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PubMed1995

Catalytic specificity of protein-tyrosine kinases is critical for selective signalling.

How do distinct protein-tyrosine kinases activate specific down-stream events? Src-homology-2 (SH2) domains on tyrosine kinases or targets of tyrosine kinases recognize phosphotyrosine in a specific sequence context and thereby provide some specificity. The role of the catalytic site of tyrosine kinases in determining target specificity has not been fully investigated. Here we use a degenerate peptide library to show that each of nine tyrosine kinases investigated has a unique optimal peptide substrate. We find that the cytosolic tyrosine kinases preferentially phosphorylate peptides recognized by their own SH2 domains or closely related SH2 domains (group I; ref. 3), whereas receptor tyrosine kinases preferentially phosphorylate peptides recognized by subsets of group III SH2 domains. The importance of these findings for human disease is underscored by our observation that a point mutation in the RET receptor-type tyrosine kinase, which causes multiple endocrine neoplasia type 2B, results in a shift in peptide substrate specificity.

Amino Acid Sequence↗

PubMed1976

The hypothalamo-hypophysial system in Acipenseridae. V. Ecological-histophysiological analysis of the neurohypophysis of the female sturgeon Acipenser güldenstädti Brandt during up-stream migration and after spawning.

Neurohypophyses from sexually mature female sturgeons, Acipenser guldenstadti Brandt, killed before, soon after spawning, and during down-stream migration were studied light and electron microscopically. Ovaries were examined only under a light microscope. A large amount of neurosecretory material (3.5-4.5 arbitrary units) is found in the neurohypophysial "roots" during up-stream migration to spawning grounds. Neurosecretory fibres and their terminals are replete with elementary neurosecretory granules. Side by side with the latter some terminals contain single residual granules and few "synaptic" vesicles. Soon after spawning the amount of neurosecretory material decreases markedly in most individuals (content up to 1-3 units), and elementary granules are few in the fibres and their terminals. The number of residual granules increases, and "synaptic" vesicles are especially numerous. Granulated, disintegrating neurosecretory granules and granule-shadows occur in the preterminal parts of the fibres. The number of pituicytes increases becuase some "light" tanycytes seem to migrate from the ependymal into the subependymal layer. The restoration of neurosecretory material (reaching again up to 3.5-4.5 units) occurs within a month after spawning. The number of elementary granules increases in fibres and terminals while that of residual granules and "synaptic" vesicles decreases. These changes in the neurohypophysis of females after spawning are due to discharge of great amounts of peptide neurohorsmones into the general circulation after spawning. Spawning is regarded as a "physiological" stress for these females.

Animals↗

PubMed1991

[Pit cells--a tissue form of large granule-containing lymphocytes with natural killer activity].

Basing on structural and functional signs, pit cells and large granule-containing lymphocytes (LGCL) can be considered as the same type of cells. LGCL take origin from bone marrow, circulate in the blood stream and get into tissues as pit cells or as interepitheliocytic lymphocytes. Therefore, on this ground for convenience of studying and understanding these elements of cell immunity a suggestion is made to distinguish the LGCL system, possessing natural killer activity and paracrinic secretion. An increased functional load to the organism results in an intensified getting of LGCL into places of structural reconstructions of tissues (regenerative active zones). The same effector elements--LGCL (pit cells)--in dependence of necessary functional ensuring under various conditions, produce in the surrounding parenchyma both a proliferative effect and its opposite action. The latter can be manifested as a monocellular colliquational necrosis, or as apoptosis. Apoptosis (active cellular death) is of greatest interest. Disturbances of this mechanism can facilitate to an excess proliferation of cells, or to their extreme death; malignant growth and transplant-versus-host reaction can be mentioned as examples, respectively. LGCL play an important role in the immunobiological supervision and are effectors of active ageing mechanisms. Studying of this important link in regulation of quantity of cell population adequate to functional load should help to purposively influence it for stimulating or repressing cell proliferation, depending on certain concrete purposes.

