4-aminoquinoline-induced 'giant' miniature endplate potentials at mammalian neuromuscular junctions

4-Aminoquinoline (4-AQ) in concentrations around 200 micrometers induces, within minutes of its application to isolated mouse or rat neuromuscular junctions, the appearance of a population of miniature endplate potentials (m.e.p.ps) with a larger than normal amplitude, so-called giant m.e.p.ps (g.m.e.p.ps). With amplitudes 2-12 times the modal value of m.e.p.p. amplitude, the population of g.m.e.p.ps varied between 15 and 45% of the total population of m.e.p.ps. There was no increase in the frequency of m.e.p.ps but a positive correlation between the frequency of g.m.e.p.ps and the total frequency of m.e.p.ps. In many instances the rise time and decay time of g.m.e.p.ps were prolonged compared to normal. Elevated extracellular calcium concentrations increased the frequency of m.e.p.ps but had no effect on g.m.e.p.p. frequency. High extracellular potassium concentrations markedly increased m.e.p.p. frequency but failed to influence g.m.e.p.p. frequency. Similar observations were made with ethanol 0.1 M, ouabain 200 micrometers or black widow spider venom. Botulinum toxin type A markedly reduced total m.e.p.p. frequency but 4-AQ still induced g.m.e.p.ps. Nerve stimulation failed to release quanta corresponding to the g.m.e.p.ps. G.m.e.p.ps seemed to originate from quantal acetylcholine release from the nerve terminal since they were abolished by surgical denervation and by the addition of d-tubocurarine to the medium. Blockade of voltage-sensitive calcium or sodium channels by, respectively, manganese ions or tetrodotoxin failed to affect the appearance and the frequency of g.m.e.p.ps. The electrophysiological findings and a statistical analysis of the characteristics of the m.e.p.ps indicate that they belong to two populations. One population is accelerated by the depolarization-release coupling mechanism responsible for evoked transmitter release and is characterized by an amplitude distribution and a process in time that indicate that they correspond to releases occurring at 'active zones' in the nerve terminal. The second population of m.e.p.ps is uninfluenced by nerve terminal depolarization and transmembrane calcium fluxes. This population apparently originates from sites dispersed in the nerve terminal membrane and outside the 'active zones'. 4-AQ increases the frequency of this second m.e.p.p. population without affecting the first population.     

Discrepancies between spontaneous and evoked synaptic potentials at normal, regenerating and botulinum toxin poisoned mammalian neuromuscular junctions

Amplitudes and times to peak of spontaneous miniature endplate potentials (m.e.p.ps) and evoked quantal endplate potentials (e.p.ps) were compared at normal, regenerating and botulinum toxin poisoned neuromuscular junctions of the extensor digitorum longus muscle of the rat. At normal junctions the mean time to peak of m.e.p.ps was longer and more variable than that of similar-sized e.p.ps. At endplates where nerve regeneration was induced by mechanical crushing of the motor nerve the frequency of m.e.p.ps was reduced and their amplitude distribution was broader than normal. The distribution of times to peak of m.e.p.ps was considerably broader than that of quantal e.p.ps recorded at the same endplates. At neuromuscular junctions poisoned with botulinum toxin type A, spontaneous and evoked transmitter release were greatly reduced. The amplitude distribution of m.e.p.ps was wider than that of e.p.ps and the time to peak of e.p.ps was about twice as fast as and less variable than that of m.e.p.ps. To explain the observed differences in time to peak among m.e.p.ps and between m.e.p.ps and quantal e.p.ps we suggest that some m.e.p.ps, but not e.p.ps, originate from transmitter quanta released from sites at a greater distance from postsynaptic receptors or that the release or diffusion process for acetylcholine is more prolonged when producing some of the m.e.p.ps. Such mechanisms produce at normal junctions a small population of m.e.p.ps with prolonged times to peak, at regenerating junctions a greater proportion of such m.e.p.ps and in botulinum toxin poisoning a majority.     

