B-ring of colchicine and its role in taxol-induced tubulin polymerization

Taxol-induced assembly of purified tubulin is not inhibited by the colchicine analogue 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone. Colchicine analogues having intact A, C and B-rings (without NH-CO-CH3) such as desacetamidocolchicine have also been found to be inactive. It has been observed that these two colchicine analogues are incorporated into polymers when incubated in the presence of taxol. Furthermore, preformed taxol-induced polymers of tubulin have been found to bind these two colchicine analogues. These results suggest that colchicine-binding domains on the tubulin molecule are mostly (if not completely) exposed in the taxol-induced polymers.     

Sulfhydryls of platelet tubulin: their role in polymerization and colchicine binding

Sulfhydryls and disulfides of platelet tubulin have been quantified, their accessibility and reactivity measured, and their role in polymerization and colchicine binding evaluated. Platelet tubulin isolated by two cycles of temperature-dependent polymerization--depolymerization was found to contain 12 free sulfhydryl groups per tubulin monomer all of which reacted rapidly with p-chloromercuribenzoate. One sulfhydryl was inaccessible to dithiobis(nitrobenzoic acid). Under anaerobic conditions of tubulin extraction, one intrachain disulfide bridge was found per tubulin monomer. Polymerization of tubulin reduced the number of sulfhydryls by one which were able to react with p-chloromercuribenzoate or dithiobis(nicotinic acid) but did not affect the disulfide bridge. Polymerizability of platelet tubulin was very sensitive to blocking of free sulfhydryl groups. Complete inhibition of microtubule assembly was obtained when the number of free sulfhydryls per tubulin was reduced by 3 but could be reversed by the addition of dithiothreitol. Colchicine binding, on the other hand, was only minimally influenced by blocking of sulfhydryls.     

Charge effect on the colchicine binding-site of tubulin

Poly(L-lysine) was found to enhance colchicine binding activity of brain tubulin to a several folds. Bases of biological interests that were tested and found to be inactive were spermine, spermidine and even L-lysine. Part of this enhance binding is due to the increase in the affinity of colchicine-tubulin interaction in the presence of poly(L-lysine). Moreover, poly(L-lysine) stabilized the colchicine binding site of tubulin against thermal denaturation.     

Comparative molecular field analysis of colchicine inhibition and tubulin polymerization for combretastatins binding to the colchicine binding site on beta-tubulin

A molecular modeling study using Comparative Molecular Field Analysis (CoMFA) was undertaken to develop a predictive model for combretastatin binding to the colchicine binding site of tubulin. Furthermore, we examined the potential contribution of lipophilicity (log P) and molecular dipole moment and were unable to correlate these properties to the observed biological data. In this study we first confirmed that tubulin polymerization inhibition (IC50) correlated (R2 = 0.92) with [3H]colchicine displacement. Although these data correlated quite well, we developed two independent models for each set of data to quantify structural features that may contribute to each biological property independently. To develop our predictive model we first examined a series of molecular alignments for the training set and ultimately found that overlaying the respective trimethoxyphenyl rings (A ring) of the analogues generated the best correlated model. The CoMFA yielded a cross-validated R2 = 0.41 (optimum number of components equal to 5) for the tubulin polymerization model and an R2 = 0.38 (optimum number of components equal to 5) for [3H]colchicine inhibition. Final non-cross-validation generated models for tubulin polymerization (R2 of 0.93) and colchicine inhibition (R2 of 0.91). These models were validated by predicting both biological properties for compounds not used in the training set. These models accurately predicted the IC50 for tubulin polymerization with an R2 of 0.88 (n = 6) and those of [3H]colchicine displacement with an R2 of 0.80 (n = 7). This study represents the first predictive model for the colchicine binding site over a wide range of combretastatin analogues.     

