Chromophoric peptide substrates for activity determination of animal aspartic proteinases in the presence of their zymogens: A novel assay

Solid-phase synthesis was used for the preparation of pyroglutamyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (I) and glycyl-glycyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (II), two water-soluble and sensitive chromophoric substrates of chicken pepsin, hog pepsin A, and bovine spleen cathepsin D. The kinetic constants of hydrolysis of the p-nitrophenylalanyl-phenylalanyl bond of the substrates were measured by difference spectrophotometry at 308 nm (Δ? = 860 m^?1 cm^?1) and by ninhydrin colorimetry (substrate I, ?₅₇₀ = 2.31 × 10⁴m^?1 cm^?1). The pH optimum of cleavage is 5 for the pepsins and 3.7 for cathepsin D. Since all three proteinases still have a significant activity at pH 5.5–6 a new, simple assay was designed for submicrogram quantities of pepsins in the presence of pepsinogens without interference of the latter. The method is particularly suitable for the analyses of the zymogen activation mixtures.     

Buspirone: A potential atypical neuroleptic

Buspirone produces a dose-dependent but short-lived elevation in striatal dopamine (DA) metabolites in the rat. $\text{In}$$\text{vitro}$, buspirone possesses an affinity similar to sulpiride for DA receptors (3H-spiperone). A moderate affinity for α₁ receptors was also observed while buspirone was inactive at α₂, β, muscarinic and serotonin₂ receptors. This pharmacological profile as well as previous behavioral data indicate that buspirone may be a potential “atypical” neuroleptic.     

The exchange hypothesis for the formation of chromatid aberrations: an experimental test using bleomycin

The association between bleomycin-induced chromatid aberrations and BUdR-label exchange between sister chromatids was investigated in order to evaluate Revell's exchange hypothesis for the formation of chromatid aberrations. The results of this study indicate that a larger than expected proportion of chromatid breaks can be accounted for by the exchange hypothesis though not all breaks are the result of incomplete exchange.     

Unresponsiveness in hapten-specific cytotoxic lymphocytes. III. Influence of H-2 and non-H-2 gene loci on the in vitro trinitrophenyl-specific CTL response following either acute or chronic in vivo treatment with trinitrobenzene sulfonic acid

The regulation of the in vitro generation of cytotoxic T lymphocytes (CTLs) directed against hapten-modified syngeneic cells has been investigated. The results indicate that acute intravenous pretreatment with water-soluble hapten, trinitrobenzene sulfonic acid (TNBS), can either positively or negatively affect the in vitro generation of trinitrophenyl (TNP)-specific CTLs. In general, mice bearing the H-2d haplotype are most likely to develop a reduced in vitro response pattern following a single acute in vivo TNBS treatment, wheras mice bearing the H-2k or H-2b haplotypes display either unchanged or augmented in vitro response patterns. We have shown that, in addition to the influences of H-2 genes, non-H-2 genes can also influence the in vitro hapten-specific CTL response following intravenous pretreatment with water-soluble hapten. Further, in two (H-2k X H-2d) F1 combinations between an H-2k strain displaying an unchanged in vitro response pattern following acute in vivo TNBS treatment and an H-2d strain displaying a reduced in vitro response pattern following similar treatment, it was observed that a single in vivo TNBS pretreatment did not induce the unresponsive state when F1-TNP stimulator cells were used. These results suggest that the mechanisms responsible for the reduced in vitro response pattern are not dominant within the F1 environment. However, when TNP-modified parental stimulators are used, a split-response pattern is observed in cells from TNBS-treated F1 mice which reflect the response patterns of the respective parents. These latter results again emphasize the influence of gene loci on the in vitro response patterns following acute TNBS treatment. In contrast to the significant influence of H-2 and non-H-2 genes on the in vitro TNP-specific response following acute in vivo TNBS treatment, these genes do not appear to significantly influence the in vitro TNP-specific response pattern following chronic TNBS treatment. Chronic TNBS treatment renders all strains tested specifically unresponsive.     

