotoxic activity may be present in serum.
Misawa
N
N
Department of Veterinary Public Health, Faculty of Agriculture, Miyazaki University, Japan.
Ohnishi
T
T
Itoh
K
K
Takahashi
E
E
D000818:Animals; D001681:Biological Assay / Q000379:methods; D001798:Blood Proteins / Q000032:analysis / Q000494:pharmacology*; D016466:CHO Cells; D016123:Campylobacter jejuni / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D003429:Cross Reactions; D003470:Culture Media; D016895:Culture Media, Serum-Free; D003603:Cytotoxins / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000494:pharmacology; D008970:Molecular Weight; D009439:Neuraminidase / Q000494:pharmacology; D010053:Ovary / Q000166:cytology; D014357:Trypsin / Q000494:pharmacology; D001616:beta-Galactosidase / Q000494:pharmacology
D001798:Blood Proteins; D003470:Culture Media; D016895:Culture Media, Serum-Free; D003603:Cytotoxins; D008049:Lipase; D009439:Neuraminidase; D001616:beta-Galactosidase; D014357:Trypsin