Identification, chromosomal arrangements and expression analyses of the evolutionarily conserved prmt1 gene in chicken in comparison with its vertebrate paralogue prmt8.

Nine protein arginine methyltransferases (PRMTs) are conserved in mammals and fish. Among these, PRMT1 is the major type I PRMT for asymmetric dimethylarginine (ADMA) formation and is the most conserved and widely distributed one. Two chicken prmt1 splicing variants were assembled and confirmed by RT-PCR experiments. However, only two scaffolds containing single separate prmt1 exon with high GC contents are present in the current chicken genome assembly. Besides, prmt1 exons are scattered in separate small scaffolds in most avian species. Complete prmt1 gene has only been predicted from two falcon species with few neighboring genes. Crocodilians are considered close to the common ancestor shared by crocodilians and birds. The gene arrangements around prmt1 in American alligator are different from that in birds but are largely conserved in human. Orthologues of genes in a large segment of human chromosomal 19 around PRMT1 are missing or not assigned to the current chicken chromosomes. In comparison, prmt8, the prmt1 paralogue, is on chicken chromosome 1 with the gene arrangements downstream of prmt8 highly conserved in birds, crocodilians, and human. However, the ones upstream vary greatly in birds. Biochemically, we found that though prmt1 transcripts were detected, limited or none PRMT1 protein was present in chicken tissues. Moreover, a much higher level of PRMT8 protein was detected in chicken brain than in mouse brain. While PRMT8 is brain specific in other vertebrate species studied, low level of PRMT8 was present in chicken but not mouse liver and muscle. We also showed that the ADMA level in chicken was similar to that in mouse. This study provides the critical information of chicken PRMT1 and PRMT8 for future analyses of the function of protein arginine methyltransferases in birds.

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.

Yeast two-hybrid analysis of PriB-interacting proteins in replication restart primosome: a proposed PriB-SSB interaction model.

PriB is one of the components of the replication restart primosome. The activity mediated by the primosomal proteins, including PriB, is required for reinitiating chromosomal DNA replication in bacteria after DNA damage. As such, the study of the interactions between PriB and members in the primosome is essential to better understand their mechanism of action. In this study, we investigated PriB-interacting proteins in the primosome through the yeast two-hybrid system. Yeast two-hybrid analysis using two strains, Y187 and AH109, revealed that PriB interacts with PriA, DnaT, SSB, and itself and does not interact with DnaA, DnaB, DnaC, or DnaG. In addition, mutational analysis showed that PriB may bind to SSB via K82, K84, and K89 in the L(45) loop. Based on these preliminary data, we proposed a PriB-SSB interaction model. Further work can focus on determination of how PriB binds to the PriA-SSB complex in replication restart.

The Effects of the KCNQ Openers Retigabine and Flupirtine on Myotonia in Mammalian Skeletal Muscle Induced by a Chloride Channel Blocker.

The purpose of this study was to investigate the effect of KCNQ (potassium channel, voltage-gated, KQT-like subfamily) openers in preventing myotonia caused by anthracene-9-carboxylic acid (9-AC, a chloride channel blocker). An animal model of myotonia can be elicited in murine skeletal muscle by 9-AC treatment. KCNQ openers, such as retigabine and flupirtine, can inhibit the increased twitch amplitude (0.1 Hz stimulation) and reduce the tetanic fade (20 Hz stimulations) observed in the presence of 9-AC. Furthermore, the prolonged twitch duration of skeletal muscle was also inhibited by retigabine or flupirtine. Lamotrigine (an anticonvulsant drug) has a lesser effect on the muscle twitch amplitude, tetanic fade, and prolonged twitch duration as compared with KCNQ openers. In experiments using intracellular recordings, retigabine and flupirtine clearly reduced the firing frequencies of repetitive action potentials induced by 9-AC. These data suggested that KCNQ openers prevent the myotonia induced by 9-AC, at least partly through enhancing potassium conductance in skeletal muscle. Taken together, these results indicate that KCNQ openers are potential alternative therapeutic agents for the treatment of myotonia.

Study of the reversal effect of NF449 on neuromuscular blockade induced by d-tubocurarine.

