The murine lymphocyte receptor for IgE. V. Biosynthesis, transport, and maturation of the B cell Fc epsilon receptor

The post-translational processing and maturation of the receptor for IgE (Fc epsilon R) on murine hybridoma B cells were studied to determine the carbohydrate content and the importance of processing events in cell surface expression and ligand (IgE) binding ability. Endo and exoglycosidase treatment demonstrated that the mature receptor is composed of two to three complex-type N-linked oligosaccharides and contains sialic acid. Pulse-chase experiments indicated that the receptor is synthesized as a 44,000 dalton precursor that begins to be processed by 1 hr to the mature 49,000 dalton form, and the latter is expressed at the cell surface by 2 hr. It was determined that the processing included the conversion of N-linked oligosaccharides to the complex type as well as an additional processing event, because in the presence of tunicamycin, the receptor is synthesized as a 36,000 dalton precursor that is processed to a 38,000 dalton species. Analysis of the effects of tunicamycin treatment and endo F digestion on soluble Fc epsilon R isolated from cell supernatants demonstrated the existence of several m.w. species of Fc epsilon R fragments, and indicated that only the higher m.w. fragments were N-glycosylated. The use of several inhibitors of the N-linked carbohydrate processing pathway demonstrated that the addition of core N-linked side-chains, but not their processing to the complex type, is required for cell surface expression of Fc epsilon R. Also, processing of N-linked carbohydrate is not required for ligand binding activity. Finally, IgE affinity chromatography indicated that the 49,000 and 38,000 dalton (tunicamycin) Fc epsilon R bind IgE more effectively than their precursor forms, 44,000 and 36,000 daltons, respectively, indicating that a processing event independent of N-linked glycosylation is necessary for optimal ligand binding activity.     

Monoclonal antibody specific for T cell-derived human IgE binding factors

A B cell hybridoma secreting monoclonal antibody against human IgE binding factors was obtained by immunization of BALB/c mice with partially purified IgE binding factors, and fusion of their spleen cells with SP-2/0-AG14 cells. The monoclonal antibody bound all of the 60,000, 30,000, and 15,000 dalton IgE binding factors from two T cell hybridomas and those from activated T cells of a normal individual. The antibody bound both IgE-potentiating factors, which had affinity for lentil lectin, and IgE-suppressive factors, which had affinity for peanut agglutinin. However, the monoclonal anti-IgE-binding factor bound neither Fc epsilon R on RPMI 8866 cells nor IgE binding factors from the B lymphoblastoid cells. A monoclonal antibody against Fc epsilon R on B cells (H107) bound the 60,000 and 30,000 dalton IgE binding factors from both T cell hybridomas and RPMI 8866 cells but did not bind the 15,000 dalton IgE binding factors from either T cells or B cells. The results indicate that T cell-derived IgE binding factors have a unique antigenic determinant that is lacking in both Fc epsilon R on B cells and B cell-derived IgE binding factors. The anti-IgE binding factor and anti-Fc epsilon R monoclonal antibodies both failed to stain cell surface components of IgE binding factor-producing T cell hybridomas. However, both antibodies induced the T cell hybridoma to form IgE binding factors. The results suggest that the T cell hybridomas bear low numbers of Fc epsilon R that share antigenic determinants with IgE binding factors secreted from the cells.     

5-aza-C-induced changes in the time of replication of the X chromosomes of Microtus agrestis are followed by non-random reversion to a late pattern of replication

Treatment with 5-azacytidine (5-aza-C) causes an advance in the time of replication and enhances the DNase-I sensitivity of the inactive X chromosome in Gerbillus gerbillus fibroblasts. We found that these changes were not stably inherited and upon removal of the drug the cells reverted to the original state of one active and one inactive X chromosome. In order to determine whether this reversion was random, we used a cell line of female Microtus agrestis fibroblasts in which the two X chromosomes are morphologically distinguishable. In this work we show that the reversion to a late pattern of replication is not random, and the originally late replicating X chromosome is preferentially reinactivated, suggesting an imprinting-like marking of one or both X chromosomes. The changes in the replication pattern of the X chromosome were associated with changes in total DNA methylation. Double treatment of cells with 5-aza-C did not alter this pattern of euchromatin activation and reinactivation. A dramatic advance in the time of replication of the entire X linked constitutive heterochromatin (XCH) region was however, observed in the doubly treated cells. This change in the replication timing of the XCH occurred in both X chromosomes and was independent of the changes observed in the euchromatic region. These observations suggest the existence of at least two independent regulatory sites which control the timing of replication of two large chromosomal regions.Deceased on 2 Jan. 1987     

Cloning of a human S-phase cell cycle gene: use of transient expression for screening.

