Apical Scaffolding Protein NHERF2 Modulates the Localization of Alternatively Spliced Plasma Membrane Ca2+ Pump 2B Variants in Polarized Epithelial Cells

The membrane localization of the plasma membrane Ca²⁺-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expression of NHERF2 greatly enhanced the apical localization of GFP-tagged PMCA2w/b in polarized Madin-Darby canine kidney cells. GFP-PMCA2z/b was also redirected to the apical membrane by NHERF2, whereas GFP-PMCA2x/b remained exclusively basolateral. In the presence of NHERF2, GFP-PMCA2w/b co-localized with the actin-binding protein ezrin even after disruption of the actin cytoskeleton by cytochalasin D or latrunculin B. Surface biotinylation and fluorescence recovery after photobleaching experiments demonstrated that NHERF2-mediated anchorage to the actin cytoskeleton reduced internalization and lateral mobility of the pump. Our results show that the specific interaction with NHERF2 enhances the apical concentration of PMCA2w/b by anchoring the pump to the apical membrane cytoskeleton. The data also suggest that the x/b splice form of PMCA2 contains a dominant lateral targeting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features.     

Advances in salt tolerance

Summary Advances in and prospects for the development of salt tolerant crops are discussed. The genetic approach to the salinity problem is fairly new, but research has become quite active in a short span of time. Difficulties and opportunities are outlined. Salinity varies spatially, temporally, qualitatively, and quantitatively. In addition, the responses of plants to salt stress vary during their life cycle. Selection and breeding, including the use of wide crosses, are considered the best short-term approaches to the development of salt tolerant crops, but the new biotechnological and molecular biological techniques will make increasingly important contributions. Cooperation is called for among soil and water scientists, agronomists, plant physiologists and biochemists, cytologists, and plant geneticists, breeders, and biotechnologists. Given such cooperation and adequate support for these endeavors, the potential for increasing productivity in salt-affected areas can be realized.     

FSH-induced aromatase activity in hamster granulosa cells: effect of estradiol-17β in vitro

Summary We have shown previously that estradiol-17 (E₂) reduces number of ovulations in cyclic rats, induces atresia of the dominant preovulatory follicle in monkeys, and that the initial effects of this treatment include reduced viability and estrogen accumulation in vitro by aspirated granulosa cells (GC) from monkeys and hamsters. The present experiment was designed to determine whether the reduction in estrogen accumulation can be ascribed to a direct action of E₂ on the aromatization of androgen to estrogen in vitro. Female hamsters were injected with 30 I.U. pregnant-mare serum gonadotropin i.p. and sacrificed 3 days later. GC were aspirated from the largest follicles and incubated for 48 h (initial incubation period) in the presence of human pituitary follicle-stimulating hormone (hFSH, 100 ng/ml). Following initial incubation, GC were further incubated for up to 24 h (secondary incubation period). During this subsequent incubation, medium was supplemented with 100nM ³H-1-androstenedione (³H-A₄). Initial incubation with E₂ at doses of 10 ng/ml, 100 ng/ml and 1 m E₂/ml induced variability in GC response, and a maximal depression of 70%. The inhibition by E₂ of hamster GC function in vitro parallels that shown in vivo for both hamsters and monkeys, but contrasts with that shown for rats. Thus, hamsters may represent an appropriate model in which to study the atretogenic effects of E₂ directly on antral follicle development.     

Interaction between biotin lipids and streptavidin in monolayers: formation of oriented two-dimensional protein domains induced by surface recognition

Highly specific ligand-receptor interactions generally characterize surface recognition reactions. Such processes can be simulated by streptavidin-biotin-specific binding. Biotin lipids have thus been synthesized, and their interaction with streptavidin (or avidin) at the air-water interface was directly shown by measurement of surface pressure isotherms and fluorescence microscopy. These proteins interact with the biotin lipid monolayer via specific binding or nonspecific adsorption. Both phenomena were clearly distinguished by use of the inactivated form of streptavidin. The binding of fluorescein-labeled streptavidin to monolayers was also directly observed by fluorescence microscopy. The fluorescence of the protein domains is directly related to the state of polarization of the exciting light. This anisotropy can only be explained by the formation of oriented two-dimensional biotin lipid-streptavidin domains.     

Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit

We have used a gene transfer system to investigate the relationship between expression of the rat Na,K-ATPase alpha 1 subunit gene and ouabain-resistant Na,K-ATPase activity. A cDNA clone encoding the entire rat Na,K-ATPase alpha 1 subunit was inserted into the expression vector pSV2neo. This construct (pSV2 alpha 1) conferred resistance to 100 microM ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of transfected clones revealed the presence of both rat-specific and endogenous Na,K-ATPase alpha 1 subunit DNA and mRNA sequences. A single form of highly ouabain-sensitive 86Rb+ uptake was detected in CV-1 cells, whereas two distinct classes of ouabain-inhibitable uptake were observed in transfectants. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K-ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Examination of the ouabain-sensitive, sodium-dependent ATPase activity of the transfectants also revealed a low affinity component of Na,K-ATPase activity characteristic of the rodent kidney enzyme. These results suggest that expression of the rat alpha 1 subunit gene is directly responsible for ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells.     

Regional assignment of the gene for human liver/bone/kidney alkaline phosphatase to chromosome 1p36.1-p34

We have used three different methods to map the human liver/bone/kidney alkaline phosphatase (ALPL) locus: (1) Southern blot analysis of DNA derived from a panel of human-rodent somatic cell hybrids; (2) in situ hybridization to human chromosomes; and (3) genetic linkage analysis. Our results indicate that the ALPL locus maps to human chromosome bands 1p36.1-p34 and is genetically linked to the Rh (maximum lod score of 15.66 at a recombination value of 0.10) and fucosidase A (maximum lod score of 8.24 at a recombination value of 0.02) loci. These results, combined with restriction fragment length polymorphisms identified by ALPL DNA probes, provide a useful marker for gene mapping studies involving the short arm of chromosome 1. In addition, our results help to elucidate further the structure and evolution of the human alkaline phosphatase multigene enzyme family.     

Nucleotide and deduced amino acid sequence of bovine adrenal medulla chromogranin B (secretogranin I)

1. A novel 1745-dalton pyroglutamyl peptide (BAM-1745)6 was recently isolated and characterized from bovine adrenal medulla chromaffin granules. Its amino acid sequence was found to be 93% identical to residues 580-593 of human chromogranin B (secretogranin I). 2. Based on this sequence a degenerate oligonucleotide probe was synthesized and used to identify a 2.4-kb bovine adrenal medulla chromogranin B cDNA. 3. The deduced polypeptide is 647 amino acids long and begins with a putative signal sequence of 20 residues as in the human, rat, and mouse proteins. Also conserved in the bovine protein is a tyrosine residue which may be sulfated, two N-terminal cysteines, and many paired basic amino acids which may serve as sites of posttranslational processing. The peptide BAM-1745 is flanked by paired basic amino acids and therefore is most likely a product of posttranslational processing. Bovine chromogranin B is 67, 58, and 58% identical to the human, rat, and mouse chromogranin B proteins, respectively. 4. The carboxyl terminus of bovine chromogranin B, including BAM-1745, was found to be the most conserved region of the polypeptide and may identify it as an important functional domain.     

A human monoclonal antibody to cytomegalovirus (CMV)

We report the development of a human monoclonal antibody to cytomegalovirus (CMV) produced from a human X human hybridoma. This hybrid was developed by fusion of an EBV-transformed cell line making antibody to CMV and a human lymphoblastoid cell line WI-L2. The antibody is directed to a CMV-specific antigen primarily in the nucleus of CMV-infected human fibroblasts. It cross-reacts with at least 10 different strains of CMV and may provide a method for the rapid in vitro diagnosis of CMV infections. The production of CMV-specific human man monoclonal antibodies from human-human hybridomas for future therapeutic use is now technically feasible with this specific method of production.