Suppressor T cells induced by soluble immune complexes can adoptively transfer inhibition of cytophilic antibody receptors on macrophages.

Immune complexes (soluble antigens of L1210 and antibody to L1210) when given to allogeneic C3H mice generated suppressor cells that inhibited receptors for cytophilic antibody on macrophages. Thymocytes or nylon-nonadherent splenic T cells (4 × 10⁷) from immune-complex-treated mice transferred this suppressive activity when injected into normal syngeneic mice. Maximal suppression of macrophages occurred 4 to 6 days after transfer. In contrast, even 5 × 10⁷ nylon-adherent, non-T spleen cells from immune-complex-treated (“suppressed”) mice failed to induce macrophage suppression in the syngeneic recipients. When T-cell-depleted “B” mice were used as recipients, neither thymocytes nor splenic T cells from suppressed mice were able to transfer suppressive activity. However, the admixture of 2 × 10⁷ normal syngeneic thymocytes with 4 × 10⁷ thymocytes from suppressed mice restored the latter's ability to elicit suppression of macrophages in T-cell-deprived recipients. Peritoneal monocytes from recipients of suppressor thymocytes (to L1210) could not attach cytophilic antibody to L1210 but could attach cytophilic antibody to EL-4 and sheep erythrocytes. Thus, suppressor T cells induced by immune complexes can transfer immunologically specific macrophage suppression (inhibition of cytophilic antibody receptors) to syngeneic recipients. The suppressor cells required the cooperation of normal T cells, suggesting either recruitment of suppressor cells from, or a helper effect by, the normal T cells, in order to produce their effect.     

Communication of gender from human breath odors: Relationship to perceived intensity and pleasantness

During five consecutive daily test sessions, 10 men and women rated the relative intensity and pleasantness of breath odors from 14 males and 19 females on a no-oral-hygiene regimen. In addition, the likely gender of the donor of each odor was also estimated. The breath odors of males were rated, on the average, as more intense and less pleasant than the breath odors of females. Women consistently gave lower pleasantness ratings to the odors than did men. Both the male and female judges assigned the breath odors to the correct gender classes at a frequency unlikely due to chance, although the females were more accurate in this regard. An inverse relation between breath odor intensity and pleasantness was noted. Systematic changes in the rated intensity and pleasantness of the odors were present across the 5 days of the study period. These data suggest that differences exist between the breath odors of men and women, and that humans, like many other mammals, may be capable of assessing gender from oral odors. However, such assignments conceivably reflect the strategy of assigning stronger and less pleasant odors to the male category, and weaker and less unpleasant odors to the female category, regardless of the true sex of the odor donor.     

Modification of the C-terminal octapeptide of cholecystokinin with a high-specific-activity iodinated imidoester: preparation, characterization, and binding to isolated pancreatic acinar cells

Proteoglycans were separated by high-performance liquid chromatography (HPLC), using two coupled Aquapore columns containing glycerylpropylsilane groups covalently linked to large-pore (50–100 nm) silica spheres. This two-column HPLC system was effective in separating cartilage proteoglycan aggregates and monomers, without altering their biochemical integrity. This system was also effective in resolving small amounts of isotopically labeled proteoglycans synthesized by cultured mammalian cells. The small sample size, short analysis time, and high reproducibility represent improvements in the study of proteoglycans over conventional soft-gel chromatography.     

