Location of ligand-binding sites on the nicotinic acetylcholine receptor alpha-subunit

The portions of the Torpedo californica nicotinic acetylcholine receptor (AChR) alpha-subunit that contribute to the allosteric antagonist-binding site and to the agonist-binding site have been localized by affinity labeling and proteolytic mapping. [3H]Meproadifen mustard was employed as an affinity label for the allosteric antagonist-binding site and [3H]tubocurare as a photoaffinity label for the agonist-binding site. Both labels were found in a 20-kDa proteolytic fragment generated from the AChR alpha-subunit by Staphylococcus aureus V8 protease. This 20-kDa peptide also contains the 3H-labeled 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide-reactive site and binds 125I-alpha-bungarotoxin. N-terminal sequencing established that the 20-kDa fragment began at Ser-173 of the alpha-subunit. Fluorescein isothiocyanate-conjugated concanavalin A could be bound to the second of the two major V8 cleavage products, an 18-kDa peptide. This peptide was also sensitive to treatment with endo-beta-N-acetyl-glucosaminidase H, consistent with the presence of N-linked carbohydrate on this fragment. The N terminus of this peptide was found to be Val-46 of the alpha-subunit sequence. Experiments designed to map disulfide bonds within the AChR alpha-subunit indicate that no bonds exist between the 18-kDa fragment (containing Cys-128 and Cys-142) and the 20-kDa fragment (containing Cys-192, Cys-193, and Cys-222). These results establish that the 20-kDa fragment contributes to both the acetylcholine and the allosteric antagonist-binding sites, whereas there is no evidence that the 18-kDa fragment is part of either site.     

Upper airway stability and apnea during nasal occlusion in newborn infants

Brief end-expiratory airway occlusions were performed in 22 preterm babies, 17 with and 5 without clinical apnea, and 4 full-term babies, 1 with Pierre-Robin syndrome. Airway stability was evaluated by comparing pressures measured simultaneously in the chest and nasal passages during occluded inspiratory efforts. The airway remained patent throughout all 301 trials in 20 babies during rapid-eye-movement (REM) and quiet sleep. Airway closure occurred during 31/102 trials in 6 babies (5 preterm and 1 term with Pierre-Robin syndrome), more commonly in quiet than in REM sleep. Overall and within individuals, mean closing pressures were significantly lower than the mean maximum falls in airway pressure recorded during occlusions without closure. Mixed-obstructive and obstructive apnea was significantly more frequent in babies with airway closure than in those without (5.3 +/- 4.0 vs. 0.4 +/- 0.8 episodes/h). Pauses in breathing greater than or equal to 3 s occurred during 28% of occlusions in preterm infants and 2% of occlusions in full-term babies. There was no significant difference between the mean frequency of pauses during occlusion and during the preceding control period or in the incidence of pauses in occlusions with vs. those without closure. It is concluded that the airway of most preterm and full-term babies is remarkably stable under load. Intermittent closure occurs in certain infants and may be related to airway muscle dysfunction.     

Differential inhibition of tryptophan synthase and of tryptophanase by the two diastereoisomers of 2,3-dihydro-L-tryptophan. Implications for the stereochemistry of the reaction intermediates

Oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan, which are analogs of a proposed reaction intermediate, are potent competitive inhibitors of both tryptophanase and the alpha 2 beta 2 complex of tryptophan synthase (Phillips, R. S., Miles, E. W., and Cohen, L. A. (1984) Biochemistry 23, 6228-6234). Since these inhibitors can exist in two diastereoisomeric forms, which we expected to differ in inhibitory potency, we have separated the diastereoisomers of 2,3-dihydro-L-tryptophan by preparative high performance liquid chromatography. These diastereoisomers were designated "A" and "B" in order of elution from the high performance liquid chromatography column. Diastereoisomer B is a potent competitive inhibitor of the alpha 2 beta 2 complex of tryptophan synthase with KI = 6 microM at pH 7.8 and 25 degrees C. In contrast, diastereoisomer A is a weak competitive inhibitor, with KI = 940 microM under these conditions. With tryptophanase, the situation is reversed; diastereoisomer A is a potent slow-binding competitive inhibitor of tryptophanase with KI = 2 microM at pH 8.0 and 25 degrees C, while diastereoisomer B is much weaker with KI = 1600 microM under these conditions. These results not only provide additional support for the proposal that the indolenine tautomer of tryptophan is an intermediate in the reactions catalyzed by both enzymes but also suggest that these enzymes catalyze their respective reactions via enantiomeric indolenine intermediates.     