Differentiation antigens in human T-cell activation: Evidence that anti-VH antibodies react with a membrane structure on human T Lymphocytes distinct from the antigens detected by monoclonal antibodies of the OKT and Leu series

The effect of a panel of monoclonal antibodies and heteroantibodies on T-cell proliferation in various assay systems has been examined. The antibodies tested were directed against T-cell differentiation antigens, HLA-DR antigens, and structures defined by an anti-human V_H antiserum. As the test cell system highly purified subpopulations of T-cell growth factor (TCGF)-dependent T-cell lines activated either by mitogen or antigen were used. A survey of the data indicates the following: (1) Mitogenic and antigenic triggering of T lymphocytes are mediated through partly different membrane structures. (2) Antigenic stimulation by purified protein derivative (PPD) as well as polyclonal activation induced by OKT3/anti-Leu 4 monoclonal antibodies can be inhibited by heteroantibodies raised against human immunoglobulin V_H fragments thus pointing to a possible connection between the antigens detected by these antisera. (3) There does not seem to be differences between the two major subpopulations of T lymphocytes (i.e., helper/inducer and suppressor/cytotoxic cells) as to how they respond to antigens or mitogens in the investigated assay systems. (4) A clear distinction was found between T blasts specific for PPD and allogeneic cells as compared to cytotoxic T cells (CTL), as the T4 and T8 antigens seem to be functionally important for antigen recognition among CTL but not for the blasts proliferating in response to PPD and allogeneic cells. (5) An inhibitory effect of OKT3/anti-Leu 4, OKIal, and anti-HLA-DR on TCGF-dependent growth was detected, possibly indicating a steric relationship between these antigens and TCGF receptors on mitogen-induced T blasts. (6) Soluble factors obtained after incubating adherent cells with OKIal and anti-HLA-DR antibodies seemed to have an inhibitory effect on overall T-cell proliferation stressing the importance of studying the T-cell activation process at different levels in these kinds of experiments. (7) The results further suggest a complexity in the build up of antigen receptors on the various T-effector cells, perhaps also involving receptors for growth factors, HLA-DR antigens, and receptors for the latter.     

Comparative effectiveness of lymphotoxin anticarcinogenic and tumor cell growth-inhibitory activities

Prevention of ultraviolet radiation- or chemical carcinogen-induced morphologic transformation and inhibition of tumor-producing transformed cell growth by lymphotoxin and by normal spleen leukocytes were quantitatively compared to define the antineoplastic activity spectra of these natural immune mediators. When Syrian golden hamster embryo cells seeded for colony formation in culture dishes were treated simultaneously with carcinogen and lymphotoxin, the number of morphologically transformed cell colonies was irreversibly reduced by 50% in the presence of 6 units of lymphotoxin/ml. Lymphotoxin inhibition of tumor cell growth, however, was reversible and 50% reduction in tumor cell growth in three transformed lines required 124, 330, and 477 units/ml. Thus, the anticarcinogenic activity of lymphotoxin can be 20-fold or more greater than its tumor growth-inhibitory activity. Similarly spleen leukocytes also were more effective as an anticarcinogen than as an inhibitor of tumor cell growth, consistent with previous observations that naturally occurring spleen leukocyte antineoplastic activity may result from lymphotoxin secretion.     

Modulation of endogenous prostaglandins by thymosin-α1 in lymphocytes

The effects of thymosin-α₁ on the stimulation of specific release of prostaglandin E₂ (PGE₂) from splenic lymphocytes and thymocytes were studied. Experiments were also performed to study in parallel the absolute levels of thymosin-α₁ in the blood and the induction of serum FTS activity and of azathioprine sensitivity of spleen cells from adult thymectomized (ATx) mice. A significant difference in the release of PGE₂ between normal splenocytes and splenocytes from ATx mice was observed. Thymosin-α₁ at certain concentrations was able to stimulate PGE₂ release from lymphocytes of ATx mice while inhibiting release in lymphocytes of normal mice. Also, thymocytes were stimulated to release PGE₂ after incubation with α₁ in a manner similar to that seen in spleen cells of ATx mice. Approximately the same concentration of α₁ was found to also correct the low azathioprine sensitivity of splenocytes from ATx mice. Determinations of FTS-like activity in the blood and the pharmacokinetics of α₁ after administration of this synthetic molecule show a clear dissociation. A maximum peak of α₁ activity was obtained after 1 hr, while maximal FTS-like activity was observed after 24 hr. The inhibition of the induction by α₁ of FTS-like activity and of Thy 1.2 antigen by indomethacin suggests that the action of α₁ requires prostaglandin biosynthesis.     