Transmitter release in botulinum-poisoned muscles

Examination of miniature end-plate potentials (m.e.p.ps) in rat skeletal muscle poisoned in vivo by botulinum toxin type A reveals the presence of two populations of potentials. One population which corresponds to m.e.p.ps in unpoisoned muscles and to quantal end-plate potentials. The frequency of these m.e.p.ps is greatly reduced by botulinum toxin. The second population of m.e.p.ps has quite different characteristics. These m.e.p.ps have a more variable, but generally much larger amplitude, and their time to peak is longer than normal m.e.p.ps. The frequency of these m.e.p.ps increases during poisoning and reaches 0.3-1 Hz after 10-14 days. In addition to the variability in amplitude and time-to-peak these m.e.p.ps differ from those at unpoisoned junctions by being unaffected by procedures which alter extra- or intracellular Ca2+ concentrations. The appearance of this Ca2+-insensitive spontaneous quantal secretion of acetylcholine is apparently not a direct effect of the toxin but secondary to blockade of impulse transmission since it also appears at unpoisoned end-plates when transmission is impaired for other reasons. Procedures which increase the intracellular Ca2+ concentration in nerve terminals restore transmitter release from botulinum toxin poisoned nerves. Furthermore, the block caused by the toxin is very temperature-dependent, a reduction in temperature relieving the block. Since presynaptic Ca2+ currents are unaltered by the toxin it is proposed that the block of transmission is due to a reduction in the calcium content of the nerve terminal to a level where the amount of Ca2+, which normally enters, is insufficient to activate transmitter release.     

Structure and stability of the consecutive stereoregulated chiral phosphorothioate DNA duplex

The duplex structures of the stereoregulated phosphorothioate DNAs, [R(p),R(p)]- and [S(p),S(p)]-[d(GC(ps)T(ps)ACG)] (ps, phosphorothioate; PS-DNA), with their complementary RNA have been investigated by combined use of (1)H NMR and restrained molecular dynamics calculation. Compared to those obtained for the unmodified duplex structures (PO-DNA.RNA), the NOE cross-peak intensities are virtually identical for the PS-DNA.RNA hybrid duplexes. The structural analysis on the basis of the NOE restraints reveals that all of the three DNA.RNA duplexes take a A-form conformation and that there is no significant difference in the base stacking for the DNA.RNA hybrid duplexes. On the other hand, the NOE cross-peak intensities of the protons around the central T(ps)A step of the PS-DNA.DNA duplexes are apparently different from those of PO-DNA. DNA. The chemical shifts of H8/6 and H1' at the T(ps)A step are also largely different among PS-DNA.DNAs and PO-DNA.DNA, suggesting that the DNA.DNA structure is readily changed by the introduction of the phosphorothioate groups to the central T(p)A step. The structure calculations indicate that all of these DNA.DNA duplexes are B-form although there exist some small differences in helical parameters between the [R(p),R(p)]- and [S(p),S(p)]PS-DNA.DNA duplexes. The melting temperatures (T(m)) were determined for all of the duplexes by plotting the chemical shift change of isolated peaks as a function of temperature. For the PS-DNA.RNA hybrid duplexes, the [S(p),S(p)] isomer is less stable than the [R(p),R(p)] isomer while this trend is reversed for the PS-DNA.DNA duplexes. Consequently, although the PS-DNA.RNA duplexes take the similar A-form structure, the duplex stability is different between PS-DNA.RNA duplexes. The stability of the DNA.RNA duplexes may not be governed by the A-form structure itself but by some other factors such as the hydration around the phosphorothioate backbone, although the T(m) difference of the DNA.DNA duplexes could be explained by the structural factor.     

Ionic basis of different synaptic potentials mediated by an identified dopamine-containing neuron in Planorbis.

