Purification of two muscle enzymes by chromatography on immobilized ferric ions

Two enzymes, glycogen phosphorylase and lactate dehydrogenase, were purified simultaneously in a single step. Ferric ions immobilized on a chelating gel were used as the adsorbent. Adsorption and desorption steps were accomplished by changes in buffer composition. The recoveries were better than 80% and the capacities were about 5 mg of protein per milliliter of adsorbent. The procedure worked well both on a small and on a preparative scale. The homogeneity of the purified enzymes was checked by FPLC.     

Heart and the peritoneal cover of the gut sinus in the polychaete Arenicola marina: an ultrastructural and autoradiographic study

To elucidate the cellular mechanism underlying the growth of the peritoneal cover of the gut sinus and the heart in the polychaete Arenicola marina, cellular organization of these structures and proliferative potential of their cells were investigated using electron microscopy and electron microscopic autoradiography. Arenicola has a pair of dorsolaterally situated hearts connected to the gut sinus via a short duct and composed of two muscular layers separated by a layer of the extracellular matrix (ECM). The peritoneal cover of the gut sinus and the outer muscular layer of the heart present a myoepithelial layer resting on the ECM. The inner muscular layer of the heart is composed of myofibril-containing cells lacking well-defined polarity in arrangement of organelles. However, their persistent connection to branches of the ECM and the adherens-like intercellular junctions allow for considering the inner layer a modified myoepithelium. In the peritoneal cover of the gut sinus and in both myoepithelial layers of the heart, noncontractile epithelial cells have been observed. As determined by thymidine labeling, these epithelial cells are capable of DNA synthesis, while myoepithelial cells are not. Some suggestions are made about the myogenic nature of the epithelial cells in the investigated structures of A. marina.     

Determination of relative protein abundance by internally normalized ratio algorithm with antibody arrays

In this paper, we report an experimental setup and mathematical algorithm for determination of relative protein abundance from directly labeled native protein samples applied to an array of antibodies. The application of the proposed experimental system compensates internally at each array element for a number of deficiencies in array experiments such as differential labeling efficiency in dual color assay systems, differential solubility of protein molecules in dual color assay systems, and differential affinity of capture reagents toward proteins labeled with two different fluorescent dyes. This system offers full compensation for variable amounts of capture reagents on separate array structures, as well as limited compensation for nonspecific interactions between capture reagents and analytes. The proposed experimental strategy enables the use of a large number of capture reagents to develop a true multiplex analysis system that will yield complete relative protein abundance information in two biological systems.     

Lamellipodial actin mechanically links myosin activity with adhesion-site formation

Cell motility proceeds by cycles of edge protrusion, adhesion, and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction, and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process.     

Larval ontogenetic stages of Chaetopterus: developmental heterochrony in the evolution of chaetopterid polychaetes

Seven post-gastrulation larval stages are described for the sedentary polychaete Chaetopterus. Analysis of larval anatomy and morphology through ontogeny reveals significant differences in the temporal sequence of segmentation, and in the character of segments formed, from the typical embryological pattern described for other polychaete families, such as nereidids or spionids. When compared in alternative phylogenetic schemes, these differences represent significant developmental heterochrony, among other evolutionary transitions, which has arisen in the chaetopterid lineage. The heterochrony is correlated with the extreme morphological regionalization along the anterior-posterior body axis, a feature that is also characteristic of chaetopterids.     

Mechanism of filopodia initiation by reorganization of a dendritic network

Afilopodium protrudes by elongation of bundled actin filaments in its core. However, the mechanism of filopodia initiation remains unknown. Using live-cell imaging with GFP-tagged proteins and correlative electron microscopy, we performed a kinetic-structural analysis of filopodial initiation in B16F1 melanoma cells. Filopodial bundles arose not by a specific nucleation event, but by reorganization of the lamellipodial dendritic network analogous to fusion of established filopodia but occurring at the level of individual filaments. Subsets of independently nucleated lamellipodial filaments elongated and gradually associated with each other at their barbed ends, leading to formation of cone-shaped structures that we term Lambda-precursors. An early marker of initiation was the gradual coalescence of GFP-vasodilator-stimulated phosphoprotein (GFP-VASP) fluorescence at the leading edge into discrete foci. The GFP-VASP foci were associated with Lambda-precursors, whereas Arp2/3 was not. Subsequent recruitment of fascin to the clustered barbed ends of Lambda-precursors initiated filament bundling and completed formation of the nascent filopodium. We propose a convergent elongation model of filopodia initiation, stipulating that filaments within the lamellipodial dendritic network acquire privileged status by binding a set of molecules (including VASP) to their barbed ends, which protect them from capping and mediate association of barbed ends with each other.     