Suppression of natural killer activity in human blood and bone marrow cultures by bone marrow-adherent OKM1-positive cells

Maintenance and regulation of natural killer (NK) cell activity in human bone marrow cultures were studied using K562 leukemia cells as targets. Culture of bone marrow cells in medium supporting long-term generation of myeloid cells resulted in a rapid loss of NK activity in 1-3 days. In contrast, antibody-dependent cytotoxicity to an NK-resistant tumor was maintained for more than 7 weeks. Horse serum, a component of the myelopoietic culture medium, was found to diminish NK cytotoxicity of blood and bone marrow cultures whereas hydrocortisone supplement did not. In addition, an adherent cell is present in bone marrow which greatly inhibits NK activity. Nonadherent bone marrow cells exhibited higher cytotoxicity than unfractionated cells at all days of culture; adherent cells were not cytotoxic to K562. Purified adherent marrow cells inhibited the cytotoxic capacity of nonadherent blood or marrow mononuclear cells during coculture. Indomethacin, an inhibitor of protaglandin synthesis, augmented levels of NK activity in cultures of bone marrow cells, indicating that macrophages may be suppressing this effector function via prostaglandins. Further identification of the adherent suppressor cells came from experiments in which suppression was prevented by treatment of the adherent cells with monoclonal OKM1 antibody plus complement. This study shows that bone marrow-adherent OKM1-positive cells, presumably macrophages, negatively regulate NK activity, and it defines conditions for analysis of the generation and/or positive regulation of NK cells in human bone marrow.     

Rabbit kidney function in vitro: the effect of colloids, energy substrate, a vasodilator, perfusion pressure, and bovine serum albumin

An in vitro perfusion system at 37 degrees C for the assessment of rabbit kidney function is described. The purpose of this assay system is to evaluate the effects of cryobiological manipulation on kidney function. The effect of the colloids dextran (MW = 70,000, 80,000, and 180,000) in the perfusate at 110 mm Hg were compared to a reduced perfusion pressure, colloid-free perfusate. Better function was obtained at lower perfusion pressure with the colloid-free perfusate. Less damage was noted histologically on light and electron microscopy. Investigation of energy substrates on rabbit kidney function demonstrated that butyrate, or lactate, in addition to glucose resulted in increased sodium and glucose reabsorption over glucose alone. Substrate-free perfused kidneys exhibited depressed Na transport. Lactate, and to some extent butyrate, decreased net glucose utilization. An alpha-adrenergic blocking agent, isoxsuprine, in the initial flush solution did not appear to be beneficial. An increase of perfusion pressure from 50 to 75 mm Hg resulted in an increase in GFR. Tubular function was enhanced by inclusion of small amounts of BSA in the perfusate.     

Presence of mast cell precursors in the yolk sac of mice

Concentration of mast-cell precursors in hematopoietic tissues of mouse embryos was evaluated by a limiting dilution method. Cells from yolk sacs, livers, and bodies of (WB x C57BL/6)F1 (hereafter called WBB6F1)- +/+ embryos were injected directly into the skin of adult WBB6F1-W/Wv mice which were genetically depleted of tissue mast cells. Concentration of mast-cell precursors was calculated from the proportion of injection sites at which mast cells did not appear. Since the concentration of mast-cell precursors in the yolk sac was about 30 times as great as that of embryonic body at Day 9.5 of the pregnancy, the mast-cell precursors seemed to be generated within the yolk sac. The concentration in the yolk sac reached the maximum level at Day 11, and then dropped markedly at Day 13. In contrast, mast-cell precursors increased from Day 11 to Day 15 in the fetal liver. As a result, the concentration of 11-day yolk sacs was comparable to that of 15-day fetal liver. Although intravenous injection of 15-day fetal liver cells (2 x 10(6)) rescued the general mast-cell depletion of WBB6F1-W/Wv mice, the intravenous injection of the same number of 11-day yolk sac cells did not rescue it. In contrast with fetal livers, yolk sacs scarcely contained hematopoietic stem cells which were measured by spleen colony formation. Therefore, the mast-cell precursors of the yolk sac may not originate from such stem cells.