Hapten-mediated immunopurification of membrane proteins labeled with fluorescein derivatives

The behavior of cell surface components labeled with fluorochromes can be studied by fluorescence microscopy and spectroscopy; further structural analyses would be facilitated by purification of the labeled components. We have developed a protocol for identifying the targets for labeling with fluorescein derivatives, by using 125I- diiodofluorescein isothiocyanate ( 125IFC ) and for isolating the labeled components with anti-IFC immunoadsorbents. Anti-IFC antibodies obtained from rabbits immunized with IFC-hemocyanin were purified by affinity chromatography and coupled to CNBr-activated Sepharose 4B. The anti-IFC immunoadsorbents could then be used to isolate the entire set of 125IFC -proteins from crude detergent extracts of labeled sea urchin sperm, with a 70% yield and a purification of more than 250 fold. Nonspecific binding of unlabeled proteins to the immunoadsorbent was insignificant. When the immunoadsorbent IFC-protein complex was used directly as an immunogen, antibodies were obtained that reacted with the underivatized proteins that were targets for IFC labeling, as indicated by immunoblotting after gel electrophoresis. The antibodies also reacted with the surface of unlabeled sperm as shown by immunofluorescence. Thus, by treating the IFC-sperm proteins as a class, we obtained antibodies that recognized the unlabeled proteins in situ or in cell extracts. This approach should be generally useful in obtaining reagents directed against specific cell surface components.     

Multiphasic Uptake of Amino Acids by Barley Roots

Concentration-dependence and other characteristics of uptake of ³H-labeled l -lysine, l -methionine and l -proline by excised roots of barley (Hordeum vulgare L.) were studied. Use of relatively short uptake and wash periods and low solute concentrations ensured good estimates of influx across the plasmalemma. Uptake in the range of 10^?7M– 6.3 × 10^?3M can be precisely represented by four or five phases of single, multiphasic mechanisms. The mechanisms appear to be relatively specific as judged from the competition by unlabeled analogues. Structural requirements for interaction of a compound with the uptake site for methionine are given, as are the effects of analogues on the phase pattern for this amino acid. There is no indication of separate uptake and transition sites for methionine or lysine. i.e. phase transitions seem in this case to be caused by binding of molecule(s) to the uptake site. Uptake, but not phase patterns, was highly pH-dependent. The optima were pH 5 for lysine, pH 3–5 (a broad peak) for methionine and about pH 5.5 for proline. Uptake of the three amino acids was strongly inhibited by 2,4-dinitrophenol. sulfhydryl reagents and deoxycholate.     

High performance liquid chromatographic analysis of catecholamines in growing and non-growing Tetrahymena pyriformis

Tetrahymena pyriformis, strains NT-1 and W, harvested in logarithmic (growing) and stationary (non-growing) phases, were found by high-performance liquid chromatography to contain considerable quantities of dopamine. In addition, small amounts of epinephrine and norepinephrine were detected. Logarithmic-phase strain NT-1 cells contained 249±44 pg dopamine/10⁶ cells compared to 477±42 pg/10⁶ cells for logarithmic-phase strain W cells for logarithmic-phase strain W cells. The dopamine content of stationary-phase cells was approximately half the value of the logarithmic-phase cells. There was a significant amount of dopamine in the growth medium from stationary-phase cultures and, to a lesser extent, logarithmic-phase cells.     

9-Benzylpurines with inhibitory activity against Mycobacterium tuberculosis

9-Benzylpurines with a variety of substituents in the 2-, 6- and/or 8-position have been prepared and screened for antimycobacterial effects. High inhibitory activity against Mycobacterium tuberculosis was found for 9-benzylpurines carrying a phenylethynyl-, trans-styryl or aryl substituents in the 6-position and generally chlorine in the 2-position tends to increase activity.     

Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization

In migrating adherent cells such as fibroblasts and endothelial cells, the microtubule-organizing center (MTOC) reorients toward the leading edge [1-3]. MTOC reorientation repositions the Golgi toward the front of the cell [1] and contributes to directional migration [4]. The mechanism of MTOC reorientation and its relation to the formation of stabilized microtubules (MTs) in the leading edge, which occurs concomitantly with MTOC reorientation [3], is unknown. We show that serum and the serum lipid, lysophosphatidic acid (LPA), increased Cdc42 GTP levels and triggered MTOC reorientation in serum-starved wounded monolayers of 3T3 fibroblasts. Cdc42, but not Rho or Rac, was both sufficient and necessary for LPA-stimulated MTOC reorientation. MTOC reorientation was independent of Cdc42-induced changes in actin and was not blocked by cytochalasin D. Inhibition of dynein or dynactin blocked LPA- and Cdc42-stimulated MTOC reorientation. LPA also stimulates a Rho/mDia pathway that selectively stabilizes MTs in the leading edge [5, 6]; however, activators and inhibitors of MTOC reorientation and MT stabilization showed that each response was regulated independently. These results establish an LPA/Cdc42 signaling pathway that regulates MTOC reorientation in a dynein-dependent manner. MTOC reorientation and MT stabilization both act to polarize the MT array in migrating cells, yet these processes act independently and are regulated by separate Rho family GTPase-signaling pathways.     