Migration of recently divided B and T lymphocytes to peritoneum and lung

It is recognized that a population of newly divided (or young) cells migrate preferentially to inflamed foci. It has been shown that a large proportion of lymphocytes residing in the bronchoalveolar airspaces of rat are recently divided cells and that blood may be an important source of these cells. To further delineate how blood may contribute to lymphocyte subpopulations in inflamed peritoneum and lung, a comparison of the capacity of recently divided T and B cells to migrate from blood to inflamed peritoneum and lung was made. To label young lymphocytes, DA strain donor rats were given Initiated thymidine by vein in vivo for 7 days. After thoracic duct drainage, the following labeled cell populations were adoptively transferred by vein into syngeneic recipients: (i) unseparated thoracic duct lymphocytes (TDL), (ii) enriched T cells (>90%) or B cells (>80%) recovered after passage of TDL through nylon columns, and (iii) thoracic duct lymphocytes (> 99% B cells) obtained from “B rats” that were prepared by X irradiation, thymectomy, and bone marrow reconstitution. T and B cells were identified by specific heterologous antisera. The percentage recovery of labeled lymphocytes in the recipients with inflamed peritoneum or lung aspirates was determined from cell counts and autoradiographs. The studies indicated that (a) both labeled T and B cells migrated to inflamed peritoneum and lung; (b) labeled B cells migrated to peritoneum and lung better than did labeled TDL or T cells; and (c) labeled lymphocytes did not migrate to unstimulated peritoneum. The enhanced migration of newly divided B lymphocytes to inflamed peritoneum and normal lung (a site that is likely under chronic antigenic stimulation) was unexpected, but may provide additional information on the relative contribution of these subpopulations in the immune inflammatory response.     

Differential response of satellite cells and embryonic myoblasts to a tumor promoter

Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) reversibly suppressed myotube formation and expression of acetylcholine receptors in cultures of Day 15 mouse embryo presumptive myoblasts, but was totally ineffective in cultures of adult mouse satellite cells. A subpopulation of TPA-resistant myogenic cells became apparent in cultures prepared from older embryos or newborn mice. Thus, limb presumptive myoblasts are a heterogeneous population, and part of the distinct TPA-resistant subpopulation may represent satellite cell precursors.     

The role of Ia antigens and Fc receptor-bearing T-cells in the inhibition of macrophage receptors for cytophilic antibody induced by soluble immune complexes

Soluble antigen-antibody complexes composed of 3 M KCl-extracted L1210 antigens and alloantibody to L1210 given to C3H mice caused immunosuppression in the mice. This was reflected in part by the inhibition of cytophilic antibody receptors on macrophages which could be used as a measure of the suppression. Thymocytes or splenic T cells from mice treated with immune complexes could adoptively transfer the suppression to normal syngeneic mice. These cells, which we have termed suppressor inducers, were found to be Ia positive: specifically, I-A⁺, I-J^?. Thus, treatment of the inducers with anti-la or anti-I-A antibodies and complement in vitro abrogated their ability to transfer the suppression to normal mice. In contrast treatment with anti-I-J serum and complement had no effect. Through a similar approach, the cooperating (acceptor) T cells were found to be I-A⁺, I-J^?. Pretreatment of mice with anti-Ia or anti-I-A serum before the administration of antigen-antibody complexes prevented the inhibition of macrophages. This was due at least in part to steric hindrance of adjacent Fc receptors on the FcR⁺ T cells with which the complexes interacted. Early interaction of immune complexes with FcR⁺ T cells was in fact demonstrated directly by the inability of the complexes to induce suppression when FcR⁺ T cells were depleted. The thymocytes or splenic T cells from anti-Ia-pretreated mice failed to transfer the suppression to recipient mice. In contrast, treatment with either anti-Ia or anti-I-A after the immune complexes did not abrogate the generation of suppressor inducers. Treatment of normal recipient mice with anti-Ia serum in vivo before they received the suppressor inducer cells did not prevent cooperation between the two types of cells. By the same token, blocking of Ia antigens of the inducers in vitro with anti-Ia serum (without complement) also did not impair the cooperative interaction. These results indicate that antigen-antibody complexes generate I-A-positive, I-J-negative T-suppressor inducer cells from FcR⁺ naive T cells. These in turn interact with Ia-positive (I-A⁺ and I-J^?) normal thymocytes or spleen T cells. This interaction most likely generates the ultimate suppressor T cells that suppress cytophilic antibody receptors on macrophages in vivo. However, the I-region determined antigens did not appear to be directly involved in the T-T interaction of suppressor inducer and acceptor cells.     