A specified dopamine neuron in Planorbis corneus produces dopamine-mediated e.p.s.ps, i.p.s.ps or biphasic, depolarizing-hyperpolarizing p.s.ps in different follower neurons. The excitatory potentials were of three types. Some follower neurons exhibited slow e.p.s.ps (ca 1 s), and a long-lasting, slowly desensitizing, depolarizing response to iontophoresed dopamine. Others showed rapid (ca. 150 ms) e.p.s.ps, often of variable amplitude, and a rapid, quickly desensitizing, response to iontophoresed dopamine. The rapid e.p.s.ps were sometimes followed by the inhibitory response (biphasic potential). The e.p.s.ps were potentiated by hyperpolarization and reduced by depolarization, though they could not be inverted. The slow e.p.s.p. was shown to be associated with an increase in membrane conductance, but it has proved difficult to elucidate the ions involved. A third type of e.p.s.p. was produced by electrical transmission. The inhibitory potentials were generally reduced in amplitude by artificial hyperpolarization but could rarely be inverted. This is probably due in part to the presence of of electrotonic coupling between these follower neurons. The i.p.s.ps were associated with an increase in conductance which appeared small when measured in the cell body. However, the i.p.s.ps produced considerable shunting of electrotonic transmission between coupled followers indicating a large increase in conductance at the synapse. I.p.s.ps were unaffected by Cl-free solution but they were greatly reduced, though rarely inverted, by increasing the external K concentration. They were blocked by intracellular tetraethylammonium, or cooling. The effects on corresponding responses to iontophoresed dopamine were in each case the same as on the i.p.s.ps. It is concluded that the i.p.s.ps mediated by the dopamine neuron are produced by an increase in permeability to K+. On a few occasions i.p.s.ps mediated by the dopamine neuron were potentiated by hyperpolarization. This appeared to be caused by a sharp increase in membrane resistance with hyperpolarization of these particular neurons. However, mediation by a mechanism of conductance decrease could not be completely excluded.     

Capturing escape in infectious disease dynamics

Identifying the causes of interannual variability in disease dynamics is important for understanding and managing epidemics. Traditionally, these causes have been classified as intrinsic (e.g. immunity fluctuations) or extrinsic (e.g. climate forcing); ecologists determine the relative contributions of these factors by applying statistical models to time series of cases. Here we address the problem of isolating the drivers of pathogen dynamics that are influenced by antigenic evolution. Recent findings indicate that many pathogens escape immunity in a punctuated manner; for them, we argue that time series of cases alone will be insufficient to isolate causal drivers. We detail observations that can reveal the presence of punctuated immune escape, and which can be used in new statistical approaches to identify extrinsic and intrinsic regulators of disease.     

Predicted Delays in the Activation of the Contractile System

The capacitance C'(e), presumed to be located across the walls of the transverse tubules of twitch fibers, was identified in earlier impedance measurements by virtue of having a resistance in series with it. When the voltage V(m) across the surface membrane is made to vary, the voltage V(c) across C'(e) will be delayed with respect to V(m), the extent of the delay depending on the location of the series resistance. Model 1 assumes that the resistivity of the lumen of the tubules is negligible; model 2 assumes that the series resistance arises entirely in the tubular lumen; model 3 assumes that the resistivity of the tubular lumen is small, but not negligible and that the bulk of the resistance arises in a structure directly in series with C'(e) and having a similar geometric distribution. If V(m) varies sinusoidally, the relative value of V(c(max)) will fall with increasingly higher powers of the frequency at the center of the fiber if model 2 is applicable, whereas models 1 and 3 predict that V(c(max)) will fall at high frequency only in proportion to the frequency everywhere in the cross-section of the fiber. Equations have been derived for the voltage change V(c) in response to a step change of V(m) and during an action potential. On the assumption that contraction is initiated when V(c) reaches mechanical threshold, the delay between the activation of myofibrils on the axis of the fiber and at the surface would amount to 2.6 msec in model 2 and 0.25 msec in model 3 for frog fibers of about 100 mum diameter during a twitch.     

NMR structure and backbone dynamics of the extended second transmembrane domain of the human neuronal glycine receptor alpha1 subunit

The structure and backbone dynamics of an extended second transmembrane segment (TM2e) of the human neuronal glycine receptor alpha(1) subunit in sodium dodecyl sulfate micelles were studied by (1)H and (15)N solution-state NMR. The 28-amino acid segment contained the consensus TM2 domain plus part of the linker between the second and third transmembrane domains. The presence of a well-structured helical region of at least 13 amino acids long and an unstructured region near the linker was evident from the proton chemical shifts and the pattern of midrange nuclear Overhauser effects (NOE). (15)N relaxation rate constants, R(1) and R(2), and (15)N-[(1)H] NOE indicated restricted internal motions in the helical region with NOE values between 0.6 and 0.8. The squared order parameter (S(2)), the effective correlation time for fast internal motions (tau(e)), and the global rotational correlation time (tau(m)) were calculated for all TM2e backbone N-H bonds using the model-free approach. The S(2) values ranged about 0.75-0.86, and the tau(e) values were below 100 ps for most of the residues in the helical region. The tau(m) value, calculated from the dynamics of the helical region, was 5.1 ns. The S(2) values decreased to 0.1, and the tau(e) values sharply increased up to 1.2 ns at the linker near the C-terminus, indicating that the motion of this region is unrestricted. The results suggest a relatively high degree of motional freedom of TM2e in micelles and different propensities of the N- and C-terminal moieties of the transmembrane domain to assume stable helical structures.     

