[Immunohistochemical analysis and immuno-gold localization of ECBP21 in Angelica dahurica].

ECBP21 is a calmodulin binding protein (CaMBP) purified from extracellular extracts of suspension-cultured cells of Angelica dahurica, and it is the first reported extracellular CaMBP in plant kingdom. We have recently cloned the full-length cDNA for ECBP21. In this work, using recombinant ECBP21 we prepared rabbit antiserum with high specificity and high titer against ECBP21, and investigated the organ-specific distribution of ECBP21 in Angelica dahurica. ECBP21 was found in all organs examined, particularly abundant in the leaves flowers, and raches, and less in the roots. It was also found in all cells examined, and particularly enriched in the cell wall. These data support the notion that ECBP21 is specifically localized extracellularly, and imply that it may be involved in plant growth and development. In addition, using immunogold transmission electron microscopy method, we studied the subcellular localization of ECBP21 in rachis cells of Angelica dahurica. The results indicated that the ECBP21 was mainly localized in cell wall; this provided a direct evidence of the extracellular existence of ECBP21.

Anaplastic lymphoma kinase (ALK 1) staining and molecular analysis in inflammatory myofibroblastic tumours of the bladder: a preliminary clinicopathological study of nine cases and review of the literature.

Inflammatory myofibroblastic tumours (IMFT) may arise at any anatomical site, including lung, soft tissues, retroperitoneum and bladder. Although morphologically similar, these lesions encompass a spectrum of entities with differing aetiology, ranging from reactive/regenerative proliferations to low-grade neoplasms with a risk of local recurrence, but no significant metastatic potential. Vesical IMFT usually presents as a polypoid mass with a pale firm cut surface and can be of considerable size, mimicking a malignant tumour clinically and radiologically. Its good outcome, however, warrants conservative surgical excision, emphasising the importance of identification and distinction from malignant tumours of the bladder that may require more radical surgery and/or adjuvant therapy. We conducted a preliminary retrospective, comparative immunocytochemical study of 20 bladder tumours, including nine IMFTs, five spindle cell (sarcomatoid) carcinomas, two rhabdomyosarcomas, two leiomyosarcomas and two neurofibromas. The results confirmed IMFT positivity for smooth muscle actin, desmin and cytokeratin in 78-89% cases, resulting in potential confusion with sarcomatoid carcinoma or leiomyosarcoma. In contrast, cytoplasmic anaplastic lymphoma kinase (ALK 1) staining was present in eight IMFT (89%), but was not seen in any other lesion examined. The ALK 1 staining was confirmed by fluorescence in situ hybridisation, with translocation of the ALK gene present in 15-60% tumour cells in four of six IMFT examined, but not in four cases of sarcomatoid carcinoma or three of leiomyosarcoma. In conclusion, ALK 1 staining may be of value in the distinction of vesical IMFT from morphologically similar entities, and often reflects ALK gene translocations in these lesions.

CaMBOT: profiling and characterizing calmodulin-binding proteins.

Calmodulin (CaM) is an essential calcium-binding protein that binds to and activates a diverse population of downstream targets (calmodulin-binding proteins; CaMBPs) that carry out its critical signalling functions. In spite of the central importance of CaM in Ca(2+)-mediated signal transduction pathways in all eukaryotes, many CaMBPs remain to be identified and characterized. SDS-PAGE followed by gel overlay with recombinant, metabolically radiolabelled CaM (Calmodulin-binding Overlay Technique, CaMBOT) is a valuable method for following behavioural, developmental, forensic and physiological changes in total CaMBP populations and to identify candidate CaMBPs for further study. CaMBOT has also been adapted to isolate cDNAs encoding novel CaMBPs in various organisms. Recently, the method was used to examine the CaMBP complement encoded by the Arabidopsis genome and to identify a new family of transcription activators. To add to its diversity, CaMBOT may be useful for finding target proteins for work on phytoremediation and for the screening of pharmaceuticals and toxic agents that, directly or indirectly, affect CaM and its target proteins. This review discusses all of these topics and the role of CaMBOT in characterizing a functional unit of the proteome-proteins regulated by calmodulin.

Squid p196, a new member of the myosin-V class of motor proteins, is associated with motile axoplasmic organelles.

Axoplasmic organelles obtained from the squid giant axon move on actin filaments at an average velocity of 1 microm/s [Nature 356 (1992) 722]. The unconventional myosins, in particular, the myosin-V class of motor proteins, represent the most likely candidates to have a role in this motility. Experiments were performed to determine whether a member of the myosin-V class of unconventional myosins is present in axoplasm and optic lobes. Western blots of axoplasm probed with an affinity purified antibody to chicken brain myosin-V (CBM-V) showed cross-reactivity with a protein of Mr 196 kD (p196) which was subsequently purified from squid optic lobes using a modification of a protocol for the purification of CBM-V [Methods Enzymol. 298 (1998) 3; Cell 75 (1993) 215]. Western blots of CBM-V probed with an alpha-p196 polyclonal IgG showed cross-reactivity with CBM-V. Purified p196 has been found to be a calmodulin (CaM) binding protein that possesses calcium-stimulated actin-activated ATPase activity. Equilibrium density fractionation of motile axoplasmic organelle preparations has revealed that p196 cosedimented with the peak organelle fraction into Percoll gradients in the presence of cytochalasin B and ATP. Based on this evidence, we conclude that the p196 present in axoplasm and purified from optic lobes is a squid homolog of CBM-V and functions as a motor for fast transport of membranous organelles on actin filaments in neurons.