Ultrastructural study of the heart and its relationship with the connective tissue sheath of the stomach in Rhynchonella psittacea (Tentaculata,Brachiopoda)

The fine structure of the heart and connective tissue sheath surrounding the stomach of the brachiopod Rhynchonella psittacea has been studied. The stomach wall is lined externally with peritoneal epithelium. Between the bases of the peritoneal epithelial cells and those of the stomach epithelial cells is an extracellular amorphous matrix. The exterior part of the matrix is occupied by smooth muscle cells and the interior part by fibroblasts. The heart wall shows continuity with the peritoneal epithelium covering the stomach wall and consists of three layers: an outer layer of smooth myoepithelial and epithelial cells, an intermediate thick layer of extracellular matrix, and an inner discontinuous layer of fibroblasts. In myoepithelial cells, nucleated heads protruding freely into the coelom and contractile parts embedded in the extracellular matrix can easily be distinguished. These cells contain no sarcoplasmic reticulum or any elements of a T system. The epithelial cells are non-muscular mononucleated cells scattered among the myoepithelial cells and closely associated with these basally. They possess a well-developed rough endoplasmic reticulum. In rare cases, a small amount of myofibrils occurs basally in the epithelial cells. Morphologically the epithelial cells in the myocardium are very similar to the peritoneal epithelial cells covering the stomach wall. Both epithelial and myoepithelial cells are ciliated. No nerve elements have been found in the brachiopod heart. The structure of the brachiopod heart is compared with that of other invertebrates; similarity of cellular composition of the brachiopod heart and stomach cover is considered evidence of origin of the heart cells from the cells of the connective tissue sheath of the stomach. The myogenic role of the peritoneal cells and epithelial cells of the myocardium is suggested. J. Morphol. 234:69–77, 1997. © 1997 Wiley-Liss, Inc.     

Bifunctional phosphoramidite reagents for the introduction of histidyl and dihistidyl residues into oligonucleotides.

The synthesis and characterization of reagents for the incorporation of histidyl residues into oligonucleotides by automated chemical synthesis is described. Automated oligonucleotide synthesis utilizing a bifunctional reagent for the incorporation of a dihistidyl residue into oligonucleotides is described. Oligonucleotides incorporating one to three dihistidyl residues were prepared and characterized. The interaction of these oligonucleotides with a metal chelating IMAC matrix was explored.     

Hemocytes of Calliphora vicina larvae. I. Histological analysis

The rational classification of blood cells of a blowfly Calliphora vicina larva has been worked out basing on the studies of hemocyte morphology and their dynamics while ageing. Eight morphological cell types can be found during the third age in larva's hemolymph. Hemocytes of type I (prohemocytes) are undifferentiated cells, and, probably, serve as cambial elements of hemocytes of other types. Hemocytes of types II (thrombocytoids), III (cell platelets), IV, and V (early- and late post-thrombocytoids) possibly correspond to consecutive stages of a certain differentiation pattern. Hemocytes of types VI (filopodocytes), VII (crystal cells), and VIII (histolysocytes) posses a number of specific features and represent three independent cell lines. The existence of four independent cell lines in C. vicina hemolymph supports the polygenetic conception of hematopoiesis in insects. We compared our classification with other existing classifications of C. vicina larva blood cells, and proposed a new nomenclature of these cells. The existence of cells with crystal inclusions (type VII hemocytes) in C. vicina larva hemolymph has been shown for the first time. Data on two generations of blood cells during larval development of C. vicina were obtained. Hemocytes of types II, VI and VII belong to the first generation. These cells disappear completely at the stage of empty-poropped larva, when hemocytes of the second generation i.e. those of types III and VIII, appear in the hemolymph.