Evidence for "diminishing returns" from the scaling of stem diameter and specific leaf area

Research indicates that increases in total leaf area (A(T)) may fail to keep pace with increases in total leaf mass (M(L)) across plants differing in size (e.g., as measured by stem diameter, D). This "diminishing returns" hypothesis predicts that the scaling exponent for A(T) vs. M(L) will be less than one and that the exponent for specific leaf mass (i.e., A(T) / M(L)) vs. D will be negative. These predictions were examined using data from 46 plants ranging between 0.125 cm ≤ D ≤ 0.485 m across 25 woody dicot species. Standardized major axis slopes were used to quantify scaling exponents and random effects models were used to quantify species and size effects on the numerical values of exponents. The exponents for A(T) vs. M(L) and A(T) / M(L) vs. D differed among species and different species groupings. In general, the exponent for A(T) vs. M(L) was less than one and the exponent for A(T) / M(L) vs. D was negative, as predicted. However, random effects models indicated that species effects overshadowed size effects, although size effects were statistically significant. The diminishing returns hypothesis therefore receives statistical support, i.e., although the numerical values of exponents are "species-dependent," they are less than unity, as predicted by theory.     

Self-interaction, nucleic acid binding, and nucleic acid chaperone activities are unexpectedly retained in the unique ORF1p of zebrafish LINE

Long interspersed elements (LINEs) are mobile elements that comprise a large proportion of many eukaryotic genomes. Although some LINE-encoded open reading frame 1 proteins (ORF1ps) were suggested to be required for LINE mobilization through binding to their RNA, their general role is not known. The ZfL2-1 ORF1p, which belongs to the esterase-type ORF1p, is especially interesting because it has no known RNA-binding domain. Here we demonstrate that ZfL2-1 ORF1p has all the canonical activities associated with known ORF1ps, including self-interaction, nucleic acid binding, and nucleic acid chaperone activities. In particular, we showed that its chaperone activity is reversible, suggesting that the chaperone activities of many other ORF1ps are also reversible. From this discovery, we propose that LINE ORF1ps play a general role in LINE integration by forming a complex with LINE RNA and rearranging its conformation.     

Design, synthesis and biological activities of thiourea containing sorafenib analogs as antitumor agents

A novel series of diaryl thiourea containing sorafenib derivatives 9a-t was designed and synthesized. The structures of all the newly synthesized compounds were determined by (1)H NMR, (13)C NMR and HRMS. Their antiproliferative activities against HCT116 and MDA-MB-231 cell lines, and their inhibitory activities against the phosphorylation of VEGFR were evaluated and described. Some of the compounds showed significant activities against both cell lines and VEGFR. Compounds 9g, 9m, 9o and 9p demonstrated competitive antiproliferative activities to sorafenib, the reference standard, while compounds 9d, 9m, and 9p showed significant inhibitory activities against the phosphorylation of VEGFR.     

Folding thermodynamics of peptides

A simplified interaction potential for protein folding studies at the atomic level is discussed and tested on a set of peptides with approximately 20 residues each. The test set contains both alpha-helical (Trp cage, F(s)) and beta-sheet (GB1p, GB1m2, GB1m3, Betanova, LLM) peptides. The model, which is entirely sequence-based, is able to fold these different peptides for one and the same choice of model parameters. Furthermore, the melting behavior of the peptides is in good quantitative agreement with experimental data. Apparent folded populations obtained using different observables are compared, and are found to be very different for some of the peptides (e.g., Betanova). In other cases (in particular, GB1m2 and GB1m3), the different estimates agree reasonably well, indicating a more two-state-like melting behavior.     

