Temperature dependent binding of mebendazole to tubulin in benzimidazole-susceptible and -resistant strains of Trichostrongylus colubriformis and Caenorhabditis elegans

The binding of [³H]mebendazole ([³H]MBZ) to tubulin in benzimidazole-susceptible (BZ-S) and benzimidazole-resistant (BZ-R) strains of Trichostrongylus colubriformis and Caenorhabditis elegans was examined in order to investigate the biochemical changes to tubulin that result in BZ resistance in parasitic and free-living nematodes. In both species the extent of [³H]MBZ binding to tubulin was significantly reduced in the BZ-R strain compared with the BZ-S strain. The decrease in [³H]MBZ binding in the BZ-R strain of each species was the result of a significant reduction in the amount of charcoal stable [³H]MBZ-tubulin complexes and was not related to a change in the association constant of the [³H]MBZ-tubulin interaction. [³H]MBZ binding to tubulin was temperature dependent, reaching maximum levels at 37°C in BZ-S T. colubriformis and 10°C in BZ-R T. colubriformis. Both the BZ-S and BZ-R strains of C. elegans displayed maximum [³H]MBZ binding at 4°C. Resistance ratios derived from the amount of [³H]MBZ binding in the BZ-S and BZ-R strains and in vitro development assays demonstrated that the temperature dependence and extent of drug binding was indicative of BZ resistance status and was species specific in the BZ-S isolates. These results indicate that biochemical differences exist in the binding of benzimidazole carbamates to tubulin in nematode species, and suggest that the susceptibility of the parasitic nematodes to the benzimidazole anthelmintics is the result of a unique high affinity and/or high capacity interaction ofbenzimidazole carbamates with tubulin.     

Differential stability of the benzimidazole (BZ)-tubulin complex in BZ-resistant and BZ-susceptible isolates of Haemonchus contortus and Trichostrongylus colubriformis.

The binding of [3H]mebendazole ([3H]MBZ) to tubulin from BZ-susceptible (BZ-S) and BZ-resistant (BZ-R) isolates of Haemonchus contortus and Trichostrongylus colubriformis was investigated using charcoal extraction and gel filtration techniques. The amount of [3H]MBZ bound at infinite free ligand concentration (Bmax) was significantly reduced for the BZ-R isolate compared with the BZ-S isolate in both species when assayed by charcoal extraction. However, Bmax was increased to comparable levels for both BZ-S and BZ-R isolates of each species when assayed by the less stringent gel filtration technique. These results indicate that the BZ-tubulin interaction in trichostrongylid nematodes is comprised of a minimum of two components. As similar levels of total [3H]MBZ binding were observed for both BZ-S and BZ-R isolates of each species when assayed by gel filtration, it is suggested that the reduction in the pseudo-irreversible BZ binding component in BZ-R isolates results in an increase in the level of reversible BZ binding and therefore provides a survival advantage to BZ-R nematodes.     

Cytoplasmic dynein is a vesicle protein.

Microtubule-based organelle transport is thought to be mediated by the force-generating proteins cytoplasmic dynein and kinesin. These motor proteins have been characterized based on their ability to associate with and translocate microtubules. We show here that cytoplasmic dynein is also present as a peripheral membrane protein of purified synaptic vesicles. The vesicle-associated cytoplasmic dynein is identified by its photo-induced cleavage in the presence of ATP and vanadate. Purified, soluble cytoplasmic dynein is competent to bind to vesicle membranes stripped of endogenous peripheral membrane proteins by alkaline pH. Dynein binding to membranes is saturable at a concentration of 1.00 +/- 0.15 pmol/micrograms vesicle protein and has a dissociation constant of 22.3 +/- 2.4 nM. The association of cytoplasmic dynein with the membrane cannot be reversed by incubation with ATP. Furthermore, following binding to membranes, dynein retains its ability to bind ATP and to be photo-cleaved in the presence of vanadate. The presence of cytoplasmic dynein on synaptic vesicles and its ability to bind to extracted membranes supports current models of microtubule-based organelle translocation.     

