Close linkage of MEN2A with RBP3 locus in Japanese kindreds

Summary The gene responsible for multiple endocrine neoplasia type 2A (MEN2A) has recently been assigned to the pericentromeric region of chromsome 10 in European Caucasian kindreds by linkage analysis using a DNA marker, interstitial retinol-binding protein 3 (RBP3). We have found tight linkage between the MEN2A and RBP3 loci in Japanese MEN2A kindreds. The maximum lod score is 5.19 at a recombination fraction of 0.00. This result suggests that mutation of a certain gene close to RBP3 is responsible for MEN2A irrespective of ethnic backgrounds.     

Purification and characterization from bovine brain cytosol of two GTPase-activating proteins specific for smg p21, a GTP-binding protein having the same effector domain as c-ras p21s

The smg-21 GTP-binding protein (smg p21) has the same effector domain as the ras proteins (ras p21s) and is identical with the proteins of the rap1A and Krev-1 genes. In this paper, two proteins stimulating the GTPase activity of smg p21 are partially purified from bovine brain cytosol. These proteins, designated as smg p21 GTPase-activating protein (GAP) 1 and 2, are separated from a c-ras p21 GAP described previously by column chromatographies. smg p21 GAP1 and -2 stimulate the GTPase activity of only smg p21 but not that of c-Ha-ras p21 or the rho and smg-25A GTP-binding proteins. smg p21 GAP1 or -2 does not stimulate the dissociation of guanosine 5'-3-O-(thio)triphosphate or GDP from smg p21. smg p21 GAP1 or -2 themselves do not have GTP/GDP binding or GTPase activity. The Mr values of smg p21 GAP1 and -2 are estimated to be 250-400 x 10(3) and 80-100 x 10(3) by gel filtration and sucrose density gradient ultracentrifugation, respectively. The activity of smg p21 GAP1 and -2 is killed by tryptic digestion or heat boiling. These results indicate that bovine brain contains two smg p21 GAPs in addition to c-ras p21 GAP.     

Small molecular weight GTP-binding proteins in human platelet membranes. Purification and characterization of a novel GTP-binding protein with a molecular weight of 22,000

When guanosine 5'-(3-O-[35S]thio)triphosphate (GTP gamma S)-binding activity was assayed in the particulate and cytosol fractions of human platelets, most activity was found in the particulate fraction. GTP-binding proteins (G proteins) were extracted from the particulate fraction by sodium cholate and purified by several column chromatographies. At least three G proteins with Mr values of about 21,000, 22,000, and 24,000 (21K G, 22K G, and 24K G, respectively) were separated in addition to the stimulatory (Gs) and inhibitory (Gi) regulatory GTP-binding proteins of adenylate cyclase. Among them, the amount of 22K G was more than 10-fold of those of other G proteins. 22K G was purified to near homogeneity and characterized. 22K G specifically bound GTP gamma S, GTP, and GDP, with a Kd value for GTP gamma S of about 50 nM. [35S]GTP gamma S binding to 22K G was inhibited by pretreatment with N-ethylmaleimide. 22K G hydrolyzed GTP to liberate Pi, with a turnover number of 0.01 min-1. 22K G was not copurified with the beta gamma subunits of Gs and Gi and was not recognized by the antibodies against the ADP-ribosylation factor for Gs and the ras protein. The peptide map of 22K G was different from those of the smg-25A and rho proteins, which we have purified from bovine brain membranes. 21K G was identified to be the c-ras protein, but 24K G was unidentified. These results indicate that there are multiple G proteins in platelet membranes and that a novel G protein (22K G) is a major G protein in platelets.     

Analysis of the topological changes induced on cells exposed to adhesive or mechanical stimuli

Fluorescent probes are widely used to study cell structure and function. However, few reports were devoted to a quantitative analysis of the intracellular distribution of fluorescent markers. In the present work, we describe the topographical changes of surface and cytoskeletal markers on individual cells subjected to adhesive or mechanical interaction. Conjugates were prepared with a cytotoxic T-lymphocyte clone and target cells. Specific antigens, membrane phospholipids, surface glycoconjugates, and polymerized actin were labeled with fluorescent antibodies or biochemical probes. The analysis of fluorescence distributions in conjugates demonstrated a selective reorganization of the plasma membrane with a gathering of some molecular species in the intercellular adhesion area. Furthermore, individual phagocytic cells were sucked into glass micropipets, then stained with fluorescent phallacidin to analyze the effect of mechanical efforts on the cytoskeleton organization. The concentration of polymerized actin was found to be similar in mechanicallyinduced protrusions and whole cells. It is concluded that adhesive interactions may result in marked cell polarization and formation of membrane zones with a particular biochemical composition. The submembranar cytoskeleton might play a role in this process.     

