of cwllagenous fillers, as seen for example in peridorit it is, results fro111 pririury alteratiori by the iriflariirnzatory dary attack by bacterial proteases.'I' r ea tm en t w it h ethylene oxicl c rendered reconstituted acid-soluble collagen readily digestible by colmibined pure cultures of human oral strains of fusiforrn bacilli, diphtheroid bacilli, streptococci, and veillonellae. The fact that only a few of these strains individually attacked such altered collagen to a slight extent indicates digestion by synergistic and symbiotic reactions between members of the oral microbiota. Concomitantly with increased digestibility from treatment with ethylene oxide, reconstituted collagen exhibited marked morphological changes as revealed by electron microscopy. S u jyc w? N v y . Mayo Clinic and M a y o Fn.,t Rochester, Ninn.The enzyme glutamic-oxalacetic transaminase (GOT) has attracted considerable interest recently because its concentrations in blood and tissue are altered characteristically in a numiber of disease states. Methods of purification devised up to now( 1,2) have produced no indication that this enzyme may occur in more than one form. We observed that crude GOT in an extract of cardiac muscle from dog, when chromatographed on certain ion exchangers, behaves as a mixture of 2 enzymes. When CM-cellulose was employed with phosphate buffer of pH 7.0 and r/2 = 0.01 or less, one component was adsorbed and could be eluted a t higher ionic strength and lower pH, while the other was adsorbed very little if at all. With DEAE-cellulose the be--*This investigation was supported in part by Research Grant from Nat. Tnst. of Arthritis and Metab. Diseases, P.H.S. t The Mayo Foundation, Rochester, Minn., is part of Graduate School of Univ. of Minnesota.havior olf the 2 fractions was reversed. These findings suggested that the 2 GOT'S may have opposite electrical charges at p H 7. To test this assumption, the paper electrophoresis of crude preparations of GOT was investigated.Methods and materials. Tissues were minced and extracted in a blendor with 3 volumes of cold water. The centrifuged extracts were concentrated several fold by ultrafiltration at 0°C. The solutions were recentrifuged at 30,000 g. They were then applied, in proportions of 10 to 40 pl, containing about 1 to 5 mg of protein, to the paper strips in a Spinco electrophoretic apparatus Model R. Phosphate buffer of pH 7.4, r/2 = 0.075, was used, often with the addition of one per cent bovine serum albumin as a stabilizer of enzymatic activity. Stabilization could be achieved also by applying quantities of tissue extracts corresponding to the larger amount of protein. Current was 5 milliamperes, temperature 8"C, at UNSW Library on July 18, 2015 ebm.sagepub.com Downloaded from

L-forms of a strain of Pseudomonas aeruginosa were produced by serial subculture of the bacterial form on agar medium containing sucrose as an osmotic stabilizer and carbenicillin. L-forms eventually became stable, i.e., would not revert in the absence of antibiotic, and were adapted to grow well in broth with the osmotic stabilizer. Gross morphology and light microscopic colony morphology were typical of an L-form. L-form cells were approximately spherical and bounded in part by a plasma membrane; they lacked the triple-layer cell wall structure and coarse, electron-dense nucleoidal granules of the parent bacterial form. The L-form, but not the bacterial form, contained cores, organelles previously reported only in group D streptococci. Antibiotic disc-sensitivity studies showed the stable L-form to be as sensitive as, or more sensitive than, the bacterial form to most antibiotics. Exceptions were polymyxin B, colimycin sulfate, and gentamicin, which were more active against the bacterial form. The remainder of the aminoglycosides and cell wallactive antibiotics showed no inhibition of either form. The L-form was more susceptible to cidal activity of normal human serum than the parent form. The L-form exhibited fewer biochemical activities than the parent bacteria or bacterial forms derived by reversion at a time when the L-form was still unstable. L-form colonies appeared colorless, and chemical analysis demonstrated that, if the L-form produces pigment at all, which was not demonstrated, it could not have been more than 3.6%o of that produced by the bacterial form. The role of L-forms in infectious diseases in general and in urinary tract infections in particular has not been clarified. However, there is inferential evidence that these wall-deficient variants may be significant, not only as primary etiologic agents, but particularly may account for relapse after apparent cure (23). Postulated mechanism for production in vivo is based on the in vitro demonstration that wall-deficient variants can be induced by serum action on the parent form (1, 37). L-forms have been isolated from urine of patients with urinary tract infection (14) and inferential evidence has been obtained by treatment of experimental animals with antibiotics (15). Pseudomonas infections present an important problem, particularly in urinary tract infections and burn patients, because antibiotics in general use are not active against this organism. The semisynthetic penicillin, carbenicillin (disodium a-carboxybenzylpenicillin; BRL 2064), has been demonstrated to possess significant activity against strains of Pseudomonas aeruginosa, both in vitro (40) and in vivo (21, 39, 44). In view of this activity, it was felt that carbenicillin provided an opportunity to determine whether L-forms could be produced in vitro from P. aeruginosa and to study some biological properties of the L-forms. This study details initial production of the Lform of P. aeruginosa and the achievement of nonreversion in the absence of antibiotic. Comparisons of morphology,...

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