Chemical and immunological characterization of developmentally expressed chicken erythroid surface membrane antigens

The expression of two hematopoietic-lymphoid membrane antigens, referred to as chicken fetal antigen (CFA) and chicken adult antigen (CAA) were investigated on primitive and definitive peripheral red blood cells (RBC) from different-aged chickens using chemical and immunological techniques. Differential adsorptions of antisera specific for adult RBC membrane antigens permitted the serological dissection of CAA into eight antigenic determinants. CFA and CAA were assayed by hemagglutination, hemolysis, and immune precipitation of radioiodinated surface antigens of RBC from different-aged chickens. Primitive RBC express CFA, while definitive RBC express three distinct phenotypes: CFA, both CFA and CAA, or CAA, depending on the developmental age of the chicken from which the RBC were obtained. When solubilized membrane extracts of radioiodinated peripheral RBC from chickens at 5 and 18 days embryonic development (E5 and E18, respectively), 13 days posthatch development (H13), and adult chickens were immunoprecipitated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the major antigen detected by anti-CFA sera was associated with proteins having apparent molecular weights (M_r) of 50,000 daltons (50 kd). The antigens detected by anti-CAA sera were associated with proteins having apparent M_r of 102, 81, 48, and 43 kd.     

Glutamine is a powerful effector of heat shock protein expression in Drosophila Kc cells.

We have investigated the effects of extracellular anions on the regulation of expression of the heat shock response in Drosophila Kc cells incubated in defined balanced salt solutions. Widely varying chloride concentrations had no effect on normal or heat shock protein (hsp) expression. Increasing glutamate concentrations from zero to 15 mM increased hsp expression more than 100-fold while affecting expression of non-heat-shock proteins minimally. Glutamine was 20-100-fold more potent than glutamate in supporting hsp expression, while other amino acids were less effective or supported no detectable hsp synthesis in heat shock. Inhibition of glutamine synthetase with methionine-sulfoximine resulted in very low hsp expression with glutamate and normal high level expression with glutamine, confirming the importance of glutamine. The absence of glucose and treatment with 2-deoxyglucose did not change the requirement for adequate glutamine for hsp expression. Cells heat shocked under conditions which gave very low hsp expression resumed growth when returned to normal medium as well as cells which expressed normal levels of hsps. Measurements of free amino acid levels in cells heat shocked in the presence and absence of glutamine showed a correlation between glutamine levels and amount of hsp expression. We conclude that a physiological process regulated by glutamine or a glutamine metabolite is important for normal hsp expression in heat shock conditions in Drosophila.     

B cell-associated LFA-1 and T cell-associated ICAM-1 transiently cluster in the area of contact between interacting cells.

TNP-specific B cells interact with carrier-specific T hybridoma cells in an antigen-specific, MHC-restricted manner. The formation of T cell/B cell conjugates is time and temperature dependent and results in the formation of a broad area of close contact between the interacting cells. In order to determine which surface molecules on the two cells cluster at the interaction site. T cell/B cell conjugates were formalin-fixed at different times following conjugation and were stained with antibodies directed against cell surface molecules. Results of these studies indicate that the alpha- and beta-subunits of LFA-1 on B cells transiently cluster in the area of cell contact. Maximum clustering of LFA-1 occurs at 45 min, after which time LFA-1 redistributes on the surface of the B cells. Several other B cell-associated molecules (MHC Class II, ICAM-1, Ig, B220, J11D, or CD23) do not cluster at the interaction site at any time point. T cell-associated LFA-1 does not cluster with any specific pattern, but ICAM-1 does. Maximum clustering of ICAM-1 occurs 60 to 90 min after intercellular contact. After this time, ICAM-1 redistributes on the surface of the T cells. Although both the alpha- and beta-subunits of LFA-1 cluster at the interaction site on B cells, antibodies recognizing these subunits differ in their ability to affect conjugation. One antibody recognizing the alpha chain of LFA-1 (M17/4.2) inhibits T-cell/B cell conjugation, whereas another antibody that also recognizes the alpha chain-(G-48) enhances conjugation. In contrast, an antibody that recognizes LFA-1 beta (M18/2.a.8) has no effect. An antibody that recognizes ICAM-1 (YN/1.7), the ligand for LFA-1, inhibits conjugation. These data show that, during T cell/B cell interaction. LFA-1 on B cells and ICAM-1 on T cells transiently cluster with similar, albeit not identical, kinetics to the site of cell-cell contact.     