Regulation of mouse oocyte meiotic maturation: implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis

Mouse oocytes are reversibly inhibited from resuming meiotic maturation in vitro by cAMP phosphodiesterase inhibitors such as 3-isobutyl-1-methyl xanthine (IBMX) and cAMP analogs such as dibutyryl cAMP (dbcAMP). Oocytes cultured in IBMX-containing medium were transferred to and cultured in IBMX-free medium for various periods of time prior to their return to either IBMX- or dbcAMP-containing medium. Results from these experiments defined a period of time in which oocytes became committed to resuming meiosis. Forskolin, which elevated the intracellular oocyte cAMP concentration, transiently inhibited oocytes from resuming meiosis. Levels of cAMP were determined in oocytes incubated in medium that allows resumption of meiosis. The level of oocyte cAMP decreased significantly during the time in which oocytes become committed to resuming meiosis. This decrease in oocyte cAMP was not observed in oocytes inhibited from resuming meiosis by IBMX. In addition, cAMP levels were determined in preovulatory antral follicles, cumulus cell-oocyte complexes, and oocytes during gonadotropin-induced resumption of meiosis in vivo. A decrease in oocyte cAMP preceded resumption of meiosis as manifested by germinal vesicle breakdown (GVBD). This decrease apparently occurred before or during a period of time in which follicle and cumulus cell cAMP were increasing. Associated with commitment to resume meiosis was a characteristic set of changes in oocyte phosphoprotein metabolism that preceded GVBD. These changes are, to date, some of the first reported biochemical changes that precede GVBD. Results from these experiments are discussed in terms of a possible role cAMP may play in regulation of resumption of meiosis in mammals.     

A kinetic study comparing the light-reversal properties of carbon monoxide inhibition of ethylmorphine, benzphetamine, and 7-ethoxycoumarin dealkylation with those of hydroxylation of 17-hydroxyprogesterone and testosterone

The light-reversal properties of carbon monoxide (CO) inhibition of the dealkylation of benzphetamine, ethylmorphine, and 7-ethoxycoumarin by microsomes from phenobarbital (PB)-induced rat livers were compared with those of the 6 beta-, 7 alpha-, and 16 alpha-hydroxylations of testosterone by the same rat hepatic microsomes and C-21 hydroxylation of 17-OH progesterone by steer adrenal microsomes. CO inhibited all reactions studied to essentially the same degree. The significant finding was that the dealkylations were reversed most effectively by light of wavelengths between 440 and 445 nm, rather than around 450 nm, the optimal wavelength for steroid hydroxylations. Moreover, the dealkylations required several-fold higher light intensities for equivalent light reversal. These studies suggest that the heme protein-CO complex responsible for dealkylations has a spectrum corresponding to the shape of the pass band of the 445-nm filter, whereas that of the steroid hydroxylations has its light-reversal maximum at 450 nm and appears to be broader. The measurable differences in the light-reversal properties between the monooxygenations of two groups of substrates, (i) dealkylations and (ii) hydroxylations of lipid substrates, furnish biophysical properties that allow a better characterization of microsomal monooxygenases which should be of value in forwarding progress in the study of these systems.     

Lever pressing for pups: Evidence for hormonal influence upon maternal behavior of mice

Five experiments were performed to determine the extent to which hormones influence maternal behavior of the female mouse. The number of pup-reinforced lever presses served as the measure of maternal behavior. In Experiment 1 it was found that postpartum mice performed significantly more pup-reinforced lever presses than intact or ovariectomized virgins. The second experiment showed that the augmentation of responding was unique to the postpartum period since responding was not facilitated during the last three days of pregnancy. Experiment 3 established that the postpartum augmentation of responding was mediated by the termination of pregnancy and not by parturition per se. It also was observed that the earlier during gestation pregnancy was terminated the greater the decline in response rate. Experiment 4 proved that ovarian secretions play a critical role in the pregnancy-terminated facilitation of pup-reinforced lever pressing. The final experiment demonstrated that the termination-of-pregnancy effect on responding in all likelihood is mediated by the synergistic actions of estrogen stimulation and progesterone withdrawal. The results show that the maternal behavior of the mouse, like that of the rat, is responsive to hormonal stimulation.     