We report here the cloning of a human cell cycle gene capable of complementing a temperature-sensitive (ts) S-phase cell cycle mutation in a Chinese hamster cell line. Cloning was performed as follows. A human genomic library in phage lambda containing 600,000 phages was screened with labeled cDNA synthesized from an mRNA fraction enriched for the specific cell cycle gene message. Plaques containing DNA inserts which hybridized to the cDNA were picked, and their DNAs were assayed for transient complementation in DNA transformation experiments. The transient complementation assay we developed is suitable for most cell cycle genes and indeed for many genes whose products are required for cell proliferation. Of 845 phages screened, 1 contained an insert active in transient complementation of the ts cell cycle mutation. Introduction of this phage into the ts cell cycle mutant also gave rise to stable transformants which grew normally at the restrictive temperature for the ts mutant cells.     

A cyanogen bromide fragment of S4 that specifically rebinds 16S RNA.

Escherichia coli ribosomal protein S4 was subjected to cyanogen bromide cleavage and was found to generate a complete cleavage product capable of rebinding 16S rRNA. This fragment, consisting of residues 1-103, was found to bind with an apparent association constant of 11 microM-1. This fragment was used in place of S4 in an in vitro reconstitution experiment. Although the particles formed had a protein composition not significantly different from reconstituted 30S ribosomal subunits, their sedimentation behavior was more like that of particles reconstituted without S4. These results indicate to us that, although residues 104-203 of S4 are involved in the assembly of the 30S ribosome, they are not necessary for the binding of S4 to 16S RNA. Taken with previous results, the domain of S4 involved in specific binding of 16S RNA can be confined to residues 47-103.     

Treatment of asthma by a controlled-release theophylline tablet formulation: a review of the North American experience with nocturnal dosing

As many as 80 percent of asthmatics experience nighttime or early-morning episodes, which are difficult to treat and potentially fatal. The greater-than-normal amplitude of circadian airflow variation in many asthmatics contributes heavily to the genesis of the early 'morning dip'. Beta-agonists and corticosteroids are of limited usefulness in nocturnal asthma, and slow-release theophylline drugs, while potentially effective, vary in 24-hr blood profile and hence their influence on nocturnal episodes. Traditional 12-hr 'symmetric' theophylline regimens, instead of meeting increased nocturnal demands, may actually produce lower night- than daytime blood levels. On the other hand, appropriately timed administration of a once-daily theophylline drug might provide maximum blood levels when needed and help stabilize 24-hr airflow. Accumulated data, summarized in this review, demonstrate the chronotherapeutic potential of single-daily evening doses of a controlled-release theophylline preparation (Uniphyl 400-mg tablets) in nocturnal and early morning asthma. Nighttime blood concentrations with this regimen were higher than were those with Theo-Dur tablets, B.I.D., in the same total daily doses, or with once-daily morning Uniphyl administration. In fed and fasted subjects, evening administration of Uniphyl 400-mg tablets was well tolerated and did not lead to 'dose dumping.' Clinically, this treatment demonstrated advantages over B.I.D. theophylline, over single-daily morning regimens, and over prior theophylline therapy. Advantages of the evening regimen included better early-morning airflow (without significant decline later in the day), more effective symptom control, better patient acceptance, fewer night awakenings, and the obvious convenience of once-daily dosing. In addition, lung function showed greater stability, throughout the day, with once-daily evening therapy than with traditional 12 hr dosing. Uniphyl 400-mg tablets may be administered once daily to provide maximum blood levels at the time of peak bronchoconstriction, whether at night or during the day.     

