Determination of human hepatic cytochrome P4501A2 activity in vitro use of tacrine as an isoenzyme-specific probe

Oxidative metabolism of the cognition activator tacrine (1,2,3,4-tetrahydro-9-aminoacridine) is thought to be catalyzed by cytochrome P4501A2 (CYP1A2). In this study, the use of tacrine as a specific substrate to measure CYP1A2 activity in vitro was investigated. Tacrine metabolism was assessed in 16 human liver microsomal samples. Initially, the percentage conversion of tacrine to stable metabolites (i.e. 1-, 2-, 4-, and 7-hydroxytacrine) at a single time point was correlated with levels of CYP1A2 apoprotein. Apoprotein was detected by immunoquantification using a monospecific CYP1A2 antipeptide antibody. Significant correlations were seen between CYP1A2 content and the degree of 1-hydroxylation (r = 0.81, p < 0.001), 7-hydroxylation (r = 0.70, p < 0.001), and metabolism to all stable products (r = 0.82, p < 0.001). The major metabolite formed in all livers was 1-hydroxytacrine. The conversion of tacrine to this metabolite was examined in more detail. The rate of formation varied from 19.2 pmol min-1 mg-1 to 101.0 pmol min-1 mg-1. There was a significant correlation (r = 0.84, p < 0.001) between the rate of formation and CYP1A2 levels. Tacrine metabolism was also compared with the rate of formation of 3-methylxanthine, from theophylline, a reaction previously shown to be catalyzed by CYP1A2. Significant correlations were found between 3-methylxanthine formation and all quantified tacrine metabolites. The rate of 3-methylxanthine generation also correlated with CYP1A2 apoprotein levels. It is concluded, therefore, that tacrine is a valuable probe for the determination of human hepatic CYP1A2 activity in vitro.     

Studies on otitis externa in dogs. I. Survey of aetiological agents: fungi

The mechanism of the teratogenic effect of thalidomide is unknown. The hypothesis that this drug acts on the sensory nerves of the embryo was derived from radiological observations of two series of thalidomide-deformed children. Supplementary evidence was found in neurological, embryological and other literature, to support the hypothesis of embryonic neuropathy. The concept can be extended to include visceral as well as skeletal deformities. Evidence for and against the hypothesis is presented, and its application to a broad spectrum of congenital deformities provides an explanation of a number of previously unrelated facts. Areas for further research are suggested.     

Scanning electron microscopy of normoxic and hyperoxic hyperbaric exposed lungs

Lungs from adult guinea pigs exposed to 1 ATA He-O2 with 200 mm Hg PO2 and 20 ATA He-O2 with 200, 400, and 600 mm Hg PO2 were studied with scanning electron microscopy. The appearance of normal alveoli is described. Even before pulmonary O2 toxicity became symptomatic, subtle changes occurred in the alveoli, such as an increase in macrophages and a marked increase in length of alveolar type-II cell microvilli. These changes occurred in animals exposed to 400 mm Hg PO2, heretofore considered below toxic levels. With increased toxic involvement, the number of alveolar type-II cells increased. A thick layer of material appeared in some of the alveoli, obscuring the Kohns pores and type-I and -II cell surfaces. The alveolar-capillary network with underlying erythrocytes was no longer observable. Lungs with the greatest toxic involvement possessed large numbers of macrophages encompassed by a fibrin-like matrix. The alveolar walls were broken down in many instances, and the alveoli were no longer discrete units but took on the appearance of an amorphous mass of lung tissue.     

Specificity in interaction of benzo[a]pyrene with nuclear macromolecules: implication of derivatives of two dihydrodiols in protein binding

Benzo[a]pyrene (B[a]P), 7,8-dihydroxy-7,8-dihydro-B[a]P, and 9,10-dihydro-B[a]P are metabolized by hamster embryo cells to derivatives that bind to nuclear macromolecules. The selectivity for different classes of macromolecules varies depending on the compound analyzed. The ratio of DNA specific activity to protein specific activity (pmol bound/mg of macromolecules) is high (1.51) for 7,8-dihydroxy-7,8-dihydro-B[a]P, extremely low (0.03) for 9,10-dihydroxy-9,10-dihydro-B[a]P, and intermediate (0.26) for B[a]P. Histones H3 and H2A are the major targets of 7,8-dihydroxy-7,8-dihydro-B[a]P; a protein(s) with a mobility similar to that of histone H1 is heavily labeled by 9,10-dihydroxy-9,10-dihydro-B[a]P, with minor labeling of other (nonhistone) bands. The labeling pattern seen with B[a]P is a combination of the patterns seen with the two dihydrodiol metabolites studied. Analysis of the ethyl acetate-soluble metabolites suggests that hamster embryo cells produce 9,10-dihydroxy-7,8-oxy-7,8,9,10-tetrahydro-B[a]P from 9,10-dihydroxy-9,10-dihydro-B[a]P and raise the possibility that this vicinal diol epoxide is an intermediate in the binding of 9,10-dihydroxy-9,10-dihydro-B[a]P to nuclear proteins. The differences seen suggest that factors other than the intrinsic chemical reactivity of the epoxide group are extremely important in the interaction of potential ultimate carcinogens with biological systems.     