Identification of macrophage external membrane proteins and their possible role in cell adhesion

Starch-activated mouse peritoneal macrophages (STpMAC) plated on plastic demonstrate the adhesive properties typical for activated pMAC: attaching as round cells and, within 15 min, spreading out with marginal membrane ruffles. These attached STpMAC were labeled by lactoperoxidase-catalysed 125I surface iodination, sodium dodecyl- sulfate-lysed, and the lysates electrophoresed on polyacrylamide gels which were examined by autoradiography. The STpMAC morphological phenotype correlates with the labeling of a particular protein (195,000, estimated mol wt). Normal pMAC (NpMAC), from unstimulated mice, do not spread and do not display the 195,000 band. Both pMAC band patterns, including the 195,000 band, are relatively resistant to trypsin digestion, as is pMAC adhesion itself trypsin-resistant. Neither class of pMAC exhibits fibronectin (Cell Adhesion Factor, LETS protein) which is a component in the adhesive matrix of cells forming trypsin-sensitive monolayers. When pMAC are tested against antifibronectin antibody, these cells do not give immunofluorescent staining. In summary, two functions in pMAC adhesion, enzyme resistance and the ability to spread, appear related to molecular properties distinctive for pMAC surface protein.     

Podophyllotoxin as a probe for the colchicine binding site of tubulin.

The binding of [3H]podophyllotoxin to tubulin, measured by a DEAE-cellulose filter paper method, occurs with an affinity constant of 1.8 X 10(6) M-1 (37 degrees at pH 6.7). Like colchicine, approximately 0.8 mol of podophyllotixin are bound per mol of tubulin dimer, and the reaction is entropy-driven (43 cal deg-1 mol-1). At 37 degrees the association rate constant for podophyllotoxin binding is 3.8 X 10(6) M-1 h-1, approximtaely 10 times higher than for colchicine; this is reflected in the activation energies for binding which are 14.7 kcal/mol for podophyllotoxin and 20.3 kcal/mol for colchicine. The dissociation rate constant for the tubulin-podophyllotoxin complex is 1.9 h-1, and the affinity constant calculated from the ratio of the rates is close to that obtained by equilibrium measurements. Podophyllotxin and colchicine are mutually competitive inhibitors. This can be ascribed to the fact that both compounds have a trimethoxyphenyl ring and analogues of either compound with bulky substituents in their trimethoxyphenyl moiety are unable to inhibit the the binding of either of the two ligands. Tropolone, which inhibits colchicine binding competitively, has no effect on the podophyllotoxin/tubulin reaction. Conversely, podophyllotoxin does not influence tropolone binding. Moreover, the tropolone binding site of tubulin does not show the temperature and pH lability of the colchicine and podophyllotoxin domains, hence this lability can be ascribed to the trimethoxyphenyl binding region of tubulin. Since podophyllotoxin analogues with a modified B ring do not bind, it is concluded that both podophyllotoxin and colchicine each have at least two points of attachment to tubulin and that they share one of them, the binding region of the trimethoxyphenyl moiety.     

Effect of experimental colchicine encephalopathy on brain protein synthesis and tubulin metabolism

Colchicine blocks axoplasmic flow and produces neurofibrillary degeneration. Brain slices from mice injected intracerebrally with colchicine incorporated more [¹⁴C]leucine into protein and had a decreased uptake of [¹⁴C]leucine into the perchloric acid-soluble pool than did their controls. Brain RNA content was decreased and free leucine increased by colchicine-induced encephalopathy. The specific activities of proteins from subcellular fractions of colchicine-injected brain were increased in the nuclear fraction, the 100,000-g supernatant, and its vinblastine-precipitable tubulin. The ratio of the specific activity of the crude mitochondrial fraction to that of the total homogenate was decreased, as would consistent with impaired movement of newly labeled protein into synaptosomes. Colchicine-injected brain extracts contained one or more cytosol fractions that stimulated ribosomal incorporation of [¹⁴C]leucine into protein in a cell-free system. Colchicine-binding-activity measurements indicated loss of soluble and particulate tubulin in colchicine-injected brains; the decrease of soluble tubulin was verified by its selective precipitation with vinblastine. Colchicine encephalopathy did not affect the rate of spontaneous breakdown of in vitro colchicine binding activity. Similarities of colchicine encephalopathy to the neuron's response to axonal damage suggest that colchicine-induced increase in protein synthesis may, in part, reflect a neuronal response to blockage of neuroplasmic transport.     