Electrogenic properties of the cloned Na+/glucose cotransporter: I. Voltage-clamp studies

Summary Cystic fibrosis (CF) is characterized by abnormal epithelial Cl^– conductance (G_Cl). In vitro studies that have shown that cAMP regulation is an intrinsic property of the CF-affected G_Cl(CF-G_Cl) have been carried out previously on cultured secretory cells and on nonepithelial cells. Even though G_Cl in absorption is defective in CF, a clear demonstration of cAMP regulation of CF-G_Cl in a purely absorptive tissue is lacking. We studied the cAMP regulation of CF-G_Cl in the microperfused intact human reabsorptive sweat duct. About 40% of the ducts responded to cAMP (responsive) while the remainder of the ducts did not. In responsive ducts, cAMP-elevating agents: -adrenergic agonist isoproterenol (IPR), CPT-cAMP, forskolin, theophylline or IBMX increased G _tby about 2.3-fold (n = no. of ducts = 8). Removal of media Cl^–, but not amiloride pretreatment (in the lumen), abolished the cAMP response, indicating exclusive activation of G_Cl. cAMP activated both apical and basolateral G_Cl. cAMP hyperpolarized gluconate: Cl^– (lumen: bath) transepithelial bionic potentials (V _t=–20.3±5.2 mV, mean ±se, n=9) and transepithelial 3 1 luminal NaCl dilution diffusion potentials (V _t=–8.8±2.9 mV, n=5). cAMP activated basolateral G_Cl as indicated by increased bi-ionic (gluconate: Cl^–, bath: lumen) diffusion potentials (by about 12 mV). The voltage divider ratio in symmetric NaCl solutions increased by 60%. Compared to responsive ducts, nonresponsive ducts were characterized by smaller spontaneous transepithelial potentials in symmetrical Ringer's solution (V _t=–6.9±0.8 mV, n=24, nonresponsive vs. –19.4±1.8 mV, n=22, responsive ducts) but larger bi-ionic potentials (–94±6 mV, n=35, nonresponsive vs. –65±5 mV, n=17, responsive ducts) and dilution diffusion potentials (–40±5 mV, n=11, nonresponsive vs. –29±3 mV, n=7, responsive ducts). These results are consistent with an inherently (prestimulus) maximal activation of G_Cl in nonresponsive ducts and submaximal activation of G_Cl in responsive ducts. We conclude that cAMP activates CF-G _Cl which is expressed and abnormal in both apical and basal membranes of this absorptive epithelium in CF.Abbreviations CF cystic fibrosis - G _t transepithelial conductance - V _b electrical potential across the basolateral membrane - V _a electrical potential across the apical membrane - V _t transepithelial potential - V _b transepithelial currentinduced voltage deflections across the basolateral membrane - V _a transepithelial current-induced voltage deflections across the apical membrane - V _t transepithelial current-induced voltage deflection across the epithelium - VDR voltage divider ratio - G_Cl transepithelial Cl^– conductance - CF-G_Cl cystic fibrosis-affected Cl^– conductance - EMF electromotive force - IPR isoproterenol - IBMX 3-isobutyl-1-methylxanthine - CPT-cAMP chlorophenylthio-adenosine 3-5 cyclic monophosphate - PGE₂ prostaglandin E₂     

Calmodulin structure and function: Implication of arginine in the compaction related to ligand binding

Calmodulin (CAM) is a modulatory protein that regulates cellular activity by binding to a large number of proteins. Key elements in the Ca²⁺-dependent mechanism of interaction between CAM and the proteins it activates are the selectivity for Ca²⁺ ions and the requirement for Ca²⁺-dependent conformational changes. We report on results from a series of molecular dynamics simulations that identified discrete steps in the mechanism of structural rearrangement of CAM. The findings implicate the side chains of arginine residues in the bending of the central alpha helix. Structural and energetic considerations point to a dynamic hydrogen bonding pattern around the arginine residues as a ratcheting-type mechanism, causing the kinking of the central helix in consecutive steps stabilized by each new pattern of hydrogen bonds. Initial model building studies to locate potential binding sites of ligands such as trifluoperazine (TFP) indicate that the compaction of CAM results in several structural changes, that explain the selective binding of molecules such as TFP in the N-terminal domain. The present studies identify specific residues involved in the process of compaction and point to specific CAM residues involved in the binding of the ligand. These insights lead directly to propositions for experimental engineering of the molecular structure of CAM in order to probe the hypotheses and their consequences for the function of this important protein.     

MITOTIC ACTIVITY AT THE SHOOT APEX OF AZOLLA FILICULOIDES

The mosquito fern, Azolla filiculoides Lam., was grown in a growth chamber on a nitrogen-free culture solution at 24 C under the following photoperiod: 16 hr light/8 hr darkness. Shoot tips were fixed every 2 hr for 24 hr to determine the mitotic index for the apical cell, immediate derivatives, and remaining cells to the level of the first leaf or lateral shoot primordium. Mitotic indices were 6.9%, 6.5% and 6.3%, respectively. The colchicine method was employed to determine the cell-cycle durations and duration of mitosis for the same populations of cells. The cell-cycle duration and duration of mitosis of the apical cell were 28.2 hr and 2.8 hr, respectively; for the immediate derivatives, 26.7 hr and 2.5 hr; for the remaining cells, 23.6 hr and 2.1 hr. Conclusions: the apical cell is as mitotically active as its immediate derivatives, and there is no evidence of a quiescent apical cell.     

