C5-azobenzene-functionalized locked nucleic acid uridine: isomerization properties, hybridization ability, and enzymatic stability.

Oligonucleotides (ONs) modified with a locked nucleic acid (LNA) are widely used in the fields of therapeutics, diagnosis, and nanotechnology. There have been significant efforts towards developing LNA analogues bearing modified bridges to improve their hybridization ability, nuclease resistance, and pharmacokinetic profiles. Moreover, nucleobase modifications of LNA are useful strategies for the functionalization of ONs. Modifications of the C5-position of pyrimidine nucleobases are particularly interesting because they enable predictable positioning of functional groups in the major groove of the duplex. Here we report the synthesis of C5-azobenzene-functionalized LNA uridine (LNA-U(Az)) and properties of LNA-U(Az)-modified ONs, including isomerization properties, hybridization ability, and enzyme stability. LNA-U(Az) in ON is photo-isomerized effectively and reversibly by irradiation at 365 nm (trans to cis) and 450 nm (cis to trans). LNA-U(Az)-modified ONs show RNA-selective hybridization ability despite the large hydrophobic azobenzene moiety extending into the major groove of the duplex. The enzymatic stability of LNA-U(Az)-modified ONs is higher than that of natural and LNA-modified ONs with or without photo-irradiation. Our results indicate that LNA-U(Az) holds promise for RNA targeting and photo-switchable technologies.

Isolation of polymorphic microsatellite markers in the tree sparrow (Passer montanus).

To enable the study of the population genetics of the tree sparrow (Passer montanus), which is distributed in many islands of Japan, nine polymorphic microsatellite loci were isolated from 32 individuals. The number of alleles per locus ranged from 2 to 17, and observed heterozygosities ranged from 0.1250 to 0.9375. In eight of the nine loci, the heterozygosities were not significantly different from those expected from Hardy-Weinberg equilibrium. Cross-species amplification in the russet sparrow (P. rutilans) was successful with all primer sets, which were highly polymorphic, suggesting these markers are useful for the population genetics of genus Passer.

A set of polymorphic trinucleotide and tetranucleotide microsatellite markers for the Japanese flounder (Paralichthys olivaceus).

We have developed the first set of trinucleotide and tetranucleotide markers for the Japanese flounder, Paralichthys olivaceus. One hundred and sixty-seven polymorphic trinucleotide and tetranucleotide microsatellites were isolated using clones derived from two libraries. Of almost 200,000 clones analysed, 0.5% presented trinucleotide or tetranucleotide repeat regions. Among the trinucleotide repeats analysed in this study, the most frequent one was (CAG)(n) and the most common tetranucleotide repeat was (GATA)(n). The position of the new markers in the genetic linkage map was determined. Markers were evenly distributed along the P. olivaceus linkage groups, without distinction between the kinds of repeats and library of origin. The markers isolated in this study contribute significantly to the genetic linkage map of the Japanese flounder.

[Therapeutic outcome of spasmodic torticollis].

We investigated 117 patients with spasmodic torticollis who had visited us to seek for appropriate treatment in these 14 years. They were 71 men and 46 women, aged 44 +/- 14 (mean +/- SD) years, and suffered from this disorder during 4 +/- 5 years, maximum 26 years. Involuntary abnormal head positions, not only torticollis but also laterocollis and antero- or retrocollis, were contained in this study. Most of them were torticollis due to idiopathic focal dystonia. One or more courses of alcoholization therapy was accomplished in 82 patients who wished to be done. This therapy course consisted of about ten times totally of 99% ethanol injection to the motor point of two most hypertonic neck muscles, either side of the sternocleidomastoideus and the opposite side of the splenius in most cases, repeated every 2 or 3 weeks. One patient received as many as 98 times of this injection and resolved completely. Training to reinforce antagonistic muscles was also instructed. Twenty-one patients (26%) were resolved completely after this treatment. Fifty-four patients (66%) were ameliorated and satisfied partially, but 18 of them relapsed in 1 to 4 years after the treatment and were obliged to repeat one more course of this treatment. On the other hand, in five patients their torticollis improved under certain drug therapy alone. Sixteen patients (14%) gave up to continue the treatment within two months, and 14 patients (12%) dropped out before starting the therapy. This alcoholization therapy resulted in amelioration of torticollis in about 90% of the patients with a long effective period. Nevertheless, this alcohol injection is painful, and requires 5 to 6 months to be completed. In 2 patients who had already received many times of this injection, sudden hoarseness occurred one day immediately after the alcohol injection to the sternocleidomastoideus. This complication was presumably brought about by the unexpected infiltration of alcohol to the laryngeal area, located posterior to that muscle. They recovered in two months, but careful attention should be paid to the adverse effects. If botulinum toxin be available also in our country, we will be able to have another choice of therapy and the treatment of this disorder will become easier.

