Possible role of voltage-sensitive potassium channels in generation of response in Pacinian corpuscles

The effects of 20 mM tetraethylammonium (TEA) and 5 mM 4-aminopyridine (4-AP) on mechanically and electrically excitable membranes of Pacinian corpuscles were investigated using the air gap technique for producing constant superfusion and recording electrical response at the receptor. The effects of TEA led to a 150% rise in the duration of receptor potential, the amplitude of which declined by 40%. No statistically significant changes in response to mechanical stimulation could be detected after applying 4-AP to the receptor membrane. The two blockers mentioned did modify the membrane of the first nodes of Ranvier, producing a 2–3-fold increase in the duration of action potentials. Computations based on the Dodge model would indicate that the observed effects may be explained by inhibition of voltage-dependent potassium channels which help to transform receptor current into spike response in the intact receptor.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 20, No. 5, pp. 623–630, September–October, 1988.     

Sodium-dependent calcium efflux from adrenal chromaffin cells following exocytosis. Possible role of secretory vesicle membranes.

Although cytosolic Ca2+ transients are known to influence the magnitude and duration of hormone and neurotransmitter release, the processes regulating the decay of such transients after cell stimulation are not well understood. Na(+)-dependent Ca2+ efflux across the secretory vesicle membrane, following its incorporation into the plasma membrane, may play a significant role in Ca2+ efflux after stimulation of secretion. We have measured an enhanced 45Ca2+ efflux from cultured bovine adrenal chromaffin cells following cell stimulation with depolarizing medium (75 mM K+) or nicotine (10 microM). Such stimulation also causes Ca2+ uptake via voltage-gated Ca2+ channels and secretion of catecholamines. Na+ replacement with any of several substitutes (N-methyl-glucamine, Li+, choline, or sucrose) during cell stimulation inhibited the enhanced 45Ca2+ efflux, indicating and Na(+)-dependent Ca2+ efflux process. Na+ deprivation did not inhibit 45Ca2+ uptake or catecholamine secretion evoked by elevated K+. Suppression of exocytotic incorporation of secretory vesicle membranes into the plasma membrane with hypertonic medium (620 mOsm) or by lowering temperature to 12 degrees C inhibited K(+)-stimulated 45Ca2+ efflux in Na(+)-containing medium but did not inhibit the stimulated 45Ca2+ uptake. Enhancement of exocytotic secretion with pertussis toxin resulted in an enhanced 45Ca2+ efflux without affecting calcium uptake. The combined results suggest that Na(+)-dependent Ca2+ efflux across secretory vesicle membranes, following their incorporation into the plasma membrane during exocytosis, plays a significant role in regulating calcium efflux and the decay of cytosolic Ca2+ in adrenal chromaffin cells and possibly in related secretory cells.     

The role of thermal activation in motion and force generation by molecular motors

The currently accepted mechanism for ATP-driven motion of kinesin is called the hand-over-hand model, where some chemical transition during the ATP hydrolysis cycle stretches a spring, and motion and force production result from the subsequent relaxation. It is essential in this mechanism for the moving head of kinesin to dissociate, while the other head remains firmly attached to the microtubule. Here we propose an alternative Brownian motor model where the action of ATP modulates the interaction potential between kinesin and the microtubule rather than a spring internal to the kinesin molecule alone. In this model neither head need dissociate (which predicts that under some circumstances a single-headed kinesin can display processive motion) and the transitions by which the motor moves are best described as thermally activated steps. This model is consistent with a wide range of experimental data on the force-velocity curves, the one ATP to one-step stoichiometry observed at small load, and the stochastic properties of the stepping.     

Cell-cell contact mediates cAMP secretion in Dictyostelium discoideum.