Characterization of an immune suppressor from transformed human trophoblastic JEG-3 cells

Cultured human choriocarcinoma JEG-3 cells secrete an immunosuppressor that inhibits lymphocyte proliferation stimulated by either an antigen or a mitogen. In this study, the immunosuppressive factor was characterized by three methods: ion-exchange and exclusion chromatography, partition in organic solvents, and thin-layer chromatography on silicic acid. This JEG-3 cell factor appeared to be a protein complex of about 150,000–200,000 Da that contained an immunologically active polar lipid. The structural and functional characteristics of JEG-3 cell immunosuppressor are similar if not identical to those of SIF, a suppressor lymphokine derived from T cells. These secretions from transformed trophoblastic cells may correspond to normal placental products or represent a function of malignant cells.     

Biochemical and morphological characterization of a plasma membrane-enriched fraction from bovine parathyroid cells

A fraction of enriched plasma membranes from bovine parathyroid cells has been prepared by differential centrifugation. Biochemical characterization shows that this fraction has a specific activity enrichment of 7.2-fold in ouabain-sensitive Na+-K+ ATPase, and 3.5-fold in 5'-nucleotidase. Less than 4% of the total mitochondria and lysosomes are present within the plasma membranes, while microsomal contamination accounts for 14% of total specific activity. Parathyroid hormone radioimmunoassay also reveals the presence of some secretory granules within the plasma membrane fraction. The characteristic morphological aspect of the unusual surface membrane is shown by freeze-fracture electron microscopy. In the enriched pellets, vesicles identified as having a plasma membrane origin have variable sizes, and 50% show an inside-out conformation. Even though the plasma membrane fraction described herein is not absolutely free from contamination by other subcellular components, this protocol represents the first attempt to purify surface membrane from parathyroid tissue and provide the starting material for understanding, at a molecular level, the properties of extracellular Ca2+ regulation and its coupling with secretion of parathyroid hormone.     

Local production of Ia-inducing activity in experimental immunogenic uveitis

The production of soluble lymphokine products of T lymphocytes was studied in the aqueous of rabbits with uveitis induced by intravitreal injection of 2.0 mg of ovalbumin. Aqueous from uveitic eyes demonstrated Ia-inducing activity at a dilution of 1:50 which diluted out at 1:250. The control aqueous and sera from normal or uveitic animals were negative. While production of interleukin 2(IL-2) in uveitic eyes could not be demonstrated in vitro, control rabbit concanavalin A-induced supernatants of nylon-wool-purified T cells contained both Ia-inducing and IL-2 activity. These data demonstrate antigen-driven production of a lymphokine activity in the aqueous of the uveitic eye. Local production of lymphokine may be a potent mechanism for amplification of the inflammatory process in uveitis.     

Expression of creatine kinase isoenzyme during oogenesis and embryogenesis in the mouse

Creatine kinase activity was discovered in the growing mouse oocyte and in the preimplantation embryo. Changes in the enzyme activity during the growth and maturation of the egg and during the development of the embryo up to the blastocyst stage were determined. Close similarity of the protein to the brain-type isoenzyme of creatine kinase was established immunochemically. The kinetic parameters of the brain-type isoenzyme (M. R. Iyengar, C. E. Fluellen, and C. W. L. Iyengar, 1982, J. Muscle Cell Motil. 3, 231–246) and the pattern of development-associated changes in activity suggest a possible role for creatine kinase in maintaining the reported high ATP/ADP ratio (L. Ginsberg and N. Hillman, 1975, J. Reprod. Fertil. 43, 83–90), which is essential for the biosynthetic activities of the embryo.     

