Scientific and practical applications of molecular colonies

The review briefs the history of the invention of the molecular colony techique, also known as a polony technology; applications of this method to studies of the reactions between single RNA molecules, ultrasensitive diagnosis, gene cloning, and in vitro screening, as well as the concepts of the origin of life that regard molecular colonies as a prototype of living organisms.     

Amphiphilic effects of dibucaine HCl on rotational mobility of n-(9-anthroyloxy)stearic acid in neuronal and model membranes

We studied dibucaine's effects on specific locations of n-(9-anthroyloxy)palmitic acid or stearic acid (n-AS) within phospholipids of synaptosomal plasma membrane vesicles isolated from bovine cerebral cortex (SPMV) and model membranes. Giant unilamellar vesicles (GUVs) were prepared with total lipids (SPMVTL) and mixture of several phospholipids (SPMVPL) extracted from SPMV. Dibucaine.HCl increased rotational mobility (increased disordering) of hydrocarbon interior, but it decreased mobility (increased ordering) of membrane interface, in both native and model membranes. The degree of rotational mobility in accordance with the carbon atom numbers of phospholipids comprising neuronal and model membranes was in the order at the 16, 12, 9, 6 and 2 position of aliphatic chain present in phospholipids. The sensitivity of increasing or decreasing effect of rotational mobility of hydrocarbon interior or surface region by dibucaine.HCl differed depending on the neuronal and model membranes in the descending order of SPMV, SPMVPL and SPMVTL.     

Identification of neuronal pathways between the stomatogastric nervous system and the retrocerebral complex of the cockroach Periplaneta americana (L.)

Summary The neuronal pathways connecting the stomatogastric nervous system with the retrocerebral complex of the cockroach, Periplaneta americana, were investigated by means of axonal cobalt chloride iontophoresis. Somata in the hypocerebral ganglion and in the nervus recurrens sending their axons to different parts of the stomatogastric nervous system were traced. Some axons in the oesophageal nerve arise from large perikarya in the anterior part of the pars intercerebralis and pass via the NCCI to the corpora cardiaca and the oesophageal nerve. They form a profuse dendritic tree in the protocerebrum. Fibers of the NCC I and NCC II as well as the NCA I and NCA II enter the stomatogastric nervous system via the hypocerebral ganglion.     

DIDS increases K+ secretion through an I sKchannel in apical membrane of vestibular dark cell epithelium of gerbil

Vestibular dark cell epithelium secretes K⁺ via I _sKchannels in the apical membrane. The previous observation that disulfonic stilbenes increased the equivalent short circuit current (I _sc) suggested that these agents might be useful investigative tools in this tissue. The present experiments were conducted to determine if the increase in I _scwas associated with an increase in K⁺ flux and if the effect was directly on the I _sKchannel or indirectly via a cytosolic intermediary. Measurements of transepithelial K⁺ flux with the K⁺-selective vibrating probe and of changes in net cellular solute flux by measurements of epithelial cell height showed that 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) increased K⁺ flux by a factor of 1.96±0.71 and caused net solute efflux. The apical membrane was partitioned with a macropatch pipette and DIDS was applied either to the membrane outside the pipette, inside the pipette or to the entire apical membrane. DIDS inside the pipette increased the current across the patch, the membrane conductance, the slowly-inactivating (I _sK) component of the membrane current and shifted the reversal voltage toward the equilibrium potential for K⁺. DIDS outside the patch decreased the patch current and conductance, consistent with shunting of current away from the membrane patch. These findings strongly support the notion that DIDS increases K⁺ secretion through I _sKchannels in the apical membrane of vestibular dark cell epithelium by acting directly on the channels or on a tightly colocalized membrane component.We thank Dr. Peter J.S. Smith and Alan Shipley of the National Vibrating Probe Facility at the Marine Biological Laboratory at Woods Hole, MA for their support and assistance in the measurements of K⁺ flux. This work was supported by National Institutes of Health grants R01-DC00212, R29-DC1098 and P41-RR01395.     

Influence of vitamin C on alcohol binding to phospholipid monolayers

The simple model of the biological membrane is provided by well-controlled lipid monolayers at the air-water interface. The Maxwell displacement current technique (MDC) provides novel approach to conformation study of the membrane models. The effect of alcohols is interaction with membrane molecules, mainly with the lipid head group and consequent changes in physical-chemical properties of the membrane. The aim of study is to detect changes in structural, electrical and mechanical properties of dipalmitoyl-phosphatidylcholine (DPPC) monolayer on the subphase of methanol-water and ethanol-water mixtures before and after addition of antioxidant agent, vitamin C. Monolayers properties are investigated by a surface pressure analysis (including mechanical properties evaluation) and the Maxwell displacement current measurement, the dipole moment projection calculation. Surface pressure-area isotherms show similar behaviour of the DPPC monolayer on alcohol-water mixtures independently on presence of vitamin C. Binding/adsorption process induces change of electron density distribution across monolayer and thus the molecular dipole moment. We observe small or negligible binding of methanol molecules on oxygen bonds of DPPC. Thus the antioxidant, vitamin C, has no significant effect. For ethanol-water mixtures is observed recovery of electrical properties in presence of antioxidant agent. We suppose that vitamin C regulates DPPC-ethanol molecules interaction.     