APUD Cells↗

PubMed1975

Studies on intra-arterial cushions. I. Morphology of the cushions at the origins of intercostal arteries in mice.

The origins of intercostal arteries in mice are surrounded by cushions which project into the aortal lumen. A boat-like structure, whose bow is directed against the blood stream, is formed by the cushions. The shape of this formation depends on the dilatation of the aortal wall, becoming flatter when the aortal wall is stretched. Grooves direct the blood into the initial segment of the branching artery which is narrowed in moderately distended or relaxed states of the aorta. The cushions are covered by an endothelial layer which continues to the endothelium of the aorta and of the branching artery. Ramified smooth muscle cells are found in the stroma of the cushions, while connective tissue are absent. Ramifications of the lamina elastica interna project into the stroma and faint elastic fibres reach the apical margins of the cushions. Basement membranes of ramified smooth muscle cells form a three-dimensional network which is connected with the basement membrane of the endothelium. Contacts between processes of endothelial cells and those of ramified smoot; muscle cells occur. Collagen fibrols are absent or extremely rare. In the interstitial space clusters of electron dense granules (mean diameter 500 A) are found which might be secreted by the specialized ramified smooth muscle cells. This peculiar morphology characterizes the intraaortal cushions as autonomous formations and excludes their being regarded as simple retroextension of the branching artery.

Animals↗

PubMed1985

Integration of X-ray microanalysis and morphometry of biological material.

It was investigated how to extract both morphometrical and X-ray elemental information from scanning electron microscopical (SEM) or scanning transmission electron microscopical (STEM)-images and how to integrate these two information streams either on line or off-line after storage. Cytochemical reaction products in cell organelles in ultrathin sections are the biological structures of interest. In such organelles four different situations can be met: morphologically the structures are homomorph or heteromorph; chemically the elements are distributed either homogeneously or heterogeneously. A new program has been proposed and described, which permits determination of both the area and the mean net-intensity value of chemical elements, inhomogeneously distributed over heteromorph organelles. The value of this integration method is demonstrated by three examples of increasing complexity, starting with two elements which are more or less homogeneously distributed over one lysosome, the establishing of a platinum discontinuity in an acidophilic granule and finally the localization of two chemical elements inhomogeneously distributed over a rather heteromorph phagolysosome. In two examples Chelex ion exchange beads, maximally loaded with the element also present in the structure of interest, are co-embedded with the tissue as internal standards. In such cases the absolute elemental concentration in the structures analysed can be established. The presence of such cross-sectioned beads in the ultrathin sections is also used: to demonstrate their function as models to select the proper conditions for the digital-controlled raster analysis of the unknown cell- or tissue structures, to prove the value of this method.

Acid Phosphatase↗

PubMed2000

The absence of cerebellar granule cell necrosis in the mouse following L-2-chloropropionic acid administration.