Treatment of dialysis membranes for simultaneous dialysis and concentration

Dialysis membranes used for simultaneous dialysis-concentration required pretreatment to remove uv-absorbing compounds leached from the membranes and to reduce the absorption of protein to the membranes. This was accomplished with sodium carbonate and ethanol or with "sulfur-removal solutions." Protein determinations were made with a micro-Bradford protein reaction and with uv absorbance at 280 nm. Soluble membrane components contributed to aberrant uv spectra and altered the ratio of 280/260-nm absorbance. Simultaneous dialysis and concentration in the micro protein dialyzer-concentrator apparatus, combining aspects of thin-layer dialysis and ultrafiltration, resulted in rapid removal of salts from the protein solutions. Prior treatment of membranes reduced uncertainties in retentate recoveries, eliminated uv-absorbing components of membranes, and improved recoveries of protein.     

Identification of protein phosphatases dephosphorylating mRNP proteins from cryptobiotic gastrulae of the brine shrimp A. salina

In the cytosol of A. salina cryptobiotic gastrulae at least five protein phosphatases active on phosphorylase a have been detected by ion exchange chromatography on DEAE-cellulose. Only two of these enzymes (PP-X and PP-Y) are active in mRNP dephosphorylation. Both enzymes are insensitive to inhibitor-1 and -2 and stimulation of enzymatic activity (2.5-fold with PP-X and 6.5-fold with PP-Y) can be accomplished by ethanol treatment of the native enzymes, or freeze-thawing in the presence of 1.7% (v/v) 2-mercaptoethanol. These properties allow PP-X and PP-Y to be classified as type-2A enzymes according to the nomenclature of Cohen. This paper is the first report of protein phosphatases capable of dephosphorylating mRNP proteins.     

The essential role of L-glutamine in lymphocyte differentiation in vitro

The biochemistry of human B lymphocyte differentiation to plasma cells is incompletely understood. L-glutamine appears to be required for both lymphoblastic transformation and plasma cell formation in pokeweed-mitogen-stimulated human peripheral blood mononuclear cell cultures. Cells cultured with pokeweed mitogen in glutamine-deficient RPMI-1640 with 10% heat-inactivated and dialyzed fetal bovine serum were unable to incorporate 3H-thymidine or undergo morphologic lymphoblastic transformation assessed at 72 hours. However, 3H-thymidine incorporation could be maximally restored with as little as 0.08 mM L-glutamine or by using nondialyzed heat-inactivated fetal bovine serum, containing approximately. 1 mM L-glutamine. In subsequent cultures, using glutamine-deficient RPMI-1640 with 10% nondialyzed heat-inactivated fetal bovine serum, lymphoblastic transformation was equivalent with or without additional L-glutamine supplementation. However, only cultures with 2 mM L-glutamine supplementation underwent plasma cell differentiation as assessed by cytoplasmic staining with fluorescein-conjugated anti-immunoglobulin. When the kinetics of cellular immunoglobulin synthesis and secretion were analyzed by 3H- leucine incorporation into immunoglobulin, synthesis was 2-5 fold greater, and secretion 3-10-fold greater in cell cultures with 2 mM L-glutamine supplementation. By electron microscopy, only the glutamine-supplemented cells showed development of rough endoplasmic reticulum consistent with active immunoglobulin production. L-glutamine supplementation had no apparent effect on cell recovery, viability, % B cells, % T cells, % monocytes, or % helper and suppressor T cells. Thus, L-glutamine is essential for both lymphoblastic transformation and plasma cell differentiation. Future investigation of the selective nutritional requirements of cultured cells should yield further insights into the biochemical control of immune cell differentiation and function.     