Identification of phytochrome B amino acid residues mutated in three new phyB mutants of Arabidopsis thaliana

The growth and development of plants is regulated by light viathe action of photoreceptors which are responsive to the red/far-red,blue and UV regions of the spectrum. Phytochrome B (the apoproteinof which is encoded by the PHYB gene) is one of the red/far-redabsorbing photoreceptors active in this process. In this paper,the isolation and characterization of three new EMS-inducedmutations of Arabidopsis which confer phytochrome B deficiencyare described. Complementation analysis showed that these mutations(phyB-101, phyB-102 and phyB-104) were allelic with PHYB. DNAsequence analysis showed that all three mutants contain nucleotidesubstitutions in the PHYB-101 gene sequence. phyB-101 carriesa nucleotide substitution within the second exon of the PHYBgene. This G-to-A substitution is a missense mutation that convertsa glutamate residue at position 812 of the phytochrome B apoproteinto a lysine residue. phyB-102, another missense mutant, carriesa C-to-T substitution which converts a serine residue at position349 of the phytochrome B apoprotein to a phenylalanine residue.phyB-104 carries a premature stop codon as a result of a G-to-Amutation 1190 bp down-stream of the ATG start codon of the PHYBsequence. The missense mutations in phyB-101 and phyB-102 causesignificant alterations in the predicted second ary structureof their respective mutant polypeptides, and identify aminoacid residues playing crucial roles in phytochrome B function,assembly or stability. Key words: Arabidopsis thaliana, phytochromet, phyB mutants, missense mutations     

Responses of nitrate assimilation and N translocation in tomato (Lycopersicon esculentum Mill) to reduced ambient air humidity

The impact of low humidity in ambient air on water relations,nitrate uptake, and translocation of recently absorbed nitrogen,was investigated in 5-week-old tomato (Lycopersicon esculentumMill cv. Ailsa Craig) plants grown hydroponically in a completenutrient solution. Plants were subjected to dry air (relativehumidity 2–4% for 6 h. The transpiration rate increasedseveral-fold and the shoot water content decreased by almost20%, whereas root water content was unaffected. No effect onin vitro nitrate reductase (NR) activity was detected when usingan EDTA-contraining assay buffer. Replacement of EDTA with Mg²⁺revealed a significant decline in shoot NR activity, which suggestsphosphorylation of the enzyme during the stress treatment. Plantswere grown in a split-root system, in which one root half wasfed ¹⁵N-nitrate during the treatment, in order to determinenitrate uptake and translocation of recently absorbed nitrogenin the plants. Uptake of nitrate was substantially inhibited,but the proportion of absorbed ¹⁵N that was translocated tothe shoots was only slightly affected. In untreated plants,71% of the ¹⁵N recovered in roots had been retranslocated fromthe shoots, whereas in plants subjected to stress the deliveryof ¹⁵N from shoots to roots appeared to be completely inhibited.The data show that lowered humidity in air has significant effectson both uptake of nitrate as well as translocation of nitrogenwithin the plants. Some of these effects appear to be commonwith those observed in plants subjected to reduced water potentialsin the root environment and point to the possibility of theshoot water relations being highly influential on nitrogen uptakeand translocation. Key words: Air humidity, nitrate assimilation, nitrate reductase activity, nitrogen translocation, tomato, water stress     

Modulation of ζ-Protein Kinase C by Cyclic AMP in PC12 Cells Occurs Through Phosphorylation by Protein Kinase A

Abstract: Although cyclic AMP (cAMP) has been reported to cross talk with the protein kinase C (PKC) system, effects of elevated intracellular cAMP on the activities of specific PKC isoforms have not been studied. We report findings from a permeabilized cell assay that was used to examine changes in the activity of the atypical PKC isoforms brought about by exposure of PC12 cells to agents that elevate intracellular cAMP. We found that increases in intracellular cAMP led to rapid stimulation of atypical PKC activity, 40–70% above control, for a sustained period of time, a response that occurred independent of the phorbol 12-myristate 13-acetate (PMA)-sensitive PKC isoforms. Changes in intracellular cAMP levels resulted in a dose-dependent redistribution of ζ-PKC to the cytoplasm with a concomitant increase in the phosphorylation state of the enzyme. Incubation of purified ζ-PKC with increasing concentrations of PKA likewise caused a twofold increase in the phosphorylation state of ζ-PKC. In contrast to the positive effect that PKA-mediated phosphorylation had on the activity of ζ-PKC, the enzyme displayed reduced binding to ras when phosphorylated. Taken together, these findings are consistent with the hypothesis that protein phosphorylation of PKC acts as a positive effector of its enzyme activity and may serve as a negative modulator for interaction with other proteins.     

