Identification of P-glycoprotein at the membrane of mast cell secretory granules. An immunofluorescence and protein A-gold electron microscopical investigation

The presence of P-glycoprotein has been investigated in rat peritoneal mast cells by means of immunofluorescence and immunogold electron microscopy, using the specific monoclonal antibody JSB-1. Immunofluorescence studi es showed that the glycoprotein is primarily concentrated in mast cell granules, and little is localized at the plasma membrane. Electron microscope observations revealed a marked accumulation of colloidal gold particles at the granule-coating membranes, whereas decoration of the plasma membrane is much less intense. When mast cells are stimulated to exocytate with compound 48/80, both immunofluorescence and electron microscopy showed concentration of P-glycoprotein reactivity at the plasma membrane level. Indeed, fusion of the granule with the plasma membrane allowed transfer of immunoreactive P-glycoprotein material from the granule-coating membrane to the cell surface membrane. These findings confirmed the presence of P-glycoprotein in mast cells; it is predominantly localized in the granules and is exposed on the cell surface only after exocytosis, suggesting, therefore, a possible physiological role for P-glycoprotein in the secretion of certain mediators. This revised version was published online in November 2006 with corrections to the Cover Date.     

Characterization of a Small Family (CAIII) of Microsatellite-Containing Sequences with X-Y Homology

Four X-linked loci showing homology with a previously described Y-linked polymorphic locus (DYS413) were identified and characterized. By fluorescent in situ hybridization (FISH), somatic cell hybrids, and YAC screening, the X-linked members of this small family of sequences (CAIII) all map in Xp22, while the Y members map in Yq11. These loci contribute to the overall similarity of the two genomic regions. All of the CAIII loci contain an internal microsatellite of the (CA)_n type. The microsatellites display extensive length polymorphism in two of the X-linked members as well as in the Y members. In addition, common sequence variants are found in the portions flanking the microsatellites in two of the X-linked members. Our results indicate that, during the evolution of this family, length variation on the Y chromosome was accumulated at a rate not slower than that on the X chromosome. Finally, these sequences represent a model system with which to analyze human populations for similar X- and Y-linked polymorphisms. Received: 29 July 1996 / Accepted: 15 January 1997     

Characterization of Nine Novel Mutations in the CD40 Ligand Gene in Patients with X-Linked Hyper IgM Syndrome of Various Ancestry

X-linked immunodeficiency with hyper-IgM (HIGMX-1) is a rare disorder caused by defective expression of the CD40 ligand (CD40L) by activated T lymphocytes, resulting in inefficient T-B cell cooperation and failure of B cells to undergo immunoglobulin isotype switch. In the present work, we describe nine patients of various ancestry who bear different mutations in the X chromosome–specific CD40L gene. Two of the mutations were nonsense mutations, one each resulting in premature stop codons at amino acid residues 39 and 140. Three patients had single point missense mutations, one each at codons 126, 140, and 144. Another patient had a 4-bp genomic deletion in exon 2, resulting in a frameshift and premature termination. Three patients showed insertions, one each of 1, 2, and 4 nt, probably because of polymerase slippage, resulting in frameshift mutation and premature termination. Overall, these observations confirm the heterogeneity of mutations in HIGMX-1. However, the identification of two patients whose mutation involves codon 140 (previously shown to be altered in two other unrelated subjects) suggests that this may be a hotspot of mutation in HIGMX-1. In two additional patients with clinical and immunological features indistinguishable from canonical HIGMX-1, no mutation was detected in the coding sequence, in the 5' flanking region, or in the 3' UTR.     

Molecular Genetics of Cystinuria: Identification of Four New Mutations and Seven Polymorphisms, and Evidence for Genetic Heterogeneity

A cystinuria disease gene (rBAT) has been recently identified, and some mutations causing the disease have been described. The frequency of these mutations has been investigated in a large sample of 51 Italian and Spanish cystinuric patients. In addition, to identify new mutated alleles, genomic DNA has been analyzed by an accurate and sensitive method able to detect nucleotide changes. Because of the lack of information available on the genomic structure of rBAT gene, the study was carried out using the sequence data so far obtained by us. More than 70% of the entire coding sequence and 8 intron-exon boundaries have been analyzed. Four new mutations and seven intragenic polymorphisms have been detected. All mutations so far identified in rBAT belong only to cystinuria type I alleles, accounting for ~44% of all type I cystinuric chromosomes. Mutation M467T is the most common mutated allele in the Italian and Spanish populations. After analysis of 70% of the rBAT coding region, we have detected normal sequences in cystinuria type II and type III chromosomes. The presence of rBAT mutated alleles only in type I chromosomes of homozygous (type I/I) and heterozygous (type I/III) patients provides evidence for genetic heterogeneity where rBAT would be responsible only for type I cystinuria and suggests a complementation mechanism to explain the intermediate type I/type III phenotype.     