Localization of a kinesin-like calmodulin-binding protein in dividing cells of Arabidopsis and tobacco.

The cloning and characterization of a novel kinesin-like protein (kinesin-like calmodulin-binding protein, KCBP) from Arabidopsis and other plants has recently been described. Unlike all other known kinesin-like proteins, KCBP interacts with calmodulin in the presence of micromolar calcium. An antibody specific to KCBP was raised using a calmodulin-binding synthetic peptide that is unique to KCBP. The KCBP antibody detected a single protein of about 140 kDa in Arabidopsis and tobacco, the size predicted from cDNA sequences. In synchronized cell cultures, the amount of KCBP was abundant during M-phase and very low in interphase. To get some insight into the function of this novel motor protein, KCBP in Arabidopsis and tobacco cells was localized by indirect immunofluorescence microscopy using affinity-purified anti-KCBP antibody. The KCBP was localized to the preprophase band, the mitotic spindle and the phragmoplast. The association of KCBP with microtubule arrays in dividing cells suggests that this minus-end-directed microtubule motor protein is likely to be involved in the formation of these microtubule arrays and/or functions associated with these structures.

Distribution of caldesmon and of the acidic isoform of calponin in cultured cerebellar neurons and in different regions of the rat brain: an immunofluorescence and confocal microscopy study.

Caldesmon and calponin are two F-actin-binding and calcium-calmodulin-dependent proteins. In smooth muscle and nonmuscle cells both proteins are localized on actin filaments. Using one- or two-dimensional gel electrophoresis followed by the Western blot technique, and by immunofluorescence studies, we have given evidence that calponin is also present in rat and pig brain. In the present study, for the first time, we demonstrate caldesmon- and calponin-specific immunoreactivities in cerebellar cultured neurons. In the rat central nervous system these antibodies mainly stain neuronal cell bodies and dendrites. By confocal analysis we observed that calponin and caldesmon are located in the actomyosin domain although the total actin and myosin were not saturated. In many cases it is clear that these two proteins are adjacent rather than superimposed in the same domain of the cell. These results are compatible with the functional role of caldesmon and calponin in the regulation of the actomyosin activity as described by others and suggest that they are part of the contractile apparatus of neural cells.

Association of calmodulin and an unconventional myosin with the contractile vacuole complex of Dictyostelium discoideum.

mAbs specific for calmodulin were used to examine the distribution of calmodulin in vegetative Dictyostelium cells. Indirect immunofluorescence indicated that calmodulin was greatly enriched at the periphery of phase lucent vacuoles. The presence of these vacuoles in newly germinated (non-feeding) as well as growing cells, and the response of the vacuoles to changes in the osmotic environment, identified them as contractile vacuoles, osmoregulatory organelles. No evidence was found for an association of calmodulin with endosomes or lysosomes, nor was calmodulin enriched along cytoskeletal filaments. When membranes from Dictyostelium cells were fractionated on equilibrium sucrose density gradients, calmodulin cofractionated with alkaline phosphatase, a cytochemical marker for contractile vacuole membranes, at a density of 1.156 g/ml. Several high molecular weight calmodulin-binding proteins were enriched in the same region of the gradient. One of the calmodulin-binding polypeptides (molecular mass approximately 150 kD) cross-reacted with an antiserum specific for Acanthamoeba myosin IC. By indirect immunofluorescence, this protein was also enriched on contractile vacuole membranes. These results suggest that a calmodulin-binding unconventional myosin is associated with contractile vacuoles in Dictyostelium; similar proteins in yeast and mammalian cells have been implicated in vesicle movement.

Cellular distribution of calmodulin and calmodulin-binding proteins in Vicia faba L.

The distribution of calmodulin (CaM) and CaM-binding proteins within Vicia faba was investigated. Both CaM and CaM-binding proteins were found to be differentially distributed among organs, tissues, and protoplast types. CaM levels, on a per protein basis, were found to be the highest in leaf epidermis, containing 3-fold higher levels of CaM than in total leaf. Similarly, guard cell and epidermal cell protoplasts were also found to have higher levels of CaM than mesophyll cell protoplasts. 125I-CaM blot overlay assays were performed to qualitatively examine CaM-binding proteins in these protoplast types as well as in whole tissues and organs. CaM-binding proteins with Mr 52,000, 78,000, and 115,000 were common in all metabolically active plant parts. Unique CaM-binding protein bands were detected in guard cell protoplasts (Mr 39,000, 88,000), stems (Mr 45,000, 60,000, 64,000), and roots (Mr 62,000), suggesting the presence of specialized CaM-dependent processes in these cells and organs.