Cyclic adenosine 3':5' monophosphate (cAMP) and cell-cell contact regulate developmental gene expression in Dictyostelium discoideum. Developing D. discoideum amoebae synthesize and secrete cAMP following the binding of cAMP to their surface cAMP receptor, a response called cAMP signaling. We have demonstrated two responses of developing D. discoideum amoebae to cell-cell contact. Cell-cell contact elicits cAMP secretion and alters the amount of cAMP secreted in a subsequent cAMP signaling response. Depending upon experimental conditions, bacterial-amoebal contact and amoebal-amoebal contact can enhance or diminish the amount of cAMP secreted during a subsequent cAMP signaling response. We have hypothesized that cell-cell contact regulates D. discoideum development by altering cellular and extracellular levels of cAMP. To begin testing this hypothesis, these responses were further characterized. The two responses to cell-cell contact are independent, i.e., they can each occur in the absence of the other. The responses to cell-cell contact also have unique temperature dependences when compared to each other, cAMP signaling, and phagocytosis. This suggests that these four responses have unique steps in their transduction mechanisms. The secretion of cAMP in response to cell-cell contact appears to be a non-specific response; contact between D. discoideum amoebae and Enterobacter aerogenes, latex beads, or other amoebae elicits cAMP secretion. Despite the apparent similarities of the effects of bacterial-amoebal and amoebal-amoebal contact on the cAMP signaling response, this contact-induced response appears to be specific. Latex beads addition does not alter the magnitude of a subsequent cAMP signaling response.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of adherent accessory cells in the generation of effector suppressor T cells

The role of accessory cell populations in the generation of effector suppressor (Ts3) cells was studied. By using an in vitro culture system, it was previously determined that the induction of NP-specific effector suppressor activity requires T cells, antigen, and an anti-idiotypic B cell population. We now demonstrate that the generation of Ts3 cells in this system also requires accessory cells. The accessory population appears to play a role in the processing and presentation of antigen. These antigen-presenting accessory cells are required early in the induction phase of Ts3 generation. These accessory cells can present NP coupled to immunogenic or non-immunogenic polypeptide carriers, including polymers of L-amino acids. However, NP coupled to polymers of poorly metabolized D-amino acids fail to induce suppressor T cell generation. Furthermore, the data demonstrate that an H-2 homology must exist between the Ts3 precursors and the antigen-presenting cell population if suppressor activity is to be generated. We also characterize the differential genetic restrictions that govern the induction of Ts3 cells that control suppression of either T cell or B cell responses. The data suggest that although I-J region encoded gene products control the induction and effector phases of suppressor cell activity as measured on T cell responses, the suppression of B cell responses appear to be controlled by I-A gene products. Possible cellular mechanisms that might explain these findings are discussed.     

Constitutive overexpression of the contact site A glycoprotein enables growth-phase cells of Dictyostelium discoideum to aggregate.

The contact site A (csA) glycoprotein is a developmentally regulated cell adhesion molecule which mediates EDTA-stable cell contacts during the aggregation stage of Dictyostelium discoideum. A transformation vector was constructed which allows overexpression of the csA protein during the growth phase. In that stage the csA protein is normally not expressed; in the transformants it was transported to the cell surface and carried all modifications investigated, including a phospholipid anchor and two types of oligosaccharide chain. csA expression enabled the normal non-aggregative growth-phase cells to form EDTA-stable contacts in suspension and to assemble into three-dimensional aggregates when moving on a substratum. After prolonged cultivation of csA overexpressing transformants in nutrient medium the developmental program was found to be turned on, as it normally occurs only in starving cells. During later development of transformed cells, the csA glycoprotein remained present on the cell surface, while it is down-regulated in the wild type. It was detected in both the prestalk and prespore regions of the multicellular slugs made from transformed cells.     

Possible role of H1 histone in the differentiation of mouse erythroleukemia cells

Serum-starved mouse erythroleukemia cells, stationary phase cells or cells cultured in dibutyryl cAMP (1 mM) can be induced to differentiate by addition of 20% fetal calf serum plus cycloheximide. Culturing unstarved log phase cells in 20% fetal calf serum plus low levels of cycloheximide and histone H1 also causes a significant level of differentiation. These same concentrations of cycloheximide and H1 histone employed separately with 20% fetal calf serum do not induce differentiation. The role these procedures may have in causing an accumulation of histone H1 and cell differentiation is discussed.     