Oral administration of L-2-chloropropionic acid (L-CPA) to rats either as a single dose (750 mg/kg) or daily doses (250 mg/kg per day for 3 days) produces selective necrosis to the granule cell layer of the cerebellum. As part of a study to understand the mechanism of this selective toxicity, we investigated the toxicity of L-CPA and a related analogue, DL-2-bromopropionic acid to the mouse with particular emphasis on the brain. Following a single oral dose (up to 1000 mg/kg), or daily oral doses of 250 mg/kg per day L-CPA up to maximum tolerated doses, produced no evidence of neurotoxicity. Similarly, daily oral doses of DL-2-bromopropionic acid at 250 mg/kg per day produced no evidence of neurotoxicity. The basis for the lack of response was explored by examining the metabolism and disposition of L-[2-14C]-CPA in the mouse. Following a single oral dose of 250 mg/kg L-CPA, radioactivity was rapidly absorbed from the gastrointestinal tract into the blood stream. Peak plasma concentrations of radiolabel and L-CPA occurred within 2 h of dosing at about 1.8 mM, and were then lost from the plasma with a half-life of 1 h. The only metabolite detected in the plasma was 2-S-cysteinylpropanoic acid derived from the glutathione conjugate. About 39% of the dose was excreted in the urine in the first 24 h, mainly as 2-S-cysteinylpropanoic acid with only a small amount of unchanged L-CPA. The remaining radiolabel from L-CPA was excreted in the faeces (26%) and exhaled as carbon dioxide (about 14%) over 72 h. Radiolabel from L-[2-14C]-CPA was present in the cerebellum at a peak concentration of 1 mM 1-2 h after dosing and then was lost more slowly than from the plasma. Measurement of non-protein sulphydryl content in the brain, liver and kidneys showed a decrease in the liver and kidneys 4 h after dosing which recovered fairly rapidly, while a more prolonged decrease was found in the brain, especially the cerebellum. Our studies show that the mouse is refractory to cerebellar injury following treatment with L-CPA and DL-2-bromopropionic acid. The mouse appears to metabolize and excrete L-CPA as its glutathione-derived conjugate(s) more rapidly than the rat, thereby limiting the availability of L-CPA to the cerebellum, which may account for the absence of neuronal cell injury.

Animals↗

PubMed2003

A kinetic mechanism for the fast movement of Chara myosin.

Endoplasmic streaming of characean cells of Nitella or Chara is known to be in the range 30-100 microm/second. The Chara myosin extracted from the cells and fixed onto a glass surface was found to move muscle actin filaments at a velocity of 60 microm/second. This is ten times faster than that of skeletal muscle myosin (myosin II). In this study, the displacement caused by single Chara myosin molecules was measured using optical trapping nanometry. The step size of Chara myosin was approximately 19nm. This step size is longer than that of skeletal muscle myosin but shorter than that of myosin V. The dwell time of the steps was relatively long, and this most likely resulted from two rate-limiting steps, the dissociation of ADP and the binding of ATP. The rate of ADP release from Chara myosin after the completion of the force-generation step was similar to that of myosin V, but was considerably slower than that of skeletal muscle myosin. The 19nm step size and the dwell time obtained could not explain the fast movement. The fast movement could be explained by the load-dependent release of ADP. As the load imposed on the myosin decreased, the rate of ADP release increased. We propose that the interaction of Chara myosin with an actin filament resulted in a negative load being imposed on other myosin molecules interacting with the same actin filament. This resulted in an accelerated release of ADP and the fast sliding movement.

Adenosine Diphosphate↗

PubMed1998

Electrophysiology in the eukaryotic model cell Saccharomyces cerevisiae.