[Study on the latency difference between compound muscle and sensory nerve action potentials].

In motor nerve conduction studies compound muscle action potentials (CMAPs) appear later than sensory nerve action potentials (SNAPs). This time lag originates from the conduction delay at the distal motor axon, neuromuscular transmission time and muscle action potential induction time. To investigate the latency difference between CMAPs and SNAPs we studied 46 healthy individuals, 46 patients with diabetes mellitus and 33 patients with carpal tunnel syndrome, using the lumbrical and interossei recording method. In this method the recording active electrode was placed on the 2nd lumbrical muscle and the reference electrode on the proximal palmar aspect of the index finger. Supramaximal stimulation was given to the median or ulnar nerve trunk at 9-cm proximal to the recording active electrode. The CMAP from the 2nd lumbrical muscle (L) and the SNAP from the digital nerve (N) were recorded after median nerve stimulation, and the CMAP from the 2nd interossei muscles (I) was recorded after ulnar nerve stimulation. The residual latency, which is arbitrary defined as the latency difference (L-N) in this study, was 1.38 +/- 0.15 (mean +/- SD) msec in healthy individuals. About 1 msec of the residual latency is regarded as the time for neuromuscular transmission and the time to evoke muscle activities. Thus, the conduction delay at the distal motor axon was calculated as about 0.4 msec in healthy individuals. The residual latency was relatively constant in 29 diabetic patients without conduction delay across the carpal tunnel, which was defined by the latency difference (L-I) < or = 0.4 msec. Their sensory nerve conduction velocities (calculated from N latency) were always above 40 m/sec. On the other hand in diabetic patients with conduction delay across the carpal tunnel, which was defined by the latency difference (L-I) > 0.4 msec, the residual latency gradually increased as the sensory nerve conduction velocity decreased. Their sensory nerve conduction velocities were mostly less than 40 m/sec. The similar relationship was observed in patients with carpal tunnel syndrome without diabetes mellitus. We consider that the diabetic neuropathy alone doesn't cause the increase of the residual latency. Instead, severe conduction delay across the carpal tunnel decreases the N velocity and increases the residual latency. We can also regard the relationship between the latency difference (L-N) and N velocity as being in inverse proportion. Perhaps the increase of the residual latency was simply caused by the proportional decrease in the conduction velocity at the distal motor axon, not by the special mechanism concerning to the carpal tunnel syndrome. This paper presented the electrophysiological changes seen in the distal segment secondary to the proximal entrapment.

[Innervation pattern to the extensor digitorum brevis by deep peroneal nerve and accessory deep peroneal nerve].

On the electrophysiological basis, extensor digitorum brevis(EDB) muscle is innervated electrophysiologically not only by deep peroneal nerve(DPN) but also by accessory deep peroneal nerve(ADPN), an anomalous branch of superficial peroneal nerve, with a prevalence of 17-28%. We investigated 23 patients who had both DPN and sufficient ADPN innervation to the EDB on the intramuscular distribution of DPN and ADPN innervation to the medial and lateral side of the EDB. Recording electrodes were placed on the medial and lateral edges of the EDB with a supramaximal stimulation to the anterior or lateral ankle, compound muscle action potential (CMAP) of DPN or ADPN innervation was recorded. In 19 patients (83%) the DPN innervation was larger than or equal to the ADPN innervation. Only in 4 patients (17%) the ADPN innervation obviously exceeded the DPN innervation. DPN enters to the EDB from the medial side, and ADPN from the lateral side of the EDB. In 16 patients(70%) the DPN innervation was relatively large and the ADPN innervation was relatively small at the medial side of the EDB, and vice versa at the lateral side of the EDB. These distributions were almost uniform in 5 patients(22%). This study clarified that a biased larger DPN innervation and smaller ADPN innervation to the medial side of the EDB, and vice versa to the lateral side of the EDB in the majority cases. In some cases diffuse innervation to the EDB was found.

[Polyneuropathy index-revised in the evaluation of diabetic neuropathy].