pK determinations via pH-mobility curves obtained by isoelectric focusing-electrophoresis: Theory and experimental verification

Basic equations have been derived linking the electrophoretic migration in a stationary pH gradient of simple, singly charged cations or anions and of mono- mono- valent ampholytes with the pKs of their ionizable groups. In the case of diprotic ampholytes, an equation and a curve are described calculating a correction factor to be applied to the mobility measurements, accounting for the influence of the opposite charge species on the mobility curve of the ion being measured. This correction factor is a function of ΔpK and increases exponentially with decreasing values of ΔpK. These theoretical considerations have been experimentally verified by running pH-mobility curves of colored compounds, such as methyl red, neutral red and dexorubicin. The pKs thus measured were in excellent agreement with the pKs obtained independently by spectrophotometric titrations.     

Localization of HLA on the short arm of chromosome 6

Summary A detailed marker gene study in a large Dutch kindred segregating for a reciprocal translocation between the chromosomes 6 and 20, t(6;20) (p21;p13), revealed a close linkage between the HLA genes and the breakpoint on the short arm of 6. During this study an apparent peak lod score of 2.9 was obtained at a recombination value of 0.05 for a linkage between HLA and the breakpoint, indicating that the chromosomal region, carrying the HLA genes, is situated near the breakpoint in band 6p21 close to the transition to 6p22.     

Co-oligopeptides of aromatic amino acids and glycine with variable distance between the aromatic residues. VII. Enzymatic synthesis of N-protected peptide amides

Several N-protected peptide amides, containing two aromatic residues spaced by one glycyl residue, have been enzymatically synthesized starting from P-Ar-OH and H-Gly-Ar-NH₂ (P is the protecting group and Ar is the aromatic residue) and using α-chymotrypsin as the catalyst for the coupling step. Reactions have been carried out in water solution, at room temperature, and afford yields ranging between 20 and 75% ca. This coupling reaction occurs in a much more restricted set of conditions than the hydrolysis reaction, e.g., only within a small pH range (ca. 6.5–7.5) and with particular buffering agents. The advantages and limitations of this type of reaction, compared with conventional coupling procedures, are discussed.     

Spectroscopic and binding studies of azide to type-2-copper-depleted ascorbate oxidase from zucchini

Summary Binding of azide to type-2-copper-depleted (T2D) zucchini ascorbate oxidase, containing reduced type-3 Cu centers, and met-T2D ascorbate oxidase, containing oxidized type-3 Cu centers, has been studied spectroscopically. In both cases titration with azide in 0.1 M phosphate pH 6.8 produces a broad near-ultraviolet band with maximum at 455 nm (e 2500 M^–1 cm^–1, with respect to the met-T2D enzyme) and shoulder at 390 nm (e 1700 M^–1 cm^–1), that are assigned to(azide)Cu(II) ligand-to-metal charge transfer (LMCT) transitions. This is accompanied by a reduction of absorbance at 330 nm in the met-T2D) enzyme adduct (e –1400 M^–1 cm^–1). A broad circular dichroic band of negative sign between 370–480 nm corresponds to the LMCT absorption band. Analysis of the titration data indicates that one azide ion binds independently to each of the binuclear T3 Cu couples with low affinity (K = 50 M^–1). The ESR signal of the T1 Cu observed in frozen solutions of the T2D enzyme is also perturbed by the addition of azide. The analogies in the azide-binding characteristics between ascorbate oxidase and laccase are discussed.     

Comparison of the Molecular Forms of the Cholinesterases in Tissues of Normal and Dystrophic Chickens

Abstract: The levels and molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and pseudocholinesterase (ΦChE, EC 3.1.1.8) were examined in various skeletal muscles, cardiac muscles, and neural tissues from normal and dystrophic chickens. The relative amount of the heavy (H_c) form of AChE in mixed-fibre-type twitch muscles varies in proportion to the percentage of glycolytic fast-twitch fibres. Conversely, muscles with higher levels of oxidative fibres (i.e., slow-tonic, oxidative-glycolytic fast-twitch, or oxidative slow-twitch) have higher proportions of the light (L) form of AChE. The effects of dystrophy on AChE and ΦChE are more severe in muscles richer in glycolytic fast-twitch fibres (e.g., pectoral or posterior latissimus dorsi, PLD); there is no alteration of AChE or ΦChE in a slow-tonic muscle. In the pectoral or PLD muscles from older dystrophic chickens, however, the AChE forms revert to a normal distribution while the ΦChE pattern remains abnormal. Muscle ΦChE is sensitive to collagenase in a similar way as is AChE, thus apparently having a similar tailed structure. Unlike skeletal muscle, cardiac muscle has very high levels of ΦChE, present mainly as the L form; AChE is present mainly as the medium (M) form, with smaller amounts of L and H_c. The latter pattern of AChE forms resembles that seen in several neural tissues examined. No alterations in AChE or ΦChE were found in cardiac or neural tissues from dystrophic chickens.