Min-Jon Lin

Long Tract of Untranslated CAG Repeats Is Deleterious in Transgenic Mice

The most frequent trinucleotide repeat found in human disorders is the CAG sequence. Expansion of CAG repeats is mostly found in coding regions and is thought to cause diseases through a protein mechanism. Recently, expanded CAG repeats were shown to induce toxicity at the RNA level in Drosophila and C. elegans. These findings raise the possibility that CAG repeats may trigger RNA-mediated pathogenesis in mammals. Here, we demonstrate that transgenic mice expressing EGFP transcripts with long CAG repeats in the 3′ untranslated region develop pathogenic features. Expression of the transgene was directed to the muscle in order to compare the resulting phenotype to that caused by the CUG expansion, as occurs in myotonic dystrophy. Transgenic mice expressing 200, but not those expressing 0 or 23 CAG repeats, showed alterations in muscle morphology, histochemistry and electrophysiology, as well as abnormal behavioral phenotypes. Expression of the expanded CAG repeats in testes resulted in reduced fertility due to defective sperm motility. The production of EGFP protein was significantly reduced by the 200 CAG repeats, and no polyglutamine-containing product was detected, which argues against a protein mechanism. Moreover, nuclear RNA foci were detected for the long CAG repeats. These data support the notion that expanded CAG repeat RNA can cause deleterious effects in mammals. They also suggest the possible involvement of an RNA mechanism in human diseases with long CAG repeats.

MULTIPLE TYPES OF CA2+ CHANNELS IN MOUSE MOTOR NERVE TERMINALS

We measured neurotransmitter release and motor nerve terminal currents in mouse phrenic nerve‐diaphragm and triangularis sterni preparations, to evaluate the role of Ca2+‐channel subtypes in regulating transmitter release. Saturated concentrations of either ωagatoxin IVA [ω‐Aga‐IVA (0.3 μM), a blocker of P‐type Ca2+channels] or ω‐conotoxin MVIIC [ω‐CTx‐MVIIC (2 μM), a P‐and Q‐type Ca2+‐channel blocker], inhibited nerve‐evoked muscle contractions and the amplitude of endplate potentials respectively. In contrast, combined treatment with nifedipine (50 μM, a blocker of L‐type Ca2+ channels) plus ω‐conotoxin GVIA [ω‐CTx‐GVIA (2 μM), a blocker of N‐type Ca2+ channels] did not elicit inhibitory effects on nerve‐evoked muscle contractions, endplate potentials or nerve terminal waveforms. Because of the non‐linear relationship between endplate potentials and Ca2+ signals, a small decrease in presynaptic Ca2+ entry can significantly reduce the amplitude of the endplate potential. Thus, we applied 3, 4‐diaminopyridine (3, 4‐DAP, a k+‐channel blocker) or high Ca2+(10 mM) to accelerate and amplify the endplate potentials and Ca2+ currents. The endplate potentials amplified by 3, 4‐DAP or by high Ca2+ correspondingly proved to be quite resistant to both ω‐Aga‐IVA and ω‐CTx‐MVIIC; ωAga‐IVA exerted only a partial inhibitory effect on endplate potentials, and the ω‐Aga‐IVA‐resistant component was further inhibited by ω‐CTx‐MVIIC. The component that was resistant to the two toxins could be completely blocked by the non‐selective Ca2+ channel blocker Cd2+ (300 μM). A combination of the two toxins had no significant effects on either spontaneous transmitter release or postsynaptic resting membrane potentials of the diaphragm preparation and the Na+ and K+ waveforms of the triangularis sterni preparations. This finding suggests a preferential inhibitory effect at a presynaptic site. Measuring the Ca2+ currents in the triangularis sterni also revealed partial inhibition by ω‐CTx‐MVIIC with further incomplete inhibition by ω‐Aga‐IVA. Cd2+ (300 μM) abolished the toxin‐resistant component of the Ca2+ current. In contrast, a combination of nifedipine (50 μM) with ω‐CTx‐GVIA (2 μM) was without inhibitory effect. We conclude that multiple types of Ca2+channels, i.e. ω‐Aga‐IVA‐sensitive, ω‐CTx‐MVIIC‐sensitive and toxin‐resistant Ca2+ channels, coexist in mouse motor nerve terminals.

