Calcium-dependent protein kinase from apple fruit membranes is calmodulin-independent but has calmodulin-like properties

Crude Ca²⁺-activated protein kinase from membranes of apple (Malus domestica L. Borkh., Cox's Orange Pippin) fruit can be partially purified to yield a Ca²⁺-dependent protein kinase whose activity is apparently not regulated by calmodulin. The autophosphorylating catalytic subunit of this protein kinase shows a Ca²⁺-dependent mobility shift of approx. 10 kilodaltons (kDa) on sodium dodecyl sulphate-polyacrylamide gel electrophoresis; in the absence of added Ca²⁺ or ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) its apparent molecular mass is approx. 50 kDa. The Ca²⁺-dependent protein kinase is inhibited by the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide and trifluoperazine with IC₅₀ values of approx. 45 M and 15 M, respectively. These similarities between the protein kinase and calmodulin indicate that the kinase may be a calmodulin-like protein.Abbreviations DEAE diethylaminoethyl - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(-2-hydroxyethyl)-1-piperazineethanesulphonic acid - kDa kilodalton - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - W7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide - W5 N-(6-aminohexyl)-naphthalenesulphonamide     

Annexins: multifunctional components of growth and adaptation

Plant annexins are ubiquitous, soluble proteins capable of Ca(2+)-dependent and Ca(2+)-independent binding to endomembranes and the plasma membrane. Some members of this multigene family are capable of binding to F-actin, hydrolysing ATP and GTP, acting as peroxidases or cation channels. These multifunctional proteins are distributed throughout the plant and throughout the life cycle. Their expression and intracellular localization are under developmental and environmental control. The in vitro properties of annexins and their known, dynamic distribution patterns suggest that they could be central regulators or effectors of plant growth and stress signalling. Potentially, they could operate in signalling pathways involving cytosolic free calcium and reactive oxygen species.     

Two novel genes, Gpr113, which encodes a family 2 G-protein-coupled receptor, and Trcg1, are selectively expressed in taste receptor cells

To identify genes important for taste receptor cell function, we analyzed the sequences and expression patterns of clones isolated from a mouse taste receptor cell-enriched cDNA library. Here, we report the analyses of two novel genes, Gpr113 and Trcg1. Gpr113 encodes a G-protein-coupled receptor belonging to family 2B, members of which are characterized by having long N-terminal, extracellular domains. The predicted N-terminal extracellular domain of GPR113 contains 696 amino acids with two functional domains, a peptide hormone-binding domain and a G-protein-coupled receptor proteolytic site. Expression analyses indicate that Gpr113 expression is highly restricted to a subset of taste receptor cells. TRCG1 is also selectively expressed in a subset of taste receptor cells. Trcg1 is alternatively spliced and encodes Trcg1 isoforms of 209 and 825 amino acids. BLAST searches of genomic sequences indicate that a putative homolog of Trcg1 resides on human chromosome 15q22.     

Bombesin stimulates the in vitro growth of a human gastric cancer cell line

Bombesin (BBS) and its mammalian equivalent, gastrin-releasing peptide (GRP) exhibit diverse biological functions, including that of a neurotransmatter, a regulator of gastrointestinal hormone release, and a trophic factor for various normal and neoplastic tissues. Bombesin stimulates the growth of normal cells of the stomach, pancreas, and bronchial epithelium as well as cells in breast cancer, gastrinoma, and small cell lung cancer. The purpose of this study was to determine whether BBS regulates the growth of a human gastric cancer cell line (SIIA) in vitro, and if so, to examine the mechanisms of signal-transduction that are involved. We found that BBS stimulated the growth of SIIA cells in vitro. The GRP receptor antagonists, BIM 26189 and BIM 26226, had no effect on growth of SIIA cells. Although these antagonists blocked the BBS-induced increase of [Ca²⁺]_i, they failed to block the growth-stimulatory effect of BBS. BBS stimulated intracellular tyrosine phosphorylation of multiple proteins, with a predominant protein of apparent molecular weight of 125 kDa. Inhibition of intracellular tyrosine kinases by tyrphostin blocked the growth-stimulatory effect of BBS on SIIA cells. These results indicate that BBS exerts its trophic effect on SIIA cells through a receptor(s) linked to tyrosine kinase pathway, but not to the phospholipase C (PLC) pathway. © 1994 Wiley-Liss, Inc.     

LEAFY, TERMINAL FLOWER1 and AGAMOUS are functionally conserved but do not regulate terminal flowering and floral determinacy in Impatiens balsamina

In Impatiens balsamina a lack of commitment of the meristem during floral development leads to the continuous requirement for a leaf-derived floral signal. In the absence of this signal the meristem reverts to leaf production. Current models for Arabidopsis state that LEAFY (LFY) is central to the integration of floral signals and regulates flowering partly via interactions with TERMINAL FLOWER1 (TFL1) and AGAMOUS (AG). Here we describe Impatiens homologues of LFY, TFL1 and AG (IbLFY, IbTFL1 and IbAG) that are highly conserved at a sequence level and demonstrate homologous functions when expressed ectopically in transgenic Arabidopsis. We relate the expression patterns of IbTFL1 and IbAG to the control of terminal flowering and floral determinacy in Impatiens. IbTFL1 is involved in controlling the phase of the axillary meristems and is expressed in axillary shoots and axillary meristems which produce inflorescences, but not in axillary flowers. It is not involved in maintaining the terminal meristem in either an inflorescence or indeterminate state. Terminal flowering in Impatiens appears therefore to be controlled by a pathway that uses a different integration system than that regulating the development of axillary flowers and branches. The pattern of ovule production in Impatiens requires the meristem to be maintained after the production of carpels. Consistent with this morphological feature IbAG appears to specify stamen and carpel identity, but is not sufficient to specify meristem determinacy in Impatiens.     

Chromatin structure and protein binding in the putative regulatory region of the c-myc gene in Burkitt lymphoma

A chromosomal myc gene displays one of three patterns of activity depending upon the arrangement of the gene and its allelic partner. In nonmalignant B cells both myc alleles are normally expressed. In Burkitt lymphoma cells carrying both a translocated and a nontranslocated myc allele, the translocated allele is inappropriately expressed, while the nontranslocated allele is virtually inactive. Here we examine the chromatin structure of these genes using DNAase I hypersensitivity in nonmalignant lymphoblastoid cells and in the Burkitt lymphoma, BL31 . Three hypersensitivity patterns emerge that correlate with the state of the gene and reveal sites associated with putative regulatory structures. One region is associated with the two myc promoters, one with a specific nuclear protein binding site, and one--which is markedly enhanced in the inactive germline gene in the Burkitt cell--with a putative negative control region. The perturbation of the normal pattern in this particular Burkitt cell may be due to the action of an immunoglobulin enhancer.