Connexin43 Is Another Gap Junction Protein in the Peripheral Nervous System

Abstract: That many cells express more than one connexin (Cx) led us to examine whether Cxs other than Cx32 are expressed in the PNS. In addition to Cx32 mRNA, Cx43 and Cx26 mRNAs were detected in rat sciatic nerve by northern blot analysis. Cx43 mRNA, but not Cx26 mRNA, was expressed in both the primary Schwann cell culture and immortalized Schwann cell line (T93). The steady-state levels of the Cx43 mRNA in the primary Schwann cell culture increased 2.0-fold with 100 µ M forskolin, whereas that of P₀ increased 7.0-fold. Immunoreactivity to Cx43 was detected on western blots of cultured Schwann cells, T93 cells, and sciatic nerves but not on blots of PNS myelin. Immunohistochemical study using human peripheral nerves revealed that anti-Cx43 antibody stained cytoplasm around nucleus of Schwann cells but not myelin, confirming western blot results. Although P₀ expression was markedly decreased by crush injury of the sciatic nerves, Cx43 expression showed no apparent change. Developmental profiles showed that Cx43 expression in the sciatic nerve increased rapidly after birth, peaked at about postnatal day 6, and then decreased gradually to a low level. In adult rats, the Cx43 mRNA value was much lower than that of Cx32. These findings suggest that Cx43 is localized in Schwann cell bodies and that, compared with P₀, its expression is less influenced by axonal contact and cyclic AMP levels. The high expression on postnatal day 6 indicates that Cx43 may be related to PNS myelination. Cx43 is another gap junction, but its function appears to differ from that of Cx32, as judged by the differences in their localization and developmental profiles.     

Extracellular Matrix Penetration by Epithelial Cells Is Influenced by Quantitative Changes in Basement Membrane Components and Growth Factors

We have previously shown that isolated mouse fetal choroid plexus epithelial (CPE) cells penetrate a basement membrane matrix (Matrigel) substratein vitroto form single-layered epithelial vesicles embedded within the matrix. To determine which properties of the matrix are important for inducing or permitting cells to penetrate the substrate and organize into multicellular vesicles we have made quantitative changes to the basement membrane components and growth factors in cell cultures. Matrigel diluted to 33 or 10% with a collagen I gel was not permissive to cell invasion, and CPE cells formed a polarized epithelial monolayer on the substrate surface which had ultrastructural characteristics similar to those of CPE vesicles. Cells in these monolayers proliferated more rapidly than cells in epithelial vesicles. When deliberately embedded within a 33 or 10% Matrigel matrix, CPE cells were able to form vesicles, indicating that a dilute matrix is nonpermissive to cell invasion but promotes epithelial polarization and organization into vesicles. Cells embedded within a 100% collagen I matrix did not proliferate or form epithelial vesicles and the majority of cells did not remain viable. Addition of laminin to the collagen I gel promoted cell adhesion and cell survival, but did not promote the formation of extensive monolayers on the substrate nor the formation of epithelial vesicles within the matrix. Cell invasion into the 33% Matrigel matrix was induced by addition of laminin, nidogen, or a laminin–nidogen complex to the substrate or by addition of TGFβ2 to the culture medium, but not TGFβ1 or PDGF. These studies show that CPE cells are sensitive to quantitative changes in matrix composition, which influences their survival and proliferation and also their ability to penetrate the matrix and organize into multicellular epithelial vesicles.     

Linkage disequilibrium and extended haplotypes in the HLA-A to D6S105 region: implications for mapping the hemochromatosis gene (HFE)

The hemochromatosis gene (HFE) maps to 6p21.3, in close linkage with the HLA Class I genes. Linkage disequilibrium (LD) studies were designed to narrow down the most likely candidate region for HFE, as an alternative to traditional linkage analysis. However, both the HLA-A and D6S105 subregions, which are situated 2–3 cM and approximately 3 Mb apart, have been suggested to contain HFE. The present report extends our previous study based upon the analysis of a large number of HFE and normal chromosomes from 66families of Breton ancestry. In addition to the previously used RFLP markers spanning the 400-kb surrounding HLA-A, we examined three microsatellites: D6S510, HLA-F, and D6S105. Our combined data not only confirm a peak of LD at D6S105, but also reveal a complex pattern of LD over the i82 to D6S105 interval. Within our ethnically well-defined population of Brittany, the association of HFE with D6S105 is as great as that with HLA-A, while the internal markers display a lower LD. Fine haplotype analysis enabled us to identify two categories of haplotypes segregating with HFE. In contrast to the vast majority of normal haplotypes, 50% of HFE haplotypes are completely conserved over the HLA-A to D6S105 interval. These haplotypes could have been conserved through recombination suppression, selective forces and/or other evolutionary factors. This particular haplotypic configuration might account for the apparent inconsistencies between genetic linkage and LD data, and additionally greatly complicates positional cloning of HFE through disequilibrium mapping.The authors contributed equally to this work