Suramin inhibits the toxic effects of presynaptic neurotoxins at the mouse motor nerve terminals

Clinically available chemical antagonists of snake neurotoxins still await to be identified. In this study, we demonstrate that an anti-trypanosomiasis agent, suramin, is an effective inhibitor of beta-bungarotoxin isolated from the venom of Formosan Krait snake. Following intraperitoneal injection (12 ng/g) of beta-bungarotoxin in mice, the time to paralysis (loss a limb withdrawal reflex, 21. 8+/-3.4 h, n=4) was significantly prolonged after intravenous injection (16 microg/g) of suramin (35.9+/-4.0 h, n=4, P<0.05). The mechanism of this inhibitory effect of suramin was analyzed at the mouse nerve terminals. beta-Bungarotoxin (1 microg/ml) produces an irreversible blocking effect of nerve-evoked muscle contractions of mouse phrenic nerve-diaphragm (blocking time 135+/-6 min, n=6). Pretreatment with suramin (0.3 mM) significantly prolonged the blocking time by three-fold. This selective inhibitory effect of suramin was further confirmed when suramin was shown to delay the neuromuscular blocking effect of another presynaptic neurotoxin, crotoxin (from American rattlesnake venom), but not that of the postsynaptic neurotoxin, alpha-bungarotoxin. Furthermore, suramin inhibited beta-bungarotoxin in blocking transmitter release as revealed by prolonging the time to abolish the end-plate potential amplitude (with suramin, 391+/-8 min; without treatment, 141+/-5 min). K(+) current was measured in the mouse triangularis sterni preparation; suramin (0.3 mM) had no significant effect on beta-bungarotoxin in inhibiting K(+) current (77+/-3% of control; with suramin 75+/-3% of control, respectively). These findings clearly show that suramin is an inhibitor of presynaptic neurotoxins, mediated by interrupting the toxins in blocking the releasing mechanism of transmitter at the motor nerve terminals. The implication of these findings is that suramin and related compounds can become useful agents in management of snakebites.

Influence of strain rate on buckle folding of an elasto–viscous single layer

Abstract The influence of strain rate on the buckle folding behavior of an elasto–viscous layer–matrix model is explored by adopting an end-rotation method, which is capable of excluding the influence of initial geometric perturbation, and by observing the energy variation in the system. The results indicate that, if the strain rate is relatively slow, the folding behavior is in fact the result of both viscous and elastic behavior, and not just the viscosity alone. For two-stage shortening at different strain rates, the final waveform depends on either the earlier strain rate inducing buckling or the later strain rate applied in the post-buckle stage of deformation. If the later strain rate is relatively fast, the final waveform will be similar to the one yielded by the fast strain rate alone, as a substantial amount of elastic energy can be accumulated during the subsequent fast deformation. On the other hand, if the later strain rate is relatively slow, the earlier waveform is retained and further amplified during the slower post-buckle deformation. This results from the phenomenon that an initial geometric perturbation is amplified into a fold, if the applied strain rate is very slow.

Functional study of CLC-1 mutants expressed in Xenopus oocytes reveals that a C-terminal region Thr891-Ser892-Thr893 is responsible for the effects of protein kinase C activator.

