Self-catalysed, O2-independent inactivation of NADPH- or dithionite-reduced microsomal cytochrome P-450 by carbon tetrachloride.
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Biomedical subjects
Publications and source records attributed to W Haas.
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The finding that the diversity (D) and joining (JH) but not the variable (VH) DNA segments of mouse immunoglobulin heavy-chain genes are joined in the DNA of some cloned cytolytic T cells, led to identification and sequencing of three different D DNA segments. Two segments identified on the embryo DNA carry on both the 5' and 3' sides two sets of characteristic sequences separated by a 12-base pair spacer, which have been implicated as recognition signals for a recombinase. The third segment, identified in a form joined with a JHDNA segment in a T cell, carries the recognition signal on the 5' side. These results support the 12/23-base pair model for somatic generation of immunoglobulin V genes, and rule out the possibility that the cytolytic T cells use assembled VH, D and JH sequences to encode their antigen receptors.
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Following the demonstration that hybrids between normal B-lymphocytes and myeloma cell lines continue to secrete antibodies with the same specificity as those produced by the parental B-cells, many groups have tried to use this approach to obtain cell lines expressing T-lymphocyte functions by crossing thymoma lines not expressing any measurable activity with various types of T-cell populations. Although there have been reports that hybrids could be isolated which secrete T-cell products with immunological activity, efforts to produce functionally active hybrids from cytolytic T-cells have all been unsuccessful (refs 6, 7, and M. N. and H. D. Engers, unpublished). We have fused an established, T-cell growth factor (TCGF)-dependent murine cytolytic T-lymphocyte (CTL) line with a mouse thymoma line and have obtained hybrids with cytolytic activity when we selected the hybrids in TCGF-containing medium, while hybrids isolated in the absence of growth factor showed no detectable cytolytic potential.
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Various procedures were used to derive continuously growing cytotoxic T lymphocyte (CTL) clones from a primary culture containing responder cells from immunized mice and 3-(p-sulfophenyldiazo)-4-hydroxylphenyl acetic acid (SP)- or fluorescein isothiocyanate (FL)-coupled stimulator cells. It seems likely that CTL have to undergo some change, possibly genetic, to be able to grow continuously in T cell growth factor conditioned medium in the absence of any stimulator or filler cells. The most convenient and reliable procedure to generate CTL clones with different specificities was to establish from several aliquots of a primary culture cell populations continuously growing in medium conditioned with T cell growth factor(s). Clones with different specificities segregated in the different populations. SP- and FL-specific CTL clones restricted to H-2Kk and H-2Dd and two FL-specific CTL clones with no apparent H-2 restriction are described.
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Murine cytolytic T lymphocytes can be kept in continuous culture apparently indefinitely by repeated passage in a concanavalin A-induced growth promoting medium. Some of these long-term cell lines maintain their cytolytic activity. Starting from three such populations, several cloned cytolytic T cell lines were derived and subsequently subcloned one or more times. Considerable variation in the levels of cytolytic activity was observed between different subclones; some initially active subclones lost activity with prolonged culture. In addition, one of the clones appeared to progressively lose the relative specificity demonstrated during the earlier passages of the parent cell line.
The H-Y-specific cytotoxic T-cell response requires helper cells: cells from bone marrow chimeras B6 X CBA leads to B6, B6 X CBA leads to B10.A (5R), or B6 X CBA leads to CBA are each unable to respond to H-2k male cells. If, however, cells from B6 X CBA leads to B6 or B6 X CBA leads to B10.A (5R) chimeras are adoptively transferred together with cells from B6 X CBA leads to CBA chimeras, H-Y-specific CTL restricted to H-2k can be obtained. Thus, cells from B6 X CBA leads to B6 or B6 X CBA leads to B10.,A (5R) chimeras (restricted to the left end of the H-2b haplotype) can help CTL precursors from B6 X CBA leads to CBA chimeras (restricted to H-2k). The two classes of T cells required for the CTL response to H-Y antigen are controlled by different IR genes. All H-Y-specific CTL obtained from chimeras B6 + CBA leads to B6 X CBA were found to be of B6 origin. This suggests that CTL or their precursors must express antigens encoded in the left end of the H-2b haplotype for interaction with helper cells.