Vacuolar proton-pumping pyrophosphatase in Beta vulgaris shows vectorial activation by potassium.

Previous work with membrane vesicles has demonstrated an absolute dependence on K+ for proton translocation by the inorganic pyrophosphatase (H(+)-PPase: EC 3.6.1.1) from the vacuolar membrane (tonoplast) of higher plants. Using intact vacuoles from sugar beet (Beta vulgaris) storage tissue, we have monitored PP1-dependent currents by patch clamp in 'whole vacuole' mode. Serial K+ substitutions were made at both tonoplast faces. The results show that K+ activation occurs only at the cytosolic face.     

cDNA encoding the chicken ortholog of the mouse dilute gene product. Sequence comparison reveals a myosin I subfamily with conserved C-terminal domains.

We report the cDNA-deduced primary structure of the chicken counterpart of the murine dilute gene product, a member of the myosin I family. Comparison of the chicken and mouse sequences reveals a distinct pattern of domains of high and low sequence conservation. An internal deletion of 25 amino acids probably reflects differential mRNA processing. Compared with other myosin heavy chain molecules, sequence similarity is highest with the MYO2 gene product of Saccharomyces cerevisiae. The MYO2 protein, implicated in vectorial vesicle transport, is homologous to the dilute protein over practically its entire length. In addition, the C-terminal domain of the dilute protein is highly similar to a putative glutamic acid decarboxylase sequence cloned from mouse brain. Alternatively, this closely related clone might represent an isoform of the dilute protein derived from a second gene, potentially involved in genetic conditions related to dilute.     

Nucleotide sequence of a cDNA encoding porcine testis 17 alpha-hydroxylase cytochrome P-450.

We describe the isolation and characterization of a cDNA encoding the complete porcine neonatal testis 17 alpha-hydroxylase/C-17,20-lyase cytochrome P-450. The deduced amino acid sequence is 509 amino acids in length.     

Beta 2-adrenoceptor stimulation increases the number of antigen-specific precursor B lymphocytes that differentiate into IgM-secreting cells without affecting burst size.

Previous studies have shown that early addition of a beta 2-adrenergic agonist to whole splenocyte cultures immunized with SRBC induced an increase in the number of cells secreting Ag-specific antibody. Because of the low frequency of Ag-specific B lymphocytes in these cultures, it has been difficult to determine the cellular mechanism by which this increase is produced. To gain insight into this cellular mechanism, the present study was designed to evaluate the responsiveness of TNP-specific B lymphocytes cultured at both high density and limiting dilution with keyhole limpet hemocyanin (KLH)-specific, IL-4-producing Th lymphocytes, TNP-KLH, and the beta 2-adrenergic agonist, terbutaline. The results showed that a maximal twofold increase in both the number of anti-TNP IgM-secreting cells and the amount of anti-TNP IgM secretion occurred in terbutaline-exposed lymphocytes after 5 days of bulk culture. This response occurred in a concentration-dependent manner and was inhibited by concomitant culture with beta-adrenoceptor antagonists. No appreciable change was measured in the level of either IgG1 secretion in terbutaline plus Ag-exposed bulk cultures or MHC class II expression on terbutaline plus Ag-exposed TNP-specific B lymphocytes as compared with Ag alone. These data raised the possibility that beta 2-adrenoceptor stimulation induced either the differentiation of a larger proportion of TNP-specific B lymphocyte precursors into anti-TNP IgM-secreting cells, or the extensive proliferation of a constant number of TNP-specific B lymphocyte precursors, or both. Limiting dilution results showed that beta 2-adrenoceptor stimulation induced a twofold increase in the number of TNP-specific B lymphocyte precursors that differentiated into anti-TNP IgM-secreting cells, without affecting the number of anti-TNP IgM-secreting cells produced by each precursor clone.     