Since the mid-1980s, use of the budding yeast, Saccharomyces cerevisiae, for expression of heterologous (foreign) genes and proteins has burgeoned for several major purposes, including facile genetic manipulation, large-scale production of specific proteins, and preliminary functional analysis. Expression of heterologous membrane proteins in yeast has not kept pace with expression of cytoplasmic proteins for two principal reasons: (1) although plant and fungal proteins express and function easily in yeast membranes, animal proteins do not, at least yet; and (2) the yeast plasma membrane is generally regarded as a difficult system to which to apply the standard electrophysiological techniques for detailed functional analysis of membrane proteins. Especially now, since completion of the genome-sequencing project for Saccharomyces, yeast membranes themselves can be seen as an ample source of diverse membrane proteins - including ion channels, pumps, and cotransporters - which lend themselves to electrophysiological analysis, and specifically to patch-clamping. Using some of these native proteins for assay, we report systematic methods to prepare both the yeast plasma membrane and the yeast vacuolar membrane (tonoplast) for patch-clamp experiments. We also describe optimized ambient conditions - such as electrode preparation, buffer solutions, and time regimens - which facilitate efficient patch recording from Saccharomyces membranes. There are two main keys to successful patch-clamping with Saccharomyces. The first is patience; the second is scrupulous cleanliness. Large cells, such as provided by polyploid strains, are also useful in yeast patch recording, especially while the skill required for gigaseal formation is being learned. Cleanliness is aided by (1) osmotic extrusion of protoplasts, after minimal digestion of yeast walls; (2) use of a rather spare suspension of protoplasts in the recording chamber; (3) maintenance of continuous chamber perfusion prior to formation of gigaseals; (4) preparation (pulling and filling) of patch pipettes immediately before use; (5) application of a modest pressure head to the pipette-filling solution before the tip enters the recording bath; (6) optical control for debris at the pipette tip; and (7) discarding of any pipette that does not "work" on the first try at gigaseal formation. Other useful tricks toward gigaseal formation include the making of protoplasts from cells grown aerobically, rather than anaerobically; use of sustained but gentle suction, rather than hard suction; and manipulation of bath temperature and/or osmotic strength. Yeast plasma membranes form gigaseals with difficulty, but these tend to be very stable and allow for long-term cell-attached or whole-cell recording. Yeast tonoplasts form gigaseals with ease, but these tend to be unstable and rarely allow recording for more than 15 min. The difference of stability accrues mainly because of the fact that yeast protoplasts adhere only lightly to the recording chamber and can therefore be lifted away on the patch pipette, whereas yeast vacuoles adhere firmly to the chamber bottom and are subsequently stressed by very slight relative movements of the pipette. With plasma membranes, conversion from cell-attached recording geometry to isolated ISO patch (inside-out) geometry is accomplished by blowing a fine stream of air bubbles across the pipette tip; to whole-cell recording geometry, by combining suction and one high-voltage pulse; and from whole-cell to OSO patch (outside-out) geometry, by sudden acceleration of the bath perfusion stream. With tonoplasts, conversion from the vacuole-attached recording geometry to whole-vacuole geometry is accomplished by application of a large brief voltage pulse; and further conversion to the OSO patch geometry is carried out conventionally, by slow withdrawal of the patch pipette from the vacuole, which usually remains attached to the chamber bottom.

Cell Membrane↗

PubMed1984

Platelet adhesion.

Platelets do not adhere to surfaces to which flowing blood is normally exposed in vivo. When the lining of a blood vessel is altered or damaged, however, platelets do adhere to the injured site. Platelet adhesion is one of the first events in the formation of hemostatic plugs and thrombi, and plays a part in the development of atherosclerotic lesions. Other surfaces to which platelets adhere include particulate matter in the blood stream, bacteria and other microorganisms, the artificial surfaces of prosthetic devices, and some altered cells in the blood, particularly macrophages. The majority of investigators have studied the interaction of platelets with the subendothelium of normal vessels of young animals, or with isolated vessel wall constituents such as collagen. There are very few studies of platelet adhesion to repeatedly damaged or diseased blood vessels, although it is generally assumed that platelets interact with the connective tissue, fibrin, and cholesterol crystals in atherosclerotic lesions. Underlying the endothelium of blood vessel is the basement membrane, which has been shown to contain type IV collagen, elastin with its associated microfibrils, von Willebrand Factor, fibronectin, thrombospondin, laminin, and heparan sulfate. If only the endothelium is removed, the main structure exposed is the basement membrane with its associated proteins, but deeper injuries expose fibrillar type III collagen and microfibrils. In most studies in which large arteries have been injured by passage of a balloon catheter, basement membrane, type III collagen and the microfibrils around elastin have been exposed. Platelets do not react strongly with basement membrane and the type IV collagen in it is relatively inert. In contrast, platelets adhere firmly to type III (and type I) collagen and spread on it. Although in vitro studies have shown that platelets can interact with collagen in artificial media without plasma proteins, investigations of platelet adhesion at high shear rates indicate that von Willebrand Factor is necessary for firm platelet adhesion under these conditions. Fibronectin and thrombospondin may also have a role in platelet adhesion. However, platelets do not bind von Willebrand Factor or fibronectin until the platelets have been stimulated to release their granule contents, so these binding sites probably do not become available until the platelets have interacted with collagen or another release-inducing agent such as thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