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As a model of flavin-dependent biological dehydrogenation, flavin-sensitized photodehydrogenation and photodecarboxylation were studied by variation of substrate, flavin, pH and solvent. Evidence for the following rules is given. (1) When the reactive site of a photosubstrate is an alpha-carbon atom of the type CH-CO2-, decarboxylation is preferred over dehydrogenation, whereas the reverse is true for the neutral CH-CO2H. (2) Consequently these reactions do not exhibit a measurable isotope effect with C2H-CO2-, in contrast with the findings by Penzer, Radda, Taylor & Taylor [(1970) Vitam. Horm. (N.Y.) 28, 441--466], which could not be reproduced. When the substate does not contain a carboxylate group, isotope effects occur, in verification of previous reports, e.g. for benzyl alcohol C6H5-C2H20H. (3) The mechanism of flavin-sensitized substrate photodecarboxylation is assumed to consist in a primary carbanion fixation at the flavin nucleus (position 4a, 5 or 8) with concomitant liberation of CO2. This step is followed by rapid fragmentation of the adduct CH-Fl-red., provided that the substrate contains a functional and electron-donating group X, e.g. X = OH, OCH3 or NH2 (but not NH3+ !) in X CH-CO2-. (4) The minimal requirement for flavin-sensitized C-H dehydrogenation is the presence of a hydroxyl group. For example, methanol as substrate and solvent is dehydrogenated at pH sufficiently alkaline for detection of the presence of the active species CH3O-, whereas at more acidic pH substrate dehydrogenation is competing with flavin autophotolysis, which depends on the substituents in the flavin nucleus.
H-Y-specific cytotoxic T cells were first cloned in soft agar and grown over a period of 8 months in media conditioned with supernatants from mouse and rat spleen cells stimulated with concanavalin A. The specificity of cloned cells and their cytolytic potential remained essentially unchanged over the entire culture period. In addition to lysing male target cells expressing H-2Db antigens, the cytolytic cells lysed also male as well as female cells expressing H-2Dd alloantigens. Seventeen out of eighteen subclones derived from the original clone revealed the same activity. The cells divide about every 17--20 h can be obtained in large quantities.
Cytotoxic T lymphocyte (CTL) responses were obtained in vitro to cells coupled with several different haptens. The degree of lysis of target cells was dependent on the amount of hapten coupled to stimulator and target cells. Spleen cells from normal mice responded to high-hapten density cells but not to low-hapten density cells. However, spleen cells from immunized CBA mice could be stimulated in vitro by low-hapten density cells to generate effector cells able to lyse low-hapten density cells. In vitro primed responder cells could be restimulated in the presence of the original hapten-coupled stimulator cells or in the presence of supernatant from concanavalin A-stimulated mouse or rat spleen cells. Large number of hapten-specific and H-2-restricted CTL could be generated by repeated exposure to fresh supernatant.
The male-specific cytotoxic T cell response was tested in various mouse strains. We found that priming with 10(7) nonirradiated or 2 x 10(7) X-irradiated cells (2000 Rad) was similarly effective in B6 mice. We have avoided possible allogeneic effects due to priming F1 hybrids with parental cells and tested all mice after the same time interval for generation of male-specific cytotoxic T lymphocytes in vitro. Under these conditions, we found an absolute requirement for H-2b gene products, encoded left of B, in order to see male-specific responses.
The concept of satisfaction is discussed from a psychopathological viewpoint with reference to complete edentulousness or complete dentures. The authors are of the opinion that the evaluation of the results from treatment with complete dentures should allow for subjective and objective appreciations. It is suggested to classify the patients in foundedly satisfied, unfoundedly satisfied, foundedly dissatisfied, and unfoundedly dissatisfied. Suggestions for the treatment of the respective groups are deduced.
XX cells from XX/XY hemopoietic chimeras do not express male determinants in a way to render them either stimulators or targets for male-specific cytotoxic lymphocytes. XX- but not XY-responder T cells from chimeras can be activated to lyse allogeneic male target cells; T cells from normal XX mice depleted of alloreactive T cells, however, cannot be sensitized to lyse allogeneic XY targets. The results imply that T cells recognize the Y-antigen and H-2 as distinct entities, and that in chimeras, they acquire the potential to react against allogeneic XY cells.