Combinatorial properties of RNA secondary structures.

The secondary structure of an RNA molecule is of great importance and possesses influence, e.g., on the interaction of tRNA molecules with proteins or on the stabilization of mRNA molecules. The classification of secondary structures by means of their order proved useful with respect to numerous applications. In 1978, Waterman, who gave the first precise formal framework for the topic, suggested to determine the number a(n,p) of secondary structures of size n and given order p. Since then, no satisfactory result has been found. Based on an observation due to Viennot et al., we will derive generating functions for the secondary structures of order p from generating functions for binary tree structures with Horton-Strahler number p. These generating functions enable us to compute a precise asymptotic equivalent for a(n,p). Furthermore, we will determine the related number of structures when the number of unpaired bases shows up as an additional parameter. Our approach proves to be general enough to compute the average order of a secondary structure together with all the r-th moments and to enumerate substructures such as hairpins or bulges in dependence on the order of the secondary structures considered.     

Moment dependency of the series elastic stiffness in the human plantar flexors measured in vivo.

The moment dependency of the series elastic stiffness (SES) in the human plantar flexors was investigated in vivo with the quick release method. At an ankle moment of 100 N m produced with either voluntary or electrical stimulation we found non-significantly different SES of 506+/-72 and 529+/-125 N m rad(-1), respectively. It has recently been proposed that the amount of series elastic tissue involved in plantar flexion changes with the moment level produced by the plantar flexors (Hof, J. Biomech 31 (1998) 793). However, our results indicate that the amount of series elastic tissue involved in plantar flexions remained constant with changing moment levels. We therefore propose that the series elastic component (SEC) in human plantar flexors act as one structure or rather one combination of anatomical structures which is engaged at all muscle activation levels, and that the mechanical properties (i.e. the stress-strain function) are determined by the combined tissue mechanical properties. Additionally, our results demonstrated that the SES in the human plantar flexors at moments levels up to about isometric maximum did not reach an asymptote where the stiffness is independent of moment, i.e. SEC of the plantar flexors is, during many daily activities, loaded for the greatest part in the non-linear part of the stress-strain function.     

Characterization of the partially reduced cyanide-inhibited derivative of cytochrome c oxidase by optical, electron-paramagnetic-resonance and magnetic-circular-dichroism spectroscopy.

Optical. e.p.r. and near-infrared low-temperature m.c.d. (magnetic-circular-dichroism) spectroscopy were used to characterize the partially reduced cyanide-inhibited derivative of cytochrome c oxidase produced by anaerobic reductive titration with dithionite. The reductions of cytochrome a3+ and Cu2+a were followed by observation of the e.p.r. signals at g = 3.03, 2.21 and 1.5 and at g = 2.18, 2.03 and 1.99. As reduction proceeds new e.p.r. signals (g = 3.58 and 1.56) appear that quantify to give one haem per enzyme unit when a small excess of dithionite has been titrated in. The e.p.r. signal of the Cu2+a titrates in parallel with the disappearance of the band and 820nm in the optical absorption spectrum. The near-infrared m.c.d. spectrum shows the presence of the low-spin ferric haem, a3+, in the oxidized state of the enzyme, as a well-resolved positive peak at 1650nm. As reduction proceeds this band is replaced by one at 1550nm due to haem a3+(3)--CN in the partially reduced state. Hence as haem a3+(3)--CN becomes e.p.r.-detectable it also shows a near-infrared m.c.d. spectrum characteristic of a low-spin ferric haem. It is concluded that the partially reduced state of cyanide-inhibited cytochrome c oxidase contains a2+ . Cu+a . a3+(3)--CN . Cu+a3.     

Synthesis and antihyperglycemic activity of chalcone based aryloxypropanolamines

A series of aryloxypropanolamines (5a-r) of different chalcones (3a-e) were synthesized and evaluated for antihyperglycemic activity in sucrose loaded (SLM) and streptozotocin (STZ) induced diabetic animal models. Among them compounds 5a, g, m, o, p and r showed significant reduction in blood glucose levels in both SLM and STZ animal models.     