Isolation of myelin basic protein-reactive T-cell lines from normal human blood

T-Cell lines which responded by proliferation to the autoantigen, myelin basic protein (MBP), were isolated from the blood of six of nine normal humans. These T-cell lines could be maintained in in vitro culture for up to 2 months through the use of Interleukin 2 and repeated MBP stimulation. Optimal antigen-induced proliferation required both antigen and antigen-presenting cells found in the adherent cell population of autologous peripheral blood mononuclear cells (PBM). The T-cell lines were predominantly of the helper phenotype (OKT3+, OKT4+, OKT8-) and responded to both human and guinea pig myelin basic protein.     

Comparative kinetic studies of the dealkylation reaction and steroid hydroxylations in various oxygen and carbon monoxide:oxygen atmospheres

The time-course kinetics of the cytochrome P-450-catalyzed dealkylations of the exogenous compounds benzphetamine, ethylmorphine, codeine, and 7-ethoxycoumarin were compared to the hydroxylation of the endogenous compound testosterone. Using liver microsomes from phenobarbital-induced rats, the time course of the demethylations of ethylmorphine, codeine, and especially benzphetamine was characterized by a fast initial phase of enzymatic activity and then a steady decline in the rate throughout the remainder of the reaction. In contrast, under the same experimental conditions, both the dealkylation of 7-ethoxycoumarin and the hydroxylation of testosterone showed no initial fast phase of activity and a constant rate of product formation for most of the remainder of the time course. The difference also held for the carbon monoxide inhibition studies in which the degree of inhibition of the demethylation reactions by a variety of CO:O₂ mixtures was time dependent, in contrast to the constant, time-independent degree of CO inhibition of the other two reactions. The kinetics of the demethylation reactions could not be explained by enzyme destruction, back reaction, or product adduct formation and were further confirmed by measurements of the rate of O₂ utilization and NADPH oxidation. The complexity of the demethylation reaction should be taken into consideration in any detailed studies of the monooxygenation reaction system.     

Azodipyrroles of unconjugated and conjugated bilirubin using diazotized ethyl anthranilate in dimethyl sulfoxide

We have developed a diazotization technique in which both conjugated and unconjugated bilirubin react completely. The method represents a crucial modification of the ethyl anthranilate diazo reaction originally described by K. P. M. Heirwegh, J. Fevery, J. A. T. P. Meuwissen, and J. de Groote (1974, Methods Biochem. Anal.22, 205–250). In the presence of dimethyl sulfoxide (2 ml/ml of sample and diazo reagent), conjugated and unconjugated bilirubin in human serum and human, rat, and mouse bile reacted rapidly and completely. The azopigments were stable for at least 4 h. Addition of human serum to unconjugated bilirubin, bilirubin monoglucuronide, and human bile did not influence azopigment formation. Because the reaction solution was optically clear, total azopigments could be measured by spectrophotometry or separated and quantitated by high-performance liquid chromatography without prior extraction into nonpolar solvents. Alternatively, the pigments could also be extracted into 2-pentanone for analysis by thin-layer or high-performance liquid chromatography. This method allows the quantitation of total bilirubin and analysis of individual ethyl anthranilate azopigments after a single diazotization step.     

Detection of autologous mixed lymphocyte reaction responding cells and their precursor frequency in NZB mice