The polyneuropathy index-revised(PNI-R), based on 8 electrophysiological parameters(conduction velocities and F-latencies), was constructed to obtain an overall estimation of peripheral nerve conduction in diabetic patients, taking PNI as a model. PNI was calculated as a mean percentage of the normal on 12 velocity or latency parameters on motor nerve conduction studies. PNI-R is composed of 8 parameters; motor nerve conduction velocities in the forearm or leg segment and F-wave latencies after wrist or ankle stimulation concerning to the median, ulnar, peroneal and posterior tibial nerves. F-wave latencies were adjusted to 160 cm height and used reciprocals to compare with the normal values. Subjects were 101 patients with diabetes mellitus. Correlation of PNI-R or PNI with other parameters or indices on conventional sensory and intrafascicular conduction studies or items concerning to the diabetes mellitus were studied. Coefficient of correlation between PNI-R and PNI was as high as 0.97. The mean value of PNI-R was 0.6% smaller than PNI. This was presumably due to the greater influence of the peroneal parameters, weighted more in PNI-R than in PNI. Peroneal nerve is known to be sensitive to various neuropathies, and is often damaged independently. Each parameter composing PNI-R had a close relationship with PNI-R itself. Mutual independence between 8 parameters was considered to be enough. Among neuropathic signs Achilles tendon reflex in particular, and among diabetic complications retinopathy in particular, had a high degree of correlation with PNI-R. These results were identical both with PNI-R and PNI. We can save 20-30% of time in measuring PNI-R as compared to measure PNI, and the usefulness of PNI-R was as well as PNI. Therefore, using PNI-R as substitute for PNI is considered to be appropriate in the evaluation of diabetic polyneuropathy. Between parameters concerning to the median nerve F-wave latency correlated less with PNI-R than motor nerve conduction velocity in the forearm segment. Presumably this was owing to an unrecognized subclinical carpal tunnel syndrome, often observed in patients with diabetes mellitus. PNI-R will be an excellent index to express the function of peripheral nerve conduction, which can be retarded by the axonal degeneration in diabetes mellitus.

[Prevalence of Martin-Gruber anastomosis on motor nerve conduction studies].

Prevalence of median to ulnar anastomosis in the forearm(Martin-Gruber anastomosis; MGA) to the first dorsal interosseous(FDI), abductor digiti quinti (ADQ) and adductor pollicis(AP) was investigated. Subjects contained 106 patients with normal nerve conduction or patients with various neuropathies. Recording electrodes were placed on the motor point of FDI, ADQ and AP. Supramaximal stimulations were given to the median and ulnar nerves at the wrist or above the elbow. The diagnosis of MGA was made by the following criteria; amplitude of compound muscle action potential(CMAP) increased after elbow stimulation as compared with the wrist stimulation in median nerve conduction studies. The corresponding decrease in CMAP amplitude was found after above elbow stimulation as compared with the wrist stimulation in ulnar nerve conduction studies. No MGA was found in 80(75%) out of 106 patients. MGA to FDI was found in all 26 patients who had MGA. MGA to ADQ and AP was found in 11% and 10% of the patients, respectively. Only 8 out of 26 patients had MGA to all 3 muscles. In the presence of MGA median motor nerve conduction studies demonstrate larger CMAP, with a small initial positivity, after elbow stimulation than after wrist stimulation. And moreover, ulnar motor nerve conduction studies reveal a conduction block-like finding in the forearm. In this study MGA was found in 25% of the patient to FDI, in 11% to ADQ and in 10% to AP. Although a very small MGA might be overlooked in our method, such a small MGA doesn't mislead us into erroneous interpretation of motor nerve conduction studies.

Evaluation of distal and proximal axonal degeneration in patients with carpal tunnel syndrome.

In patients with carpal tunnel syndrome, varying degrees of demyelination and axonal degeneration occur in the median nerve. Only a few studies have examined axonal degeneration produced at proximal to the lesion. In this study proximal axonal degeneration was evaluated and compared with other parameters. In 40 consecutive CTS patient hands, distal latency (DL), compound muscle action potential amplitude (CMAP) and motor conduction velocity (MCV) were analyzed by conventional motor nerve conduction studies. Intrafascicular compound nerve action potential amplitude (N-CNAP) at the elbow after wrist simulation and its nerve conduction velocity (NCV) between wrist and elbow were also analyzed. The negative correlation of DL with CMAP was statistically significant (r = 0.577, p < 0.001). CMAP was correlated with either MCV (r = 0.537, p < 0.001) or N-CMAP (r = 0.710, p < 0.001). A significant correlation of MCV with NCV (r = 0.517, p < 0.001) was also indicated. There were no any other significant correlation among the parameters. In CTS the degree of demyelination and axonal degeneration influence the prognosis for nerve recovery after decompressive surgery. DL is mainly influenced by demyelination that results in conduction block and slowing at the carpal tunnel. CMAP and N-CNAP indicate the degree of axonal degeneration at distal and proximal to the compression site. As in electrophysiologic evaluation of mononeuropathies, proximal axonal degeneration is best assessed by both stimulation and recording electrode locationing proximal to the lesion. Recording of intrafascicular nerve action potential was a little invasive method, but it provided important informations. The negative correlation between DL and CMAP implies that distal axonal degeneration can occur in proportion to the conduction disturbance. Moreover, N-CNAP had a higher correlation with CMAP. The greater the distal axonal degeneration, the more the proximal axonal degeneration. Conduction velocity represents the velocity of the fastest conduction fiber, not the degree of axonal degeneration.