Poliovirus RNA-Dependent RNA Polymerase Synthesizes Full-Length Copies of Poliovirion RNA, Cellular mRNA, and Several Plant Virus RNAs In Vitro

The poliovirus RNA-dependent RNA polymerase was active on synthetic homopolymeric RNA templates as well as on every natural RNA tested. The polymerase copied polyadenylate. oligouridylate [oligo(U)], polycytidylate . oligoinosinate, and polyinosinate. oligocytidylate templates to about the same extent. The observed activity on polyuridylate. oligoadenylate was about fourfold less. Full-length copies of both poliovirion RNA and a wide variety of other polyadenylated RNAs were synthesized by the polymerase in the presence of oligo(U). Polymerase elongation rates on poliovirion RNA and a heterologous RNA (squash mosaic virus RNA) were about the same. Changes in the Mg(2+) concentration affected the elongation rates on both RNAs to the same extent. With two non-polyadenylated RNAs (tobacco mosaic virus RNA and brome mosaic virus RNA3), the results were different. The purified polymerase synthesized a subgenomic-sized product RNA on brome mosaic virus RNA3 in the presence of oligo(U). This product RNA appeared to initiate on oligo(U) hybridized to an internal oligoadenylate sequence in brome mosaic virus RNA3. No oligo(U)-primed product was synthesized on tobacco mosaic virus RNA. When partially purified polymerase was used in place of the completely purified enzyme, some oligo(U)-independent activity was observed on the brome mosaic virus and tobacco mosaic virus RNAs. The size of the product RNA from these reactions suggested that at least some of the product RNA was full-sized and covalently linked to the template RNA. Thus, the polymerase was found to copy many different types of RNA and to make full-length copies of the RNAs tested.     

Author index

(1) Cyclic AMP stimulated alanine transport in isolated hepatocytes by approx. 30%, in the range 0.2–5 mM alanine. (2) Alanine utilisation was also stimulated by cyclic AMP. The rates of transport and metabolism were comparable, both in the presence and absence of cyclic AMP. (3) At concentrations of alanine above 1 mM, addition of ouabain, or the reduction of the Na⁺ concentration, could partially inhibit transport without affecting the rate of metabolism. (4) At these alanine concentrations, stimulation of metabolism by cyclic AMP was associated with a decrease in the intracellular to extracellular alanine concentration ratio. (5) At alanine concentrations below 0.5 mM, or at higher concentrations when transport was inhibited by reducing the Na⁺ concentration, cyclic AMP caused an increase in the alanine concentration ratio. (6) It is concluded that at concentrations of alanine above 1 mM, alanine transport is not rate-limiting for alanine metabolism in hepatocytes from fed rats, and cyclic AMP stimulates alanine metabolism primarily by an effect on an intracellular reaction. At physiological concentrations of alanine, however, alanine transport appears to be rate-limiting in agreement with a previous report.     

Modification of cell membrane in variants of chinese hamster cells resistant to abrin

Stable and heritable variants of Chinese hamster ovary (CHO) cells which are resistant to different levels (0.1, 1.0 and 10 μg/ml) of the toxin abrin have been isolated and characterized. The frequency of resistant colonies to abrin was increased with the concentration of a chemical mutagen. There was no effect of cell density or cross-feeding on the recovery of variants. In experiments using fluorescein-labeled abrin and ricin which bind to terminal (non-sialylated) galactose residues of cell-surface oligosaccharides, parental cells exhibited strong binding toward both toxins, whereas no fluorescence was observed in the resistant clones. A fluorescein-conjugated lectin, BS II, which is specific for terminal N-acetyl- -glucosaminyl residues, did not interact with the parental cells, but did with the resistant clones. This suggests that on the surface of resistant cells the number of terminal galactosyl residues of oligosaccharide chains in glycoproteins was reduced, exposing the penultimate N-acetyl- -glucosaminyl residues. The number of available endogenous acceptor sites for galactosyl transferase in the abrin-resistant clones was directly proportional to the degree of resistance. In the presence of great excess of exogenous acceptor, the rates of galactosyl transfer were similar in all the abrin-resistant cell types tested, with levels ranging from 1.4 to 1.7 times parental cell values. Studies with tetraploid cell hybrids reveal that resistance was a recessive trait. Fluctuation analysis showed that abrin resistance occurred in CHO cell populations at a rate of 4−7 × 10⁻⁸/cell/generation. The system may serve as a new marker for quantitative mutagenesis studies.