Regulation of mouse and human mast cell development, survival and function by stem cell factor, the ligand for the c-kit receptor

Stem cell factor (SCF), the ligand for the receptor (SCFR) that is encoded by the c-kit proto-oncogene, has many important effects in mouse and human mast cell development, survival, and function. SCF can promote mast cell survival by suppressing apoptosis, induce mast cell hyperplasia in murine rodents, experimental primates and humans, directly induce SCFR-dependent mast cell mediator release, and significantly modulate the extent of mast cell activation by Fc epsilon RI-dependent mechanisms. These findings raise several clinical issues and, in some cases, point to potentially significant therapeutic opportunities.     

Immune responses in patients with T-cell lymphoma treated with an anti-idiotype antibody mimicking a highly restricted T-cell antigen

We generated an IgG1 murine monoclonal anti-idiotype antibody (Ab2) to a highly restricted T-cell antigen designated glycoprotein (gp) 37 that is found on T-cell malignancies but not on normal cells. gp37 is identified by the murine monoclonal antibody SN2 (Ab1) against which the Ab2 was raised. Each of four patients with T-cell lymphoma predominantly confined to the skin received a minimum of four intracutaneous injections of aluminum hydroxide precipitated anti-idiotype murine monoclonal antibody (1 mg/injection) given every 2 weeks. For responding patients, injections were continued on a monthly basis. All tumors were measured along orthogonal major and minor axes, using a ruler and/or calipers, by the same observer. Tumor sizes were documented photographically. Three of the four patients developed specific idiotypic humoral immune responses, and two of the four patients also demonstrated idiotypic cell-mediated responses. Humoral responses included binding of the patients' sera to the anti-idiotype antibody as well as specific inhibition of binding of the SN2 antibody (Ab1) to the anti-idiotype antibody (Ab2). Anti-anti-idiotypic (Ab3) antibody from one patient's serum bound specifically to the gp37-positive cell line MOLT-4 and also to semipurified gp37 antigen. Cell-mediated responses were demonstrated by specific proliferative response to the aluminum hydroxide precipitated anti-idiotype antibody by patients' peripheral blood mononuclear cells. While three of the four patients had extensive disease and did not have clinical responses, one of the patients who had nine discrete skin tumors and peripheral blood involvement without other detectable disease had virtually complete disappearance of the tumors lasting over 11 months. Our results demonstrate that this particular anti-idiotype antibody can induce humoral and cellular immune responses, and at least in one patient led to a meaningful therapeutic response. Future trials should focus on immunocompetent patients with minimal disease.     

Partitioning of amino acids flowing to the abomasum into feed, bacterial, protozoal, and endogenous fractions

We partitioned the flow of amino acids (AA) to the abomasum among rumen undegradable protein (RUP) and bacterial, protozoal, and endogenous fractions using four Holstein cows in midlactation that were equipped with ruminal and abomasal cannulas. A 2 x 2 factorial design with four diets, combinations of high or low ruminally degradable organic matter, and rumen degradable protein, was employed. Crude protein (CP) and AA contents of ruminal bacteria and protozoa and abomasal digesta were determined. Equations for the source compositions and in vivo flows of CP and 16 AA were then solved simultaneously with a linear program to estimate the contribution of RUP, bacterial, protozoal, and endogenous CP to AA flows. The flows of RUP and bacterial AA were not affected by diet. Low dietary RDP increased the flow of protozoal AA to the abomasum, but the ruminally degradable organic matter content of the diet did not affect protozoal AA flow. Across diets, RUP, bacterial, protozoal, and endogenous fractions provided 55, 33, 11, and <1% of the CP, and 62, 26, 12, and <1% of the AA that reached the abomasum. The linear program was a useful tool for partitioning AA that flows to the abomasum. The technique may also allow dietary effects on ruminal microbes and the AA profile of protein flowing to the duodenum to be better understood and perhaps manipulated.