Diagnosis of early pregnancy by ultrasound in Macaca fascicularis

Real-time ultrasonography was used to detect early pregnancy in 32 longtailed macaques (Macaca fascicularis). In 92% of the successful conceptions, a correct diagnosis was made. The earliest sign of pregnancy was an intrauterine ringlike structure (11 days). A "line swelling" (14 days) preceded definite fetal echoes (21 days), and fetal heart motion (30 days) proved fetal viability. Ultrasound is a rapid, noninvasive, and relatively cost-effective method of diagnosing and monitoring early pregnancy in M. fascicularis.     

State transitions,photosystem stoichiometry adjustment and non-photochemical quenching in cyanobacterial cells acclimated to light absorbed by photosystem I or photosystem II

Cells of the cyanobacterium Synechococcus 6301 were grown in yellow light absorbed primarily by the phycobilisome (PBS) light-harvesting antenna of photosystem II (PS II), and in red light absorbed primarily by chlorophyll and, therefore, by photosystem I (PS I). Chromatic acclimation of the cells produced a higher phycocyanin/chlorophyll ratio and higher PBS-PS II/PS I ratio in cells grown under PS I-light. State 1-state 2 transitions were demonstrated as changes in the yield of chlorophyll fluorescence in both cell types. The amplitude of state transitions was substantially lower in the PS II-light grown cells, suggesting a specific attenuation of fluorescence yield by a superimposed non-photochemical quenching of excitation. 77 K fluorescence emission spectra of each cell type in state 1 and in state 2 suggested that state transitions regulate excitation energy transfer from the phycobilisome antenna to the reaction centre of PS II and are distinct from photosystem stoichiometry adjustments. The kinetics of photosystem stoichiometry adjustment and the kinetics of the appearance of the non-photochemical quenching process were measured upon switching PS I-light grown cells to PS II-light, and vice versa. Photosystem stoichiometry adjustment was complete within about 48 h, while the non-photochemical quenching occurred within about 25 h. It is proposed that there are at least three distinct phenomena exerting specific effects on the rate of light absorption and light utilization by the two photoreactions: state transitions; photosystem stoichiometry adjustment; and non-photochemical excitation quenching. The relationship between these three distinct processes is discussed.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F relative fluorescence intensity at emission wavelength nm - F _o fluorescence intensity when all PS II traps are open - light 1 light absorbed preferentially by PS I - light 2 light absorbed preferentially by PS II - PBS phycobilisome - PS photosystem     

The disaccharide composition of heparins and heparan sulfates

Heparin and heparan sulfate can be cleaved selectively at their N-sulfated glucosamine residues by direct treatment with nitrous acid at pH 1.5. These polymers can also be cleaved selectively at their N-acetylated glucosamine residues by first N-deacetylating with hydrazine and then treating the products with nitrous acid at pH 4. These procedures have been combined and optimized for the conversion of these glycosaminoglycan chains into their disaccharide units. A modified hydrazinolysis procedure in which the glycosaminoglycans were heated with hydrazine:water (70:30) containing 1% hydrazine sulfate gave rapid rates of N-deacetylation and minimal conversion of the uronic acid residues to their hydrazide derivatives. Under these conditions, N-deacetylation was complete in 4 h and the beta-eliminative cleavage of the polymer chains that occurs during hydrazinolysis (P. N. Shaklee and H. E. Conrad (1984) Biochem. J. 217, 187-197) was eliminated. Treatment of the N-deacetylated polymer with nitrous acid at pH 3 for 15 h at 25 degrees C then gave simultaneous cleavage at the N-unsubstituted glucosamine residues and the N-sulfated glucosamine residues. These deamination conditions minimized, but did not eliminate, the side reaction in which nitrous acid-reactive glucosamine residues undergo ring contraction without glucosaminide bond cleavage. Thus, the disaccharides were obtained in a yield of 90% of those originally present in the glycosaminoglycan chains. Since the ring contraction side reaction occurs randomly at the diazotized glucosamine residues, the disaccharides formed in the pH 3 nitrous acid reaction were recovered in proportions equal to those in the original glycosaminoglycan chain.(ABSTRACT TRUNCATED AT 250 WORDS)