Pertussis Toxin-Sensitive G Protein α-Subunits: Production of Monoclonal Antibodies and Detection of Differential Increases on Differentiation of PC12 and LA-N-5 Cells

Abstract: Monoclonal antibodies were produced that are specific for the three major pertussis toxin-sensitive G protein α-subunits present in mammalian brain—αo, αi1, and αi2—using purified bovine brain G proteins, purified rat brain G proteins, and purified recombinant αi2, respectively. These monoclonal antibodies were used to monitor changes in the concentrations of the three G protein α-subunits during differentiation of PC12 cells and human neuroblastoma LA-N-5 cells. In PC12 cells, levels of αi1 but not αi2 increased during nerve growth factor-induced differentiation. In contrast, αi2 but not αi1 increased when LA-N-5 cells were differentiated with retinoic acid. The concentration of αo increased in both cell lines during differentiation. Electrophoretic resolution of αo subtypes revealed that although αo2 was the major subtype in undifferentiated cells, only the concentration of αo1 increased during differentiation of both PC12 and LA-N-5 cells. The level of 43-kDa growth-associated protein, a protein known to associate with αo, increased similarly to that of αo1. ADP-ribosylation of αo, αi1, and αi2 with pertussis toxin did not alter the reactivities of the monoclonal antibodies, but toxin treatment of cells reduced the concentrations of each protein after 24 h. There was no change in the concentration of αq, which is not ADP-ribosylated by pertussis toxin. Thus, these new monoclonal antibodies enabled the detection of differential increases in subtypes of αi and αo associated with neuronal differentiation.     

Proline 21, a residue within the α-helical domain of ΦX174 lysis protein E, is required for its function in Escherichia coli

ΦX174 lysis protein E-mediated lysis of Escherichia coli is characterized by a protein E-specific fusion of the inner and outer membrane and formation of a transmembrane tunnel structure. In order to understand the fusion process, the topology of protein E within the envelope complex of E. coli was investigated. Proteinase K protection studies showed that, during the time course of protein E-mediated lysis process, more of the fusion protein E-FXa-streptavidin gradually became accessible to the protease at the cell surface. These observations postulate a conformational change in protein E during induction of the lysis process by movement of the C-terminal end of the protein throughout the envelope complex from the inner side to the outer side spanning the entire pore and fusing the inner and outer membranes at distinct areas. The initiation mechanism for such a conformational change could be the cis–trans isomerization of proline residues within α-helical membrane-spanning segments. Conversion of proline 21, presumed to be in the membrane-embedded α-helix of protein E, to alanine, glycine, serine and valine, respectively, resulted in lysis-negative E mutant proteins. Proteinase K accessibility studies using streptavidin as a reporter fused to the P21G mutant protein showed that the C-terminal part of the fusion protein is not translocated to the outer side of the membrane, suggesting that this proline residue is essential for the correct folding of protein E within the cell wall complex of E. coli . Oligomerization of protein P21G-StrpA was not disturbed.     

XY gonadal dysgenesis and gonadoblastoma: a study in two sisters with a cryptic deletion of the Y chromosome involving the SRY gene

This paper reports a case of XY gonadal dysgenesis in two sisters. Both patients presented an eunochoid female phenotype with normal external genitalia. At laparotomy, the elder sister was found to have bilateral gonadoblastoma. Cytogenetic studies, which included G and C banding and in situ hybridization, showed that the patients had an apparently normal 46, XY karyotype. PCR analyses revealed absence of the conserved portion (HMG box) of the SRY gene and of the Y chromosome pseudoautosomal boundary region sequence in both patients. The presence of the ZFY sequence was detected by Southern hybridization in the two affected sisters. The patients' father (46, XY, no mosaicism detected in peripheral blood lymphocytes) was positive for SRY and ZFY sequences. The occurrence of gonadoblastoma is discussed in terms of the genetic factors that may lead to tumor development.     

The calcium hypothesis of cystic fibrosis

Data have been presented which suggests that various CF cell types show evidence of alterations in calcium homeostasis. The significance of these observations and the exact nature of the putative calcium defect in CF remains to be elucidated. It must also be determined whether this possible defect is primary, or is secondary or tertiary to some more basic lesion. The data reviewed suggests that altered calcium homeostasis may play some focal role in the aetiology or the pathogenesis of CF.     

Cooperativity and effects of ionic strength on the binding of sodium n-dodecyl sulphate to lysozyme

The binding of sodium n-dodecyl sulphate to lysozyme has been measured by equilibrium dialysis at 25°C and pH 3.2 over a range of ionic strengts from 0.0119 to 0.2119. Binding isotherms in the region corresponding to ionic binding between the surfactant anions and cationic amino acid residues on the protein have been interpreted in terms of the Hill equation and exhibit positive cooperativity with Hill coefficients in the region of 7–11. The Gibbs energies of binding have been calculated from the Hill binding constants and from the Wyman binding potentials. The stability of the surfactant-protein complexes is discussed in relation to the stability of surfactant micelles. Ionic binding of the surfactant is weakened and hydrophobic binding strengthened by increasing ionic strength.     

Effect of chloride withdrawal on the geometry of the T-tubules in amphibian and mammalian muscle

Summary The relative chloride permeabilities of the T-tubule membranes in mammalian (rat sternomastoid) and amphibian (toad sartorius) skeletal muscle fibers have been assessed from the change in volume of the T-tubules during chloride withdrawal from fibers exposed to low extracellular chloride concentrations. A 3.5- to 4.2-fold increase in T-tubule volume was found in mammalian fibers, and this was shown to be independent of the composition of the low chloride solution or the nature of the fixative used in preparation for electron microscopy. The increase in T-tubule volume was transient and was inhibited by factors which block chloride conductance, i.e., low pH, 2,4-dichlorophenoxyacetic acid, and nitrate ions. A small increase (1.48-fold) in T-tubule volume was seen in amphibian fibers when chloride ions were replaced by sulphate ions. No increase in volume was observed in amphibian T-tubules when methyl sulphate ions replaced chloride ions. The results support the idea that the chloride permeability of the T-tubule membrane is significantly higher in mammalian fibers than in amphibian fibers.