SMR analysis of historical follow-up studies with missing death certificates

The evaluation of epidemiological follow-up studies is frequently based on a comparison of the number O of deaths observed in the cohort from a specified cause with the expected number E calculated from person years in the cohort and mortality rates from a reference population. The ratio SMR = 100 x O/E is called the standardized mortality ratio (SMR). While person years can easily be calculated from the cohort and reference rates are generally available from the national statistical offices or the World Health Organization (WHO), problems can arise with the accessibility of the causes of death of the deceased study participants. However, the information that a person has died may be available, e.g., from population registers. In this paper, a statistical model for this situation is developed to derive a maximum likelihood (ML) estimator for the true (but unknown) number O* of deaths from a specified cause, which uses the known number O of deaths from this cause and the proportion p of all known causes of death among all decreased participants. It is shown that the standardized mortality ratio SMR* based on this estimated number is just SMR* = SMR/p. Easily computable confidence limits can be obtained by dividing the usual confidence limits of the SMR by the opposite limit of the proportion p. However, the confidence level alpha has to be adjusted appropriately.     

The calculative nature of microbial biofilms and bioaggregates

Biological proliferation is optimized at various levels of organization, including the molecule (e.g. nucleic acids, prions), the cell (e.g. prokaryotic cells, eukaryotic cells), and the community (e.g. microbial biofilms, bioaggregates). Although it was initially assumed that this occurred through the genesis of information within DNA alone, it now appears that innovative design originates at other levels of organization in addition to DNA. For example, the recombination of community structures affects the proliferation rate of genetic structures; and the recombination of genetic structures affects the proliferation rate of community structures. This feedback mechanism computes compromises between the form and function of both community and nucleic acid. A nested series of proliferating objects (e.g. genetic structure, cell structure, community structure) is thus capable of continually updating the form of each object in the series. This accounts for the calculative nature of prokaryotic cells, eukaryotic cells, biofilms, bioaggregates, microbial consortia, and most other complex adaptive systems. Electronic Publication     

A framework for controlling false discovery rates and minimizing the amount of genotyping in the search for disease mutations

OBJECTIVES: To develop a method for designing studies to find disease mutations that can achieve a set of goals with respect to proportions of false and true discoveries with the minimum amount of genotyping. METHODS: Derivation of an analytical framework supplemented with simulation techniques. The approach is illustrated for a fine mapping study and a whole-genome linkage disequilibrium scan. RESULTS: The use of multiple stages where earlier stages are characterized by very high false discovery rates (FDR) followed by an abrupt change to the required FDR in the final stage results in a 50-75% reduction in genotyping. The proportion of true discoveries is a much more important determinant of the genotyping burden than the FDR. Neither sample size nor controlling the false discoveries will present major problems in whole-genome LD scans but the amount of genotyping will be extremely large even if the study is completely designed to minimize genotyping. CONCLUSIONS: The proposed statistical framework presents a simple and flexible approach to determine the design parameters (e.g. sample size, p values at which tests need to be performed at each stage) that minimize the genotyping burden given a set of goals for the percentage of true and false discoveries.     

Actions of β-Bungarotoxin on Amino Acid Transmitter Release

Abstract: The actions of purified β-bungarotoxin (5 or 10 μg/ml) on the metabolic and transmitter-releasing properties of rat cortical synaptosomes was studied. The toxin stimulated control respiratory rates, but prevented the respiratory response to veratrine. Tissue potassium levels were greatly reduced (54%) and endogenous glutamate, aspartate and GABA showed increased levels of release (10- to 25-fold), but other amino acids were unaffected or showed much smaller changes. Tissue levels of these amino acids were reduced in proportion. The toxin inhibited the uptake of [U-¹⁴C]GABA (35%) and [U-¹⁴C]glutamate (53%) over 5-min incubation periods. This uptake-inhibition was Ca²⁺-dependent and was reduced by tetrodotoxin. Miniature-end-plate-potential (m.e.p. p.) frequencies at the locust neuromuscular junction (extensor tibialis) were greatly (four- to sevenfold) accelerated by local application of the toxin (5 μg/ml). This effect was reversible and occupied about 20 min. Amplitudes of m.e.p. p.'s were also increased and muscle membrane depolarization occurred. The results are interpreted as being due to a depolarizing action of the toxin.