A possible role for membrane depolarization in epithelial wound healing

Linear narrow wounds produced on cultured bovine corneal endothelial monolayers heal by actin cable formation at the wound border and lamellar crawling of cells into the injured area. We report the novel finding that membrane potential depolarization occurs at the leading edge of wounds and gradually extends inward toward the neighboring cells. We have determined that the replacement of extracellular Na⁺ by choline and the incorporation of phenamil, an inhibitor of the epithelial Na⁺ channel (ENaC), provoke a decrease in the actin cable and depolarization areas and in the lamellar activity of the wound edges. To the contrary, extracellular Li⁺ can successfully replace Na⁺ in the determination of the depolarization and cytoskeletal responses. This finding supports the idea that membrane depolarization, not the increase in intracellular Na⁺ concentration, is responsible for the formation of the actin cable, a result that is in agreement with previous evidence showing that nonspecific depolarization of the plasma membrane potential (PMP) of epithelial cells may promote characteristic cytoskeletal rearrangements per se (Chifflet S, Hernández JA, Grasso S, and Cirillo A. Exp Cell Res 282: 1–13, 2003). We suggest that spontaneous depolarization of the PMP of the cells at the wound borders determined by a rise in the ENaC activity of these cells constitutes an additional factor in the intermediate cellular processes leading to wound healing in some epithelia. actin; epithelial sodium channel     

A possible role of cholesterol in membrane adhesion

Calcium phosphate induced membrane aggregation was studied for erythrocyte vesicles and lipid membrane vesicles. The later lipid membrane components were similar in composition to those of erythrocyte membranes. The presence of an appropriate amount of cholesterol is an important factor in the production of the calcium phosphate dependent membrane aggregation.     

The possible role of fixed membrane surface charges in acetylcholine release at the frog neuromuscular junction

Summary Bass and Moore [Proc. Nat. Acad. Sci. 55:1214 (1966)] proposed that the vesicles containing acetylcholine undergo Brownian motion in the nerve terminals. Acetylcholine is released whenever a vesicle touches the inner face of the axolemma of the nerve terminal. The frequency at which contact is made is limited by an energy barrier that must be overcome before the vesicle can touch the axolemma. The energy barrier has two components. (1) An electrostatic repulsion between positive, fixed charges on the vesicles and a relatively positive potential at the face of the axolemma that is generated by the resting potential. (2) A layer of water molecules held to the vesicle by the surface charge. This model is inconsistent with experimental data. A modification of the model is presented. Both the vesicle and the inner face of the axolemma are assumed to have fixed, negative surface charges that are responsible for the energy barrier. By a series of simplifications, the model leads to two predictions. (1) A plot of the ln (miniature end plate potentials/sec) as a function of the concentration of ions in the axoplasm)^–0.5 should give a straight line. (2) A plot of ln (end plate potential amplitudes) as a function of (extracellular Ca⁺⁺ concentration)^–0.5 should give a straight line. These predictions are shown to agree reasonably well with experimental data.     

Evidence for a role of dopamine in cardiac function

Experiments were carried out to further investigate the possibility that dopamine may have a functional role in the heart. Measurement of the activity of the enzyme tyrosine hydroxylase showed that this enzyme was present both in control hearts and in tissue with total sympathetic denervation. Furthermore, radioligand binding studies showed that there are high-affinity binding sites for dopamine in both control and denervated hearts.Our results support the view that there is dopamine in the heart which is not associated with noradrenergic nerves.     

Regulation of mammary epithelial cell function: a role for stromal and basement membrane matrices

Summary Interactions between epithelial cells and their environment are critical for normal function. Mammary epithelial cells require hormonal and extracellular matrix (ECM) signalling for the expression of tissue specific characteristics. With regard to ECM, cultured mammary epithelial cells synthesize and secrete milk proteins on stromal collagen I matrices. The onset of function coincides both with morphogenesis of a polarized epithelium and with deposition of basement membrane ECM basal to the cell layer. Mammary specific morphogenesis and biochemical differentiation is induced if mammary cells are cultured directly on exogenous basement membrane (EHS). Thus ECM may effect function by the concerted effect of permissivity for cell shape changes and the direct biochemical signalling of basement membrane molecules.A model is discussed where initial ECM control of mammary epithelial cell function originates in the interstitial matrix of stroma and subsequently transfers to the basement membrane when the epithelial cells have accumulated and deposited an organized basement membrane matrix.Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