Monoclonal antibodies against contact sites A of Dictyostelium discoideum: detection of modifications of the glycoprotein in tunicamycin-treated cells

Tunicamycin acts on cell aggregation in Dictyostelium discoideum by changing cell movement and by inhibiting the EDTA-stable type of intercellular adhesion. Tunicamycin-treated cells show unco-ordinated pseudopodial activity such that pseudopods are simultaneously extended from all parts of the cell surface, and the cells are unable to move in straight paths. Concurrent with the inhibition of formation of EDTA-stable contacts, N-glycosylation of a glycoprotein specific for aggregation-competent cells is inhibited. This glycoprotein, previously called contact site A, has an apparent mol. wt. of 80 kilodaltons (kd). In membranes of tunicamycin-treated cells, two components are detected that react with certain monoclonal antibodies against contact sites A: one component of 66 kd, the other of 53 kd apparent mol. wt. Another group of monoclonal antibodies reacts only with the 80-kd glycoprotein and the 66-kd component. These results are in accord with the assumption that the glycoprotein carries two carbohydrate chains, and that the antibodies differ in their requirement for glycosylation of the antigen. Despite the coincidence between blockage of EDTA-stable cell adhesion and inhibited glycosylation of contact sites A, direct involvement of the carbohydrate moieties of this glycoprotein in intercellular adhesion seems questionable. EDTA-stable cell adhesion has not been blocked by Fab fragments from antibodies that specifically react with the glycosylated protein.     

A stochastic model for the detection of coherent motion

A computational model is presented for the detection of coherent motion based on template matching and hidden Markov models. The premise of this approach is that the growth in detection sensitivity is greater for coherent motion of structured forms than for random coherent motion. In this preliminary study, a recent experiment was simulated with the model and the results are shown to be in agreement with the above premise. This model can be extended to be part of a more complex and elaborate computational visual system.     

The role of BEACH proteins in Dictyostelium

The BEACH family of proteins is a novel group of proteins with diverse roles in eukaryotic cells. The identifying feature of these proteins is the BEACH domain named after the founding members of this family, the mouse beige and the human Chediak–Higashi syndrome proteins. Although all BEACH proteins share a similar structural organization, they appear to have very distinct cellular roles, ranging from lysosomal traffic to apoptosis and cytokinesis. Very little is currently known about the function of most of these proteins, few binding-partner proteins have been identified, and no molecular mechanism for any of these proteins has been discovered. Thus, it is important to establish good model systems for the study of these novel proteins. Dictyostelium contains six BEACH proteins that can be classified into four subclasses. Two of them, LvsA and LvsB, have clearly distinct roles in the cell. LvsA is localized on the contractile vacuole membrane and is essential for cytokinesis and osmoregulation. LvsB is most similar in sequence to the mammalian beige/Chediak–Higashi syndrome proteins and shares with them a common function in lysosomal trafficking. Structural and functional analysis of these proteins in Dictyostelium will help elucidate the function of this enigmatic novel family of proteins .     

Possible role of neuraminidase in activated T cells in the recognition of allogeneic Ia

In a primary MLR, predominant stimulators in spleen cells are adherent cells and not B cells, although B cells are one of the cell types expressing a large amount of Ia molecules. Our previous experiments showed that T cells treated with neuraminidase (Nase) responded to an allogeneic Ia on B cells. In our experiments, the relationship between the responsiveness to the allogeneic Ia molecules on B cells and Nase activity of T cells was examined. The results showed that T cells increased in Nase activity with the acquisition of the reactivity to Ia on B cells. T cells from normal mice increased in Nase activity after the incubation for 3 days or more in MLR, and these T cells responded to allogeneic Ia on B cells. However, T cells from mice genetically deficient in Nase responded poorly to the Ia on allogeneic B cells even after the incubation in MLR for 3 days. T cells incubated for 3 days in MLR decreased in electrophoretic mobility, indicating the decrease of net negative charge of the cells, and increased in their binding of peanut agglutinin which has been reported to bind to galactosyl residues exposed on T cell surface by removing sialic acids. These results suggest that Nase in T cells was activated by the cultivation in MLR for 3 days, and sialic acids of some molecules on T cell surface were removed by the enzyme and, in turn, T cells acquired the responsiveness to allogeneic B cells in a secondary MLR. Thus, Nase was suggested to play a regulatory role in the recognition of Ia molecules in T cells.     