Carbohydrate metabolism in transforming lymphocytes from the aged

There is an age-related decline in immune capacity which has been linked to a decreased response of lymphocytes to mitogens in vitro. During transformation, lymphocytes require a marked increase in energy production and biosynthesis which is supplied primarily by glycolysis. In the elderly, the glycolytic enzymes increase significantly in transforming lymphocytes at least 24 hr later than in the young and then at significantly reduced levels. Glucose utilization is also impaired in stimulated lymphocytes from the elderly but follows the impairment of glycolysis. In stimulated cells from the young, increases in glycolytic enzyme activity levels accompany sharp increases in blastogenesis while a delayed increase in glycolytic enzyme activity in the elderly is accompanied by a delay in blastogenesis. Maximal glycolytic enzyme activity levels are significantly reduced in transformed lymphocytes from the elderly though the number of transformed cells is also significantly reduced. However, glycolytic enzyme activity levels are significantly lower in the elderly than in the young even on a per transformed cell basis. Thus, this reduction cannot be attributed to the lower number of transformed cells that are present in the elderly. This defect in the increase of glycolysis in stimulated cells from the elderly suggests an intracellular mechanism which could be related to the impaired lymphocyte stimulation in vitro in the aged.     

H-2-linked Ir gene control of T cell recognition of the Sm nuclear autoantigen and the aberrant response of autoimmune MRL/Mp-+/+ mice

We have examined T cell recognition of the nuclear autoantigen Sm. Rabbit Sm-primed cells from autoimmune MRL/Mp-+/+ (+/+) mice and from all normal strains tested were able to proliferate to rabbit Sm in vitro. In contrast, the reactivity of normal strains to Sm of murine origin was genetically restricted; only H-2f strains B10.M and A.CA, and H-2s strains B10.S and A.SW could recognize mouse Sm, suggesting that responsiveness to mouse Sm was under the control of H-2-linked Ir genes. Although five Iak-bearing normal strains (B10.A, B10.A(2R), B10.BR, A/Sn, and CBA) did not recognize mouse Sm, autoimmune +/+ (Iak) mice were responders. The responsiveness of the +/+ mice to Sm was probably not due to differences in their Iak region, compared with other strains, because the Iak region of normal strains and the autoimmune +/+ strain were indistinguishable by interstrain MLC, immune response gene product function, and recognition by anti-Iak mAb. Inhibition of Sm-induced proliferation by mAb demonstrated that T cells from autoimmune +/+ mice, responder normal strains, and nonresponder normal strains recognized rabbit and mouse Sm in the context of I region-encoded products. The T cell response to Sm antigen in normal mice is therefore Ia region restricted and, for the murine antigen, under Ir gene control. Autoimmune mice that spontaneously make anti-Sm antibodies (+/+) also perceive Sm in an Ia-restricted manner, but their responder status abrogates H-2-linked Ir gene control.     

Antigen presentation in the rat. II. An Ia+ radiosensitive T cell can present antigen to primed Ia- T cells

We demonstrated previously the presence of an Ia+ (OX-6+) antigen-presenting cell within the rat T cell fraction that is capable of presenting antigen to antigen-primed OX-6-T cells. This antigen-presenting cell (T-APC) reacted with the monoclonal antibodies W3/25 and W3/13, which is known to react mainly with rat T cells. Further characterization of the T-APC indicated that the cell also reacted with the monoclonal antibody OX-19, which is highly specific for rat T cells. Moreover, the antigen-presenting function of the T-APC was sensitive to treatment with mitomycin C or gamma-irradiation (2000 rad). Under similar conditions, antigen presentation by partially purified dendritic cells or macrophages was totally resistant to these treatments. The antigen-presenting activity of gamma-irradiated T-APC was not reconstituted by the addition of the lymphokines IL 1, IL 2, or Con A supernatants. Although unirradiated T-APC were able to stimulate an MLR response, this function was also sensitive to gamma-irradiation, whereas the MLR-stimulating ability of macrophages and dendritic cells was resistant to gamma-irradiation. These data indicate that Ia+ T cells from the rat are capable of presenting antigen to antigen-primed T lymphocytes and that, in contrast to antigen presentation by macrophages and dendritic cells, the function of T-APC is gamma-radiation sensitive.