Regulation of vacuolar h-pyrophosphatase by free calcium : a reaction kinetic analysis

The H⁺-translocating inorganic pyrophosphatase (H⁺-PPase) associated with vesicles of the vacuolar membrane (tonoplast) isolated from beet (Beta vulgaris L.) is subject to direct inhibition by Ca²⁺ and a number of other divalent cations (Co²⁺, Mn²⁺, Zn²⁺). By contrast, the H⁺-translocating ATPase (H⁺-ATPase) located on the same membrane is insensitive to Ca²⁺. Here we examine the mechanism and feasibility of regulation of the vacuolar H⁺-PPase by cytosolic free Ca²⁺ under the conditions thought to prevail in vivo with respect to Mg²⁺, inorganic pyrophosphate (PPi), and pH. The minimal reaction scheme that satisfactorily describes the effects of elevated Ca²⁺ or CaPPi on the enzyme is one that invokes equilibrium binding of substrate (Mg₂PPi) at one site, inhibitory binding of Mg₂PPi to a lower-affinity second site, binding of activator (Mg²⁺) at a third site, and direct binding of Ca²⁺ or CaPPi to a fourth site. Changes in enzyme activity in response to selective manipulation of either Ca²⁺ or CaPPi are explicable only if Ca²⁺, rather than CaPPi, is the inhibitory ligand. This conclusion is supported by the finding that CaPPi fails to mimic substrate in protection of the enzyme from inhibition by N-ethylmaleimide. Furthermore, the reaction scheme quantitatively and independently predicts the observed noncompetitive effects of free Ca²⁺ on the substrate concentration dependence of H⁺-PPase activity. The results are discussed in relation to the previous proposal that CaPPi is the principal inhibitory ligand of the vacuolar H⁺-PPase (M. Maeshima [1991] Eur J Biochem 196: 11-17) and the possibility that in vivo modulation of cytosolic free Ca²⁺ might constitute a specific mechanism for selective regulation of this enzyme, and consequently for stabilization of PPi levels in the cytoplasm of plant cells.     

A small protein in model membranes: a time-resolved fluorescence and ESR study on the interaction of M13 coat protein with lipid bilayers

Model membranes with unsaturated lipid chains containing various amounts of M13 coat protein in the -helical form were studied using time-resolved fluorescence and ESR spectroscopy. The lipid-to-protein (L/P) ratios used were > 12 to avoid protein-protein contacts and irreversible aggregation leading to -polymeric coat protein. In the ESR spectra of the 12-SASL probe in dioleoyl phosphatidylcholine (DOPC) bilayers no second protein induced component is observed upon incorporation of M13 coat protein. However, strong effects are detected on the ESR lineshapes upon changing the protein concentration. The ESR lineshapes are simulated by assuming a fixed ratio between the parallel (D) and perpendicular (D) diffusion coefficients of 4, and an order parameter equal to zero. It is found that increasing the protein concentration from L/P to L/P 15 results in a decrease of the rotational diffusion coefficient D from 3.4 × 10⁷ to 1.9 × 10⁷ s^–1. In the time-resolved fluorescence experiments with DPH-propionic acid as a probe, it is observed that increasing the M13 coat protein concentration causes an increase of the two fluorescent lifetimes, indicating an increase in bilayer order. Analysis of the time-resolved fluorescence anisotropy decay allows one to quantitatively determine the order parameters P₂ and P₄, and the rotational diffusion coefficient D of the fluorescent probe. The order parameters P₂ and P₄ increase from 0.34 to 0.55 and from 0.59 to 0.77, respectively, upon adding M13 coat protein to DOPC bilayers with an L/P ratio of 35. The rotational diffusion coefficient D of the DPH-propionic acid probe decreases on incorporating M13 coat protein, in accordance with the ESR results. It is concluded that M13 coat protein in the -monomeric state is not able to produce a long living lipid boundary shell and consequently an immobilization of the lipids. An overall effect on the lipids is induced, resulting in a reduction in the dynamics and an increase in average lipid order. The hydrophobic region of M13 coat protein is proposed to perfectly match the lipid bilayer, resulting in a relatively small distortion of the bilayer structure of the lipid system.