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A DEAE-cellulose stationary phase in a rolled configuration was used to separate recombinant secretory leukocyte protease inhibitor (rSLPI) from denaturants and reducing agents (3 M guanidine-HCl and 5 mM DTT) in less than 5 min to promote refolding of the protein to an active form. The mobile phase consisted of buffer and 500 mM NaCl, where NaCl suppressed binding of protein to this stationary phase. Separation of an initial concentration of 2 mg/mL protein from the other constituents resulted in 96% recovery of the rSLPI at an average concentration of 1.28 mg/mL. When incubated for 4 h at 20 degrees C, the fractionated rSLPI gave a 46% yield of properly refolded protein. The protein concentration was 6.4 times higher than that reported in a previously published method, where refolding was carried out by diluting the mixture of protein, denaturants, and reducing agents by a factor of 10. The results show that a combination of rapid chromatographic separation over a cellulosic stationary phase followed by protein refolding will significantly enhance process throughput by minimizing tankage, water requirements, and process time.

AbstraetThe predominant white ceil-type in circulating blood of normal rats is the lymphocyte. Granuloeytes increase in the circulating blood of adjuvant-inoculated rats; lymphocytes remain the same or increase slightly. Some drugs, like Cytosar | (cytarabine), inhibit granuioeyte increases primarily and inhibit arthritis development completely. Pretreatment of normal rats with cytarbine also inhibits the local inflammation caused by direct injections of M. butyrieum into hindpaws of rats. Pre-treatment of rats with M.butyrieum, causing granulocytes to increase in circulating blood, produces an exaggerated response to the subsequent inoculation ofM. butyricum directly into rats' paws.Injection of M. butyrieum in non-arthrltogenic vehicles (saline) does not produce the exaggerated response of granulocytes that occurs in response to injection in effective arthritogenic vehicles (mineral oil). Granulocytes are not increased greatly using the ineffective vehicle. Finally, lysates of either rat white cells (granulocytes) or platelets, both of which increase in adjuvant-inoculated rats, produce striking inflammatory reactions when inoculated locally into rats' hindpaws. The addition of non opsonized M. butyricum to granulocytes obtained from adjuvant-inoculated rats causes a significant increase in the release oflysosomal enzymes.From all these and other indirectly derived data, it is surmised that adjuvant inoculation produces a marked increase in granulocytes of circulating blood; as these increase with time, the 114. butyricum molecules or some constituent derived from them (as determined by the distribution of tritiated M. butyricum) arrive in joints and many other tissues, there reacting with granulocytes causing release of lysosomal constituents that produce the histopathology of adjuvant-disease. These results are discussed. IntroductionThe pathways and mechanisms for development of adjuvant-arthritis in the rat are unknown. Following inoculation of certain adjuvants (organism + appropriate vehicle)intradermally, rats develop a widespread disseminated disease, involving many organs -the appendages being only one manifestation of this

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Regulation of the body burden of selenium is important because selenium deficiency and excess both lead to pathologic conditions (1, 2 ) . We showed that after intraperitoneal injection of 75Se032-urinary excretion of ""Se increased as dietary selenium was raised within the range of 0 to 1 ppm (3). Fecal and expired 75Se had no such relationship with dietary selenium under those conditions. Since our previous study showed a marked increase in urinary 75Se when only 0.1 ppm selenium was added to the diet, and since the urine seems to be the major route of selenium excretion, we decided to study even lower dietary selenium levels to determine whether there was a dietary selenium threshold above which urinary selenium began to increase.Methods and Procedures. Five groups of 4 weanling male Holtzman rats each1 were fed a vitamin E-adequate (250 IU dl-a-tocopherol/kg diet) torula yeast diet (3) with 0, 0.030, 0.060, 0.090, and 0.120 ppm selenium added as NazSe03, respectively. The basal diet without selenium supplementation contained 0.024 ppm selenium.2 Rats were weighed weekly and no differences in growth rate were observed among groups. After the rats had consumed the diets for 25 days, each animal was injected intraperitoneally with 2 , NY using the method of Olson, J. Ass. offic.Anal. Chem. 52, 62'1 (1969).pCi of 75Se032-(sp act 119 Ci/g of seleniand placed in a metabolism cage. Urine and feces were collected for 7 days and percentage of administered 75Se in them was determined as before (3). Whole-body counting was performed daily for 17 days. Then the animals were sacrificed and percentage of whole-body '%e in various organs was determined as described previously (3). Figure 1 shows the whole-body retention of 75Se. The 0 and the 0.030 ppm selenium groups retained the same percentage of the %e dose whereas differences among all other groups were highly significant. Urinary excretion accounted for the differences in whole-body retention as shown in Fig. 2. Urinary and fecal excretion accounted for all losses of ?%e as calculated from whole-body retention. Results.In contrast to the identical 75Se total body retention of the 0 and 0.030 ppm groups, tissue distribution in these groups was markedly different as is seen in Fig. 3. Testes ( p < 0.001), adrenals ( p < 0.005), spleen ( p < 0.001), thymus ( p < 0.01), and brain ( p < 0.001) all contained significantly more of the whole-body 75Se per gram in the 0 ppm group, while liver ( p < O.OOS), blood ( p < O.OOS), heart ( p < 0.05) and skeletal muscle ( p < 0.05) contained more in the 0.030 ppm group.Discussion. The whole-body and excretion results indicate the existence of a dietary selenium threshold somewhere between 0.054 ppm (0.024 + 0.030 ppm) and 0.084 ppm (0.024 + 0.060 ppm) for the forms of selenium used in this experiment below which a constant percentage of the administered T5Se is excreted in the urine. Above the threshold