The autologous mixed lymphocyte reaction (AMLR) can be detected in older NZB mice after treatment of the responding cell population with monoclonal anti-I-A^d and complement and supplementation of the culture medium with T-cell growth factor (TCGF) from young animals. The addition of TCGF to cultures containing responding cells alone that had not been pretreated with anti-I-A plus complement resulted in high levels of background proliferation. This is indicative of a high number of preexisting I-A-positive, activated, TCGF-responsive T cells in these mice. These activated cells could also be removed by treatment with anti-I-A antibody and panning on anti-mouse Ig plates, or by BUdR and light killing of those cells proliferating in the presence of TCGF or purified IL-2. Prior treatment of the responding cells with anti-Lyt 2 and complement did not effect the AMLR. An NZB AMLR responding cell line was established using these methods. This line retained haplotype specificity in a proliferation assay. Limiting dilution analysis of the precursor frequency of AMLR responding cells in the nonautoimmune C58 and BALB/C strains in culture medium with TCGF gave a frequency of between 1 in 35,000 and 1 in 88,000. In young, AMLR-positive, NZB mice, supplementation with TCGF yielded precursor frequencies within the normal range. In older NZB mice, the addition of TCGF resulted in increased background proliferation of preactivated, IA⁺ T cells. After removal of these cells with anti-I-A plus complement, AMLR responding cells were found at normal frequency levels when stimulated in the presence of TCGF. In the oldest animals tested (greater than 18–20 weeks), normal precursor frequencies could not be demonstrated even after this treatment, representing a true decline in the AMLR responding cell number. AMLR deficiency in NZB mice appears therefore to be the result of the combined effects of decreased lymphokine production, excessive T-cell activation, and finally decreased numbers of AMLR responding cells.     

Light-induced red shift of the Qx absorption band of light-harvesting bacteriochlorophyll in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides

In chromatophores from Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata, the Q_x band(s) of the light-harvesting bacteriochlorophyll (BChl) (λ_max ~590 nm) shifts to the red in response to a light-induced membrane potential, as indicated by the characteristics of the light-minus-dark difference spectrum. In green strains, containing light-harvesting complexes I and II, and one or more of neurosporene, methoxyneurosporene, and hydroxyneurosporene as carotenoids, the absorption changes due to the BChl and carotenoid responses to membrane potential in the spectral region 540–610 nm are comparable in magnitude and overlap with cytochrome and reaction center absorption changes in coupled chromatophores. In strains lacking carotenoid and light-harvesting complex II, the BChl shift absorption change is relatively smaller, due in part to the lower BChl/reaction center ratio.In the carotenoid-containing strains, the peak-to-trough absorption change in the BChl difference spectrum is 5–8% of the peak-to-trough change due to the shift of the longest-wavelength carotenoid band, although the absorption of the BChl band is 25–40% of that of the carotenoid band. The responding BChl band(s) does not appear to be significantly red-shifted in the dark in comparison to the total BChl Q_x band absorption.     

Modulation of surface proteins by calcium in Chinese hamster lung cells

Investigations on the role of calcium in regulation of cell morphology of Chinese hamster lung cells (V79) revealed that cells grown with additional calcium (5 mM) in the growth medium (Ham's F12) adhere more tightly to the substratum than those grown in F12 alone. Additional calcium in the medium did not cause any changes in the structural membrane proteins or glycoproteins. Radioiodination of the surface membrane proteins of cells grown with or without additional calcium showed distinct differences in the labeling profile. The most striking change observed in cells grown with additional calcium was a very heavily labeled protein band at 70 K molecular weight. Two bands at approx. 100 K and 42 K were also heavily labeled. In contrast, the amount of radioactivity of a protein band at 52 K decreased in the cells grown in additional calcium. In general, cells grown with additional CaCl² were better iodinated than those grown in growth medium alone. The results demonstrate that calcium modulates surface proteins of V79 cells and this modulation may account for the changes observed in the cell morphology.     

Ongoing DNA synthesis continues in young human diploid cells (HDC) fused to senescent HDC, but entry into S phase is inhibited

Previous experiments have shown that when young human diploid cells (HDC) were fused to senescent HDC, neither nucleus synthesized DNA. This paper demonstrates that when young HDC are in S phase at the time of fusion to senescent HDC, they do make DNA in heterodikaryons; therefore, ongoing DNA synthesis is not inhibited by senescent cells. On the other hand, entry into S phase is inhibited: young HDC nuclei in G1 phase do not make DNA in heterodikaryons with senescent HDC.