AIMS: The aim of this study was to investigate the mechanism for the reversal effect of NF449 (a suramin analogue) on the neuromuscular block induced by d-tubocurarine (d-TC). MAIN METHODS: Nerve-stimulated muscle contractions and end-plate potentials were performed in mouse phrenic nerve-diaphragm preparations. Acetylcholine (ACh)-induced muscle contractions were performed in the chick biventer cervicis preparations. Presynaptic nerve terminal waveform recordings were performed in mouse triangularis sterni preparations. KEY FINDINGS: Amongst the suramin analogues in this study, only the NF449 and suramin were able to reverse the blockade effect produced by d-TC on nerve-stimulated muscle contractions. Each of these suramin analogues (NF007, NF023, NF279 and NF449) alone has no significant effect on the amplitude of nerve-stimulated muscle contractions. NF449 and suramin also showed the antagonising effects on the inhibition of end-plate potentials induced by d-TC. Furthermore, pre-treatment with NF449 can antagonise the inhibition of d-TC in ACh-induced contractions of chick biventer cervicis muscle. NF449 produced a greater rightward shift of the dose-response inhibition curve for d-TC than did suramin. Because other purinergic 2X (P2X) receptor antagonists, NF023 and NF279, do not have the reverse effects on the neuromuscular blockade of d-TC, the effect of NF449 seems irrelevant to inhibition of P2X receptors. SIGNIFICANCE: These data suggest that NF449 was able to compete with the binding of d-TC on the nicotinic ACh receptors, and the effect of NF449 was more potent than suramin in reducing the inhibition of d-TC. The structure of NF449 may provide useful information for designing potent antidotes against neuromuscular toxins.

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.

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 (V(1/2)) 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 V(1/2) 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 V(1/2) 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.

Functional study of the effect of phosphatase inhibitors on KCNQ4 channels expressed in Xenopus oocytes.

AIM: KCNQ4 channels play an important part in adjusting the function of cochlear outer hair cells. The aim of this study was to investigate the effects of ser/thr phosphatase inhibitors on human KCNQ4 channels expressed in Xenopuslaevis oocytes. METHODS: Synthetic cRNA encoding human KCNQ4 channels was injected into Xenopus oocytes. We used a two-electrode voltage clamp to measure the ion currents in the oocytes. RESULTS: Wild-type KCNQ4 expressed in Xenopus oocytes showed the typical properties of slow activation kinetics and low threshold activation. The outward K(+) current was almost completely blocked by a KCNQ4 blocker, linopirdine (0.25 mmol/L). BIMI (a PKC inhibitor) prevented the effects of PMA (a PKC activator) on the KCNQ4 current, indicating that PKC may be involved in the regulation of KCNQ4 expressed in the Xenopus oocyte system. Treatment with the ser/thr phosphatase inhibitors, cyclosporine (2 micromol/L), calyculin A (2 micromol/L) or okadaic acid (1 micromol/L), caused a significant positive shift in V(1/2) and a decrease in the conductance of KCNQ4 channels. The V(1/2) was shifted from -14.6+/-0.5 to -6.4+/-0.4 mV by cyclosporine, -18.8+/-0.5 to -9.2+/-0.4 mV by calyculin A, and -14.1+/-0.5 to -0.7+/-0.6 mV by okadaic acid. Moreover, the effects of these phosphatase inhibitors (okadaic acid or calyculin A) on the induction of a positive shift of V(1/2) were augmented by further addition of PMA. CONCLUSION: These results indicate that ser/thr phosphatase inhibitors can induce a shift to more positive potentials of the activation curve of the KCNQ4 current. It is highly likely that the phosphatase functions to balance the phosphorylated state of substrate protein and thus has an important role in the regulation of human KCNQ4 channels expressed in Xenopus oocytes.

Functional studies of the effect of NO donor on human CLCN1 polymorphism/mutants expressed in Xenopus laevis oocytes.