ClC-1 plays an important part in the maintenance of membrane potential in the mammalian skeletal muscle. To investigate the phosphorylation sites responsible for the effect of PKC (protein kinase C) activator, we constructed 21 different ClC-1 mutants with mutations at predicted phosphorylation sites for PKC. The functional experiments were performed on both wild-type and mutant proteins (17 point mutants and 4 double mutants) expressed in Xenopus oocytes with two-electrode voltage-clamp recording. PMA (12-myristate 13-acetate), a PKC activator, caused a right shift of half-maximum activation potential (V1/2) significantly in the wild-type (from -42.9±4.4 to -13.7±1.7 mV; n = 8, P < 0.05) and most of the single mutants except the S892P (from -39.5±4.5 to -35.7±5.7 mV; n = 6) and S892D (from -10.2±4.9 to -9.6±3.5 mV; n = 4). S892D, a mutant mimicking PKC-mediated phosphorylation at position 892, can also mimic the effect of wild-type treated with PMA in V1/2 value (-10.2±4.9 mV vs -13.7±1.7 mV, n = 4 - 8). However, S892A still had a significant response to PMA indicating that other sites responsible for PMA might exist. Thus double mutants are generated for the following analysis. The V1/2 of double mutants, T891A/S892A, S892A/T893A and T891A/T893A, show no significant difference between before and after PMA treatment. We hypothesize that this structural modification results in the observed alteration of the gating properties of ClC-1 by PMA. In summary, our observations show that a C-terminal region Thr891-Ser892-Thr893, at least in part, responsible for the effect of PMA on ClC-1.

DnaT is a single-stranded DNA binding protein

DnaT is one of the replication restart primosomal proteins required for reinitiating chromosomal DNA replication in bacteria. In this study, we identified and characterized the single‐stranded DNA (ssDNA)‐binding properties of DnaT using electrophoretic mobility shift analysis (EMSA), bioinformatic tools and two deletion mutant proteins, namely, DnaT26‐179 and DnaT42‐179. ConSurf analysis indicated that the N‐terminal region of DnaT is highly variable. The analysis of purified DnaT and the deletion mutant protein DnaT42‐179 by gel filtration chromatography showed a stable trimer in solution, indicating that the N‐terminal region, amino acid 1–41, is not crucial for the oligomerization of DnaT. Contrary to PriB, which forms a single complex with a series of ssDNA homopolymers, DnaT, DnaT26‐179 and DnaT42‐179 form distinct complexes with ssDNA of different lengths and the size of binding site of 26 ± 2 nucleotides (nt). Using bioinformatic programs (ps)2 and the analysis of the positively charged/hydrophobic residue distribution, as well as the biophysical results in this study, we propose a binding model for the DnaT trimer–ssDNA complex, in which 25‐nt‐long ssDNA is tethered on the surface groove located in the highly conserved C‐terminal domain of DnaT. These results constitute the first study regarding ssDNA‐binding activity of DnaT. Consequently, a hand‐off mechanism for primosome assembly was modified.

Phorbol ester-induced contractions of mouse detrusor muscle are inhibited by nifedipine.

The effects of phorbol esters on contractions of detrusor strips isolated from mouse urinary bladder were studied. β-Phorbol-12,13-dibutyrate (β-PDBu, 10 nM) significantly enhances both the neurogenic and myogenic detrusor contractions to a similar extent. By contrast, an inactive isoform of protein kinase C (PKC) stimulation, α- phorbol-12,13-dibutyrate (100 nM) has no such enhancing effect on the muscle contraction. The effect of β-PDBu was dependent on the extracellular Ca2+ concentration. Nifedipine (0.3 µM, a L-type Ca2+ channel blocker), staurosporine (1 µM) and bisindolylmaleimide I ( µM, a selective PKC inhibitor) but not ω-conotoxin GVIA (an N-type Ca2+ channel blocker) abolished the enhancing effect of β-PDBu. In other words, β-PDBu failed to augment the nifedipine-insensitive component of the muscle contraction. Moreover, β-PDBu not only enhances the muscle response induced by exogenous agonists (acetylcholine or ATP) and KCl but also increases the resting tone of detrusor muscle, an effect which is also inhibited by nifedipine and bisindolylmaleimide I. From these findings, it is concluded that the enhancing effect of β-PDBu is due to activation of the L-type Ca2+ channel through phosphorylation by protein kinase C. This allows more Ca2+ influx from the extracellular medium, leading to an increase in the contractions of the mouse detrusor muscle.

Ruthenium red, a novel enhancer of K+ currents at mouse motor nerve terminals.