Cambrian high-resolution biostratigraphy and carbon isotope chemostratigraphy in Scania, Sweden: first record of the SPICE and DICE excursions in Scandinavia

A core drilling (Andrarum‐3), from the classical locality at Andrarum, Scania, southernmost Sweden, penetrated a 28.90‐m‐thick Cambrian succession. The core comprises dark grey to black, finely laminated mudstones and shales with early concretionary carbonate lenses (stinkstones or orsten) and a few primary carbonate beds. The middle Cambrian (provisional Series 3) part of the core comprises 17.35 m, whereas the Furongian Series (upper Cambrian) part covers the remaining 11.55 m. Nineteen trilobite and two phosphatocopine genera are present in the middle Cambrian, whereas the less diverse Furongian interval yielded four trilobite and three phosphatocopine genera. Other, less frequent, faunal elements include conodonts (s. l.), brachiopods, sponge spicules, bradoriids, and coprolites. Trilobites and phosphatocopines were used to subdivide the core into seven biozones ranging from the Ptychagnostus atavus Zone to the Parabolina spinulosa Zone (P. spinulosa Subzone). Carbon isotopic analyses (δ¹³C_org) through the core show two important excursions, the negative DrumIan Carbon isotope Excursion (DICE) in the Pt. atavus Zone, and the Steptoean Positive Carbon Isotope Excursion (SPICE) beginning near the first appearance of Glyptagnostus reticulatus and extending upward into the Olenus and Agnostus (Homagnostus) obesus Zone. The DICE displays a peak value, in the samples at hand, of –30.45‰δ¹³C_org in the lower part of the P. atavus Zone. The δ¹³C_org values increase through the overlying L. laevigata and A. pisiformis zones and display peak values of c. –28.00‰δ¹³C_org in the lowermost Furongian Olenus wahlenbergi and O. attenuatus subzones. Thereafter the values decrease significantly through the O. scanicus Subzone. Both isotopic excursions have been documented from several palaeocontinents, but never before from Baltica. Moreover, for the first time these excursions are recorded from organic matter in an alum shale setting. The recorded shift of +1.50–2.00‰δ¹³C_org is approximately half the magnitude of the SPICE documented from other regions. This discrepancy may be related to temporal variations in the type, origin, or diagenesis of the organic fraction analysed.     

Regional Assessment of N Saturation using Foliar and Root \varvec {\delta}^{\bf 15}{\bf N}

N saturation induced by atmospheric N deposition can have serious consequences for forest health in many regions. In order to evaluate whether foliar may be a robust, regional-scale measure of the onset of N saturation in forest ecosystems, we assembled a large dataset on atmospheric N deposition, foliar and root and N concentration, soil C:N, mineralization and nitrification. The dataset included sites in northeastern North America, Colorado, Alaska, southern Chile and Europe. Local drivers of N cycling (net nitrification and mineralization, and forest floor and soil C:N) were more closely coupled with foliar than the regional driver of N deposition. Foliar increased non-linearly with nitrification:mineralization ratio and decreased with forest floor C:N. Foliar was more strongly related to nitrification rates than was foliar N concentration, but concentration was more strongly correlated with N deposition. Root was more tightly coupled to forest floor properties than was foliar . We observed a pattern of decreasing foliar values across the following species: American beech>yellow birch>sugar maple. Other factors that affected foliar included species composition and climate. Relationships between foliar and soil variables were stronger when analyzed on a species by species basis than when many species were lumped. European sites showed distinct patterns of lower foliar , due to the importance of ammonium deposition in this region. Our results suggest that examining values of foliage may improve understanding of how forests respond to the cascading effects of N deposition.     

Myosin-1c couples assembling actin to membranes to drive compensatory endocytosis

Compensatory endocytosis follows regulated exocytosis in cells ranging from eggs to neurons, but the means by which it is accomplished are unclear. In Xenopus eggs, compensatory endocytosis is driven by dynamic coats of assembling actin that surround and compress exocytosing cortical granules (CGs). We have identified Xenopus laevis myosin-1c (XlMyo1c) as a myosin that is upregulated by polyadenylation during meiotic maturation, the developmental interval that prepares eggs for fertilization and regulated CG exocytosis. Upon calcium-induced exocytosis, XlMyo1c is recruited to exocytosing CG membranes where actin coats then assemble. When XlMyo1c function is disrupted, actin coats assemble, but dynamic actin filaments are uncoupled from the exocytosing CG membranes such that coats do not compress, and compensatory endocytosis fails. Remarkably, there is also an increase in polymerized actin at membranes throughout the cell. We conclude that XlMyo1c couples polymerizing actin to membranes and so mediates force production during compensatory endocytosis.