In this study, we investigated the effect of NO donor, diethylamine/nitric oxide (DEA/NO), on the electrophysiological behavior of human skeletal muscle chloride channel (CLCN1). The wild-type and variants of CLCN1, including one polymorphism (P727L) and four mutants (T631I, D644G, G482R, and S471F), were expressed in Xenopus oocytes and the ionic current was measured by two-electrode voltage-clamp method. Our results revealed that there is no significant difference in the current-voltage relationships and half-voltage values of open probability between wild-type and variants of CLCN1 except for G482R. Application of the DEA-NO (0.1mM) significantly increases the channel conductance of wild-type, T631I, D644G, and S471F, but not P727L. This indicates that P727L polymorphism causes loss of sensitivity of CLCN1 to the DEA/NO treatment, which could be due to a conformational change caused by proline substitution. The data suggest that the polymorphic changes may affect the function of CLCN1 in response to the treatment of chemical compounds.

Length-dependent toxicity of untranslated CUG repeats on Caenorhabditis elegans.

Expansion of CTG repeat within the 3'-untranslated region of the DMPK gene causes the most common neuromuscular disorder, myotonic dystrophy type 1 (DM1), through a RNA trans-dominant mechanism. Here, we explore Caenorhabditis elegans as a model system to investigate the repeat size-dependent toxic effect by expression of green fluorescent protein (GFP) transcripts with various lengths of untranslatable CUG repeats (CUG5, CUG30, CUG83, CUG125, and CUG213) in body wall muscles. CUG213 animals died during embryogenesis or showed retarded growth at larval stages due to defective muscle development. CUG125 animals, although can produce offspring, exhibited uncoordinated muscle function, deviated electropharyngeogram, and an age-dependent abnormality in muscle structure. Most CUG83 animals had normal muscle structure and function as those expressing 30 and shorter repeats. Our results demonstrate for the first time that the in vivo toxicity of CUG repeats is repeat length- and growth-regulated and suggest that expanded CUG repeats are sufficient to cause congenital-like phenotypes in living organisms.

Functional characterization of CLCN1 mutations in Taiwanese patients with myotonia congenita via heterologous expression.

Mutations in the CLCN1 gene frequently associate with myotonia congenita (MC). We have recently reported several CLCN1 mutants in Taiwanese patients. To further elucidate the correlation between the genotypes and phenotypes, in this study, we used Xenopus oocyte as a system to investigate the functional effects of these mutants. The fs793X and G482R mutants, which were suggested to have a dual inheritance pattern, were found to cause a functional loss of CLCN1 channels. While co-expression of fs793X and wild-type (WT) showed a reduction of chloride conductance by about half of WT channels, the activation curve of voltage-dependence was not shifted. A compound heterozygous mutant, P575S/D644G, was found in a patient. When both mutants were co-expressed in oocytes, they caused a shift of the voltage-dependence of activation curve to more positive values than individual mutant. This indicates that both P575S and D644G mutants may contribute cooperatively to change the gating property of CLCN1 channel. Interestingly, the S471F mutant did not cause significant alternation of functional properties. Consistent with the fact that T631I mutant was found in three asymptomatic individuals, the electrophysiological parameters of T631I were similar to those of WT CLCN1 channels, suggesting that T631I is a neutral mutation. These results further clarify the correlation between the mutations and their functional implications of CLCN1 channels.

Studies of the effect of ionomycin on the KCNQ4 channel expressed in Xenopus oocytes.

The effect of ionomycin on the human KCNQ4 channels expressed in Xenopus leavis oocytes was investigated. KCNQ4 channels expressed in Xenopus oocytes were measured using two-electrode voltage clamp. The activation of KCNQ4 current had slow activation kinetics and low threshold (approximately -50 mV). The expressed current of KCNQ4 showed the half-maximal activation (V(1/2)) was -17.8 mV and blocked almost completely by KCNQ4 channel blockers, linopirdine (300 microM) or bepridil (200 microM). The significant increase of KCNQ4 outward current induced by ionomycin (calcium salt) is about 1.7-fold of control current amplitude at +60 mV and shifted V(1/2) by approximately -8 mV (from -17.8 to -26.0 mV). This effect of ionomycin could be reversed by the further addition of BAPTA-AM (0.3 mM), a membrane-permeable calcium chelator. Furthermore, the increased effect of ionomycin on KCNQ4 current is abolished by pretreatment of linopirdine or bepridil. In contrast, direct cytoplasmic injection of calcium medium (up to 1 mM calcium, 50 nl) did not mimic the effect of ionomycin. In conclusion, the effect of ionomycin on enhancement of KCNQ4 current is independent of intracellular calcium mobilization and possibly acts on intramembrane hydrophobic site of KCNQ4 protein expressed in Xenopus oocytes.