Effects of ryanodine on cardiac subcellular membrane fractions

Both the rate and the steady-state magnitude of net calcium accumulation by cardiac sarcoplasmic reticulum (SR) vesicles are increased by ryanodine. Sarcolemmal calcium transport mechanisms are not affected. The apparent augmentation of calcium accumulation by membrane vesicles from junctional SR derives not from an increase in the rate at which calcium is pumped into the vesicles, but from a slowing of the rate of calcium efflux. Recent results show that decamethonium blunts these effects of ryanodine, whereas valinomycin potentiates them. The mechanisms for these latter effects are not well understood, but may involve limitation and promotion, respectively, of access of potassium ion to the interior of the membrane vesicles.     

Effect of experimental colchicine encephalopathy on brain protein synthesis and tubulin metabolism.

Colchicine blocks axoplasmic flow and produces neurofibrillary degeneration. Brain slices from mice injected intracerebrally with colchicine incorporated more [14C]leucine into protein and had a decreased uptake of [14C]leucine into the perchloric acid-soluble pool than did their controls. Brain RNA content was decreased and free leucine increased by colchicine-induced encephalopathy. The specific activities of proteins from subcellular fractions of colchicine-injected brain were increased in the nuclear fraction, the 100,000-g supernatant, and its vinblastine-precipitable tubulin. The ratio of the specific activity of the crude mitochondrial fraction to that of the total homogenate was decreased, as would be consistent with impaired movement of newly labeled protein into synaptosomes. Colchicine-injected brain extracts contained one or more cytosol fractions that stimulated ribosomal incorporation of [14C]leucine into protein in a cell-free system. Colchicine-binding-activity measurements indicated loss of soluble and particulate tubulin in colchicine-injected brains; the decrease of soluble tubulin was verified by its selective precipitation with vinblastine. Colchicine encephalopathy did not affect the rate of spontaneous breakdown of in vitro colchicine binding activity. Similarities of colchicine encephalopathy to the neuron's response to axonal damage suggest that colchicine-induced increase in protein synthesis may, in part, reflect a neuronal response to blockage of neuroplasmic transport.     

The requirement for surface Ig signaling as a prerequisite for T cell:B cell interactions. A possible role for desialylation

Models for T cell:B cell collaboration suggest that activated B cells process and present Ag to Th cells which subsequently induce B cell proliferation and differentiation. In contrast to activated B cells, resting B cells have generally been shown to be less efficient APC. If this model of T:B collaboration is physiologically correct, then resting B cells must undergo a phenotypic change that permits effective interaction with T cells. In this report, the requirement for rapid signaling through surface Ig on resting B cells for the induction of T:B interaction was investigated with an in vitro clustering assay. Resting splenic B cells were unable to form specific conjugates with T cell clones, unless the B cells were first treated with neuraminidase to remove sialic acid. In contrast, LPS-activated B cells were able to form conjugates without prior treatment. The ability of antibody against LFA-1 or L3T4 to inhibit cluster formation depended on the state of B cell activation in that anti-LFA-1 and anti-L3T4 mAb inhibited cluster formation by neuraminidase-treated resting B cells, but not by LPS-activated B cells. In addition, Ag-specific B cells which were isolated by their capacity to bind specific Ag were able to form clusters without any additional treatment. Moreover, treatment of resting splenic B cells with anti-mu-antibody induced clustering potential in B cells in as little as 10 min, suggesting that signaling through surface Ig was sufficient to induce this phenotypic change in B cells. Furthermore, activation of protein kinase C and Ca2+ mobilization were shown to be involved in that PMA and ionomycin treatment were also able to induce clustering potential in resting B cells. The rapid induction of clustering potential in resting B cells after signaling through surface Ig may represent a fundamental change in B cell physiology which occurs after recognition of specific Ag and may be required for effective cognate recognition between resting hapten-specific B cells and carrier-specific T cells. The potential role of desialylation for the induction of T:B interaction is discussed.