Histomonas meleagridis: immunohistochemical localization of parasitic cells in formalin-fixed, paraffin-embedded tissue sections of experimentally infected turkeys demonstrates the wide spread of the parasite in its host

In the present investigation, a polyclonal antibody-based immunohistochemical technique was developed to localize Histomonas meleagridis in formalin-fixed, paraffin-embedded tissues of experimentally infected turkeys. The developed technique was highly specific for histomonads as no immunohistochemical reaction was observed with cultures of Tetratrichomonas gallinarum, Trichomonas gallinae and Blastocystis sp. In addition, tissues positive for various other protozoan parasites and fungi were also tested to evaluate the specificity of the technique. It was possible to detect immunohistochemically histomonad antigens in all the tested samples (n=5) of caecum, liver, spleen and lung from infected turkeys, 3 out of 5 bursa of Fabricius, 1 out of 2 bone marrow, 2 out of 5 heart and 1 out of 5 each of proventriculus, pancreas and cerebellum. An immunohistochemical reaction indicative of presence of histomonads was also detected in blood vessels of various organs that indicated a possible hematogenous route of spread of the parasite in the host. A comparative study with routine diagnostic staining techniques indicated a high sensitivity and specificity of the newly developed immunohistochemical technique. Altogether, the technique developed can be used to study the sequential pathogenesis of histomonosis in turkeys and to obtain new insights into the mechanisms of interaction with the host tissues.     

Ion permeation through single channels activated by acetylcholine in denervated toad sartorius skeletal muscle fibers: Effects of alkali cations

Summary The gigaohm seal technique was used to study ion permeation through acetylcholine-activated channels in cell-attached patches of the extrajunctional membrane of chronically denervated, enzyme-treated cells from the sartorius muscle of the toadBufo marinus. The most frequently occurring channel type (>95% of channel openings), provisionally classified as extrajunctional, had a chord conductance of approximately 25 pS under normal conditions (–70 mV, 11°C, Normal Toad Ringer's). The less frequently observed channel type (<5% of channel openings), classified as a junctional type, had a conductance of 35 pS under the same conditions, and a similar null potential. In many patches, a small percentage (usually <2%) of openings of the extrajunctional channel displayed a lower conductance state. The shape of theI–V curves obtained for the extrajunctional channel dependend on the predominant extracellular cation. For Cs and K, theI–V curves were essentially linear over the voltage range +50 to –150 mV across the patch, suggesting that the potential independent component of the energy profile within the channel was symmetrical. For Li, theI–V curve was very nonlinear, displaying a significant sublinearity at hyperpolarized potentials. Both an electrodiffusion and a symmetrical uniform four-barrier, three-site rate-theory model provided reasonable fits to the data, whereas symmetrical two-barrier, single-site rate-theory models did not. For the alkali cations examined, the relative permeability sequence wasP _Cs>P _K>P _Na>P _Li—a proportional selectivity sequence. This was different from the single channel conductance sequence which was found to be _K> _Cs> _Na> _Li implying that ions do not move independently through the channel. The relative binding constant sequence for the channel sites was found to be a polarizability sequence, i.e.,K _Li>K _Cs>K _Na>K _K There was an inverse relationship for the cations examined. Under conditions when the single-channel conductance was relatively high, the conductance at depolarized potentials was lower than that predicted by both electrodiffusion and rate theory models, suggesting that there was a rate-limiting access step for ions, from the intracellular compartment into the channel.     

Possible contribution of pannexin-1 to capsaicin-induced ATP release in rat nasal columnar epithelial cells

Current evidence indicates that transient receptor potential (TRP) channel activity involves a relationship between opening of pannexin-1 and release of ATP into the extracellular space. We examined the effects of agonists of thermosensitive TRP channels (TRPM8, TRPA1, TRPV1, and TRPV2) on ATP release from rat nasal mucosa, and measured ciliary beat frequency (CBF) using digital high-speed video imaging. Single-cell patch clamping from dissociated rat nasal columnar epithelial cells was performed to confirm the relationship between pannexin-1 and TRP. We demonstrated that ATP release and CBF were significantly potentiated by the heat-sensitive TRPV1 agonist capsaicin (10 μM), but not by other TRP agonists. Capsaicin-induced ATP release and CBF increase were significantly inhibited by the pannexin-1 blockers carbenoxolone (10 μM) and probenecid (300 μM). In addition, the voltage step-evoked currents in the presence of capsaicin were inhibited by the pannexin-1 blockers in single-cell patch clamping. Our results suggest the participation of TRPV1 and pannexin-1 in the physiologic functions of rat nasal mucosa.