Caffeic acid phenethyl ester ameliorates cerebral infarction in rats subjected to focal cerebral ischemia.

The effects of caffeic acid phenethyl ester (CAPE), an antioxidant derived from propolis, on the infarct volume elicited by focal cerebral ischemia were studied on Long-Evans rats. Cerebral infarction was induced by microsurgical procedures with ligation of the right middle cerebral artery (MCA) and clipping of bilateral common carotid arteries (CCA) for 60 min. The rats were sacrificed 24 h later and serial brain slices of 2 mm thickness were taken and stained for the measurement of infarct area. CAPE was administered intravenously 15 min before MCA occlusion. Pretreatment of CAPE (0.1, 1 and 10 microg/kg) significantly reduced the total infarct volume from 169.6 +/- 14.5 mm3 (control) to 61.0 +/- 24.1 mm3 (0.1 microg/kg CAPE), 47.4 +/- 9.1 mm3 (1 microg/kg CAPE), and 42.4 +/- 8.7 mm3 (10 microg/kg CAPE), respectively. Plasma nitric oxide (NO) content was significantly increased in rats subjected to focal cerebral ischemia. It is concluded that CAPE possesses neuroprotective properties in focal cerebral ischemia injury in rats possibly through its antioxidant effect and/or via the upregulation of NO production.

Taicatoxin inhibits the calcium-dependent slow motility of mammalian outer hair cells.

The effects of taicatoxin on the slow motility of isolated outer hair cells of guinea pig were studied in the experiments. Pretreatment with taicatoxin (0.19 microM) was able to prevent both the cell shortening induced by high K(+) (50mM), and the cell elongation induced by ionomycin (10 microM). These effects of taicatoxin can be mimicked by pretreatment of cells with Ca(2+)-free medium on the slow motility in response to ionomycin or high K(+). Pretreatment with neither calcium channel blockers such as nifedipine (L-type blocker), omega-conotoxin GVIA (N-type blocker), and omega-agatoxin IVA (P-type blocker); nor potassium channel blockers, such as tetraethylammonium chloride (TEA) and 3,4-diaminopyridine (3,4-DAP) can antagonize the cell shortening effect induced by high K(+) and cell elongation induced by ionomycin. The calcium-imaging experiment indicated that taicatoxin, but not nifedipine, did prevent an increase of intracellular Ca(2+) level significantly induced by high K(+). These results demonstrate that the effect of taicatoxin was to block the calcium entry through calcium channels of cell membrane, without relative to its properties of potassium channel blockers. We conclude that taicatoxin-sensitive-calcium channels at least impart, play a significant role in the slow motility of outer hair cell.

PGE2 enhances cytokine-elicited nitric oxide production in mouse cortical collecting duct cells.

It has been documented that arginine vasopressin (AVP) and prostaglandin E(2) (PGE(2)) regulate water reabsorption in renal tubular cells. The present study was attempted to delineate the downstream signaling of AVP and PGE(2) in a cortical collecting duct cell line (M-1 cell). Using RT-PCR, we detected mRNA for V2 and VACM-1 but not for V1a and AII/AVP receptors of AVP. Furthermore, neither AVP nor V2 receptor agonist and antagonist alter cellular cAMP. These together with unchanged cellular Ca(2+) by AVP suggested that AVP pathway was not operating in M-1 cells. All four classical PGE(2) receptors with EP3 and EP4 as the most prominent were detected in M-1 cells. PGE(2), 11-deoxy-PGE(1) (EP2 and EP4 agonist), and 17-phenyl-trinor-PGE(2) (EP1 agonist) increased cellular concentration of cAMP. There was no effect of PGE(2) or EP1 agonist on cellular Ca(2+). These findings provide evidence of the involvement of PGE(2) cascade in M-1 cells. M-1 cells were capable of synthesizing nitric oxide (NO). Although individual cytokines did not affect NO production, a mixture of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma elevated NO concentration to 4.5-fold of the control. Addition of PGE(2) and db-cAMP to the cytokine mixture further increased NO production to 7.0- and 9.8-fold, respectively, of that seen in non-treated cells. PGE(2) or db-cAMP alone, however, had no effect on NO production. The results of the study led us to speculate that enhanced production of cAMP via PGE(2) signaling pathway in M-1 cells could either stimulate or attenuate water reabsorption in renal tubule. While an increase in cAMP alone may enhance water reabsorption, a concomitant increase in cAMP and cytokines may inhibit water reabsorption in renal tubule.