The effects of ruthenium red (RR) on transmitter release and pre-synaptic currents were studied in the mouse neuromuscular junction. The action of RR (10 microM) was shown not only in the complete suppression of nerve-evoked muscle contractions associated with the depression of endplate potential amplitude but also in the partial inhibition of the amplitude of miniature-endplate potentials. However, the other ruthenium compounds, ruthenium chloride and tris (2,2-bipyridyl) ruthenium chloride did not significantly affect the neuromuscular transmission. In pre-synaptic waveform studies, the fast K(+)-current [IK(f)] as well as the ca(2+)-activated K(+)-current [IK(ca)] was significantly enhanced by 10 microM RR. Furthermore, 10 microM RR antagonized the action of beta-bungarotoxin (a blocker of slow K(+)-channel [IK(s)] in enhancing pre-synaptic Ca2+ currents. In contrast, the typical Ca(2+)-channel blockers, omega-agatoxin (0.5 microM), Gd3+ (0.5 mM) and CD2+ (0.3 mM) all suppressed the IK(ca). Although RR (1-30 microM) inhibited the Ca(2+)-currents of the nerve terminals induced by the combined treatment with the K(+)-channel blockers, 3,4-diaminopyridine plus tetraethylammonium chloride in a concentration-dependent manner, it is considered that RR-enhanced K+ currents were responsible for, at least in part, the observed inhibition of the Ca(2+)-current which led to the blockade of transmitter release.

Use of ion channel blockers in the exploration of possible mechanisms involved in the myopathy of diabetic mice

SummaryChanges in the muscle contractions of the phrenic nerve-diaphragm preparation from the diabetic mouse were investigated by means of K+- and Cl−-channel blockers and the Ca2+-mobilizing agent, selenite. The K+-channel blockers (UO22+ and 4-aminopyridine) cooperated synergistically with the Cl−-channel blockers (Cd2+ and 9-anthracenecarboxylic acid) in increasing normal muscle contraction as described previously, but failed to induce this effect in the diaphragm of the diabetic mouse. Treatment with a Cl−-channel blocker alone in 0.25 mmol/1 Ca2+ Krebs solution induced a myotonic activity accompanied by stimulus-bound repetitive action potential firings. This effect was also diminished in the diaphragm from diabetic mice. The membrane potential of the muscle cells in the diaphragm of the diabetic mouse was slightly but significantly decreased. The membrane input resistance was also increased and was refractory to being further increased by either a Cl−-channel blocker or a low Cl−-medium. Furthermore, the membrane chloride conductance was found to be decreased, but the membrane K+ conductance remained unchanged in the muscle from diabetic mice. These changes of membrane properties in the muscles from diabetic mice were shown to be similar to those induced by either Cl−-channel blockers or a low Cl−-medium. In addition, the combined treatment of the diaphragm from diabetic mice with Cd2+ Plus UO22+ in 0.25 mmol/l Ca2+ Krebs solution and then stepwise replenishment of Ca2+ led to a greater restoration of muscle contractions at a lower cumulative Ca2+ concentration than that was found with the normal diaphragm. The sustained muscle contracture of the mouse diaphragm induced by U022+ plus selenite was partially inhibited in the diaphragm from diabetic mice, indicating that the Ca2+ mobilizing mechanism of the diaphragm of the diabetic mouse was also altered. All of these observations obtained with the diaphragm of the diabetic mouse can be attributed to the diabetic state, because most of them could be normalized by insulin administration in vivo. Therefore, it is concluded that diabetes-induced changes of sarcolemmal ion channels and ion transporters may cause inhibition of muscle contraction and eventually lead to diabetic myopathy.

Enhanced spontaneous transmitter release at murine motor nerve terminals with cyclosporine.

Cyclosporine, a calcineurin inhibitor, significantly enhances spontaneous acetylcholine release after a brief tetanus and potentiates the effect of phorbol 12,13-dibutyrate. Both actions are prevented by the protein kinase C inhibitor, bisindolylmaleimide iodide. Protein kinase C and calcineurin thus play important roles in the balance between phosphorylation and dephosphorylation regulating spontaneous transmitter release at motor nerve terminals.