Possible role of endothelium in the orientation of smooth muscle cells in experimental neointima

Myointimal cells (MIC) orientation and intimal thickening evolution, induced by grafting an autogenous venous patch onto the rat common carotid artery, were studied with light microscopy at different times until 14 months after surgery. Intimal thickenings in the venous patch were most prominent at an intermediate postoperative period, after which their mean size did not change significantly. In host artery neointima, the greatest MIC proliferation was observed at a late stage of evolution. MIC arrangement was predominantly circumferential to the blood stream in venous patch neointima, whereas it was mostly axial in the host artery. The interaction between MIC orientation and endothelial regeneration in the operated vessel is discussed.     

A possible role for DIF-2 in the formation of stalk cells during Dictyostelium development.

The differentiation inducing factor (DIF) is essential for stalk cell formation in monolayers of Dictyostelium discoideum and is necessary for the expression of several prestalk cell-specific genes. DIF activity has been fractionated into a major species, designated DIF-1, and several minor species, including DIF-2. Although DIF-1 is an excellent inducer of stalk cell formation from vegetative cells, it is a poor inducer of stalk cell formation from prestalk cells. In contrast, DIF-2 is more active for the conversion of prestalk cells into stalk cells, than for the conversion of vegetative cells to stalk cells. The same results were obtained regardless of whether chemically synthesized or naturally occurring components were utilized. In addition, stalk cell formation was three- to fourfold higher when vegetative cells were incubated with DIF-1 for a suboptimal period and then subsequently incubated with DIF-2, than when cells were incubated with DIF-2 first and then subsequently with DIF-1. These results indicate a distinct role for DIF-2 during stalk cell formation and suggest the possibility that DIF-1 and DIF-2 act sequentially.     

Possible role of stresses in inducing spinal stenosis--a long term complication following disk excision

A three-dimensional finite element model of an intact ligamentous lumbar motion segment (L3-4) was used to predict stresses in the pars interarticularis regions of the modeled vertebral bodies. The changes in stresses following disk excision, as compared to the intact model, also were computed. The predicted results show an increase in stresses in the posterior bony elements following disk excision. In some patients over a long period of time this increase in stresses, in association with other clinical factors, may lead to bony hypertrophy of the structures that surround the nerve roots. Ultimately, over a long period of time the increase in pressure on the entrapped nerve root may induce recurrent pain and other complications reported in the literature.     

Possible role of RAC-GTPase-activating protein in the termination of superoxide production in phagocytic cells.

The mechanism leading to the termination of superoxide production of phagocytes is poorly understood. The aim of the present study was to investigate the involvement of the active (GTP-bound) form of the GTP-binding proteins in maintaining continuous electron transport through the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Activation of the enzyme was carried out under in vitro conditions and a shift from the active to the inactive form of the GTP-binding protein was attained (i) by addition of an excess of GDP to the assembled enzyme complex or (ii) by variation of the Rac-GTPase activating (Rac-GAP) capacity of the constituents of the cell-free system. Significant inhibition of O2*- production was observed when guanine dinucleotides were added after the assembly of the active enzyme complex. The effect was specific for GDP and GDP,S whereas ADP, CDP and UDP were ineffective. GTP was significantly less efficient in inducing superoxide production in a cell-free system containing endogenous GAP activity than in a system devoid of GAP activity. It is suggested that the active, GTP-bound form of Rac is required for sustained catalytic function and Rac-GAP proteins are involved in the downregulation of the oxidase.