The neuroprotective effect of glial cell line-derived neurotrophic factor in fibrin glue against chronic focal cerebral ischemia in conscious rats.

Glial cell line-derived neurotrophic factor (GDNF) is a transforming growth factor-beta which has shown beneficial effects in rats after acute focal cerebral ischemia (FCI). To study the effects of GDNF on chronic FCI injury in conscious rats, we used fibrin glue (GDNF-fibrin glue) and fibrin glue free (GDNF-only)-GDNF topically applied to the ischemic brain after right middle cerebral artery (MCA) ligation. Infarct brain volume and functional motor deficits were measured before and after FCI injury. After FCI injury induced by right MCA ligation, rats were randomly assigned to one of four treatment groups: (a) sham, (b) control, (c) topically applied GDNF (1 mug)-only, and (d) topically applied GDNF (1 mug)-fibrin glue. The degree of ischemic brain injury was estimated by infarct volume of right MCA territory at 4 weeks after occlusion. The functional motor deficits were quantified with rotarod test and grasping power test once a week. Topically applied GDNF-fibrin glue at infarct brain tissue after 4 weeks FCI injury significantly reduced the total infarct volume by 44.3% and 36%, respectively, compared to that of control group and GDNF-only group. The mean latencies for rats to stay on the rotarod were 55.0%, 50.3%, and 92.2% (P < 0.05 vs. control group and GDNF-only group) of baseline, respectively, in the control, GDNF-only, and GDNF-fibrin glue groups at the end of the 1st week after FCI injury but 75.3%, 67.3%, and 106.6% (P < 0.05 vs. control group and GDNF-only group) of baseline at the end of the 4th week after FCI injury. The mean values of grasping power were 78.7%, 71.7%, and 101.2% (P < 0.05 vs. control group and GDNF-only group) of baseline, respectively, in the control, GDNF-only, and GDNF-fibrin glue groups at the end of 1st week after FCI injury but 89.6%, 97.6%, and 120.7% (P < 0.05 vs. control group) of baseline at the end of 4th week after FCI injury. These results indicate that GDNF-fibrin glue not only reduced the total infarct volume after FCI injury but can also improve motor deficits after FCI injury. We concluded GDNF-fibrin glue could facilitate delivery of GDNF to the damaged brain tissue with subsequent reduction of ischemic brain injury accompanied by enhancing functional recovery in rats with chronic FCI injury.

The effect of L-arginine on slow motility of mammalian outer hair cell.

The effect of L-arginine on the slow motility of mammalian cochlear outer hair cells was studied in this experiment. L-Arginine (3 mM) but not D-arginine (3 mM) or other amino acids (L-aspartate or L-glutamate) induced length increases of guinea pig outer hair cell. Similarly, the membrane-permeant cGMP analogues, 8-(4-chlorophenylthio)guanosine 3':5'-cyclic monophosphate (1 mM) or 8-bromo-guanosine 3':5'-cyclic monophosphate (1 mM) induced length increases of guinea pig outer hair cells. These length increases induced by L-arginine can be attenuated by a 30 min preincubation of the cells with the nitric oxide synthase inhibitors N(G)-nitro-L-arginine methyl ester hydrochloride (3 mM) or 7-nitroindazole (1 mM). Comparing the effects of L-arginine and ionomycin on cell length and intracellular calcium change in outer hair cells, both L-arginine and ionomycin were able to induce the elongation of outer hair cells but L-arginine did not change the fluorescence intensity of Fluo-3. Preincubation of the cells with EGTA (3 mM) for 40 min to reduce the extracellular calcium concentration did not influence the effect of L-arginine. This experiment demonstrated that nitric oxide/cGMP pathway involvement in regulating the slow motility of mammalian outer hair cells cannot be ruled out. The effect of L-arginine is independent of extracellular calcium concentration.