[Comparison between Dyck's criteria and the polyneuropathy index-revised (PNI-R) in the electrophysiologic evaluation of diabetic neuropathy].

In Rochester diabetic neuropathy research by Dyck et al., abnormal value in two or more nerves was introduced into the nerve conduction criteria of diabetic neuropathy. Polyneuropathy index-revised(PNI-R) is calculated as the mean percentage of the normal of 8 parameters on the motor nerve conduction studies. They were motor nerve conduction velocities in the forearm or leg segment and F-wave latencies after wrist or ankle stimulation concerning to the median, ulnar, peroneal and posterior tibial nerves. F-wave latencies were adjusted to 160 cm height and used reciprocals in comparison with normal values. To compare these two indices, first we obtained the normal limit(1st or 99th percentile value) of each parameter from the data of 62 healthy individuals. Then in 78 patients with diabetes mellitus number of abnormal nerves and the PNI-R were investigated. Abnormal values were frequently observed in the categories of motor nerve conduction velocities and F-wave latencies. Amplitude of compound muscle action potential (CMAP) or sensory nerve action potential(SNAP) in each nerve had a large standard deviation. In such parameters abnormal rate was extremely low, because the lower limit of normal being very small. Nevertheless, sigma CMAP which means the summation of amplitudes of 3 CMAPs had as high as 53% of abnormal rate. The coefficient of correlation between number of abnormal nerves and the value of PNI-R mounted up to -0.87. Instead, the coefficient of correlation of sigma CMAP or sigma SNAP, which means the summation of amplitudes of ulnar and sural SNAPs, with PNI-R were 0.65 and 0.79, respectively. In 14 patients PNI-R was normal and the number of abnormal nerves was 0 or 1. In 59 both categories were abnormal, and only in 5 they were not coincide. As to the clinical signs PNI-R had better correlation than number of abnormal nerves with vibration threshold or degree of Achilles tendon reflex. sigma CMAP is a convenient index to detect the existence and the degree of neuropathy. This index expresses the degree of neurogenic muscular atrophy, though it doesn't always advance parallel to the decrease in number of motor nerves. sigma SNAP had higher coefficient of correlation with PNI-R or number of abnormal nerves than sigma CMAP. In conclusion, abnormal PNI-R and abnormal value in two or more nerves are both useful and coincide with each other in the detection of diabetic neuropathy. The PNI-R is an excellent quantitative index, and the PNI-R corresponds well with the number of abnormal nerves. These observations indicate that the number of nerves with abnormal value is also available as a simple and semi-quantitative index of diabetic neuropathy.

[Nerve conduction velocity of repeater F-waves is identical to that of M-waves].

F-wave normally varies in latency and waveform from one response to the next. But the number of identical responses in a series of F-waves may be increased with neurogenic atrophy consistent with a decreased number of motoneurons capable of responding to antidromic stimulation. They are called "repeater F-waves". We herein demonstrate some repeater F-waves observed in three patients with moderate or slight diabetic polyneuropathy. In their motor nerve conduction studies on the peroneal nerve the maximum conduction velocity was 33 m/sec in patient 1, 36 m/sec in patient 2 and 48 m/sec in patient 3. A total of 6 delayed indirect potentials were repeatedly evoked after nerve trunk stimulation. They fulfilled the characteristics of F-wave. Their conduction velocities in the leg segment were 27, 26, 23 m/sec in patient 1, 34, 33 m/sec in patient 2 and 46 m/sec in patient 3. Repeater F-waves are occasionally observed in patients with amyotrophic lateral sclerosis, cervical spondylosis or entrapment neuropathies, in which the number of motoneuron is decreased. In diabetic polyneuropathy some repeater F-waves were also observed in patients not only with moderate to severe neuropathy but also with normal nerve conduction. F-waves are generated by an antidromic backfiring of motor neurons, and they occur preferentially in large motor neurons. Larger motor neurons inhibit smaller axons through the activation of Renshaw cells. In our 3 patients conduction velocities of the repeated F-waves were all identical to the main component of M-wave. These observations reconfirmed the hypothesis that relatively large motor neurons generating F-waves are preferentially activated also in repeater F-waves.

[Sensory nerve action potentials in the evaluation of diabetic polyneuropathy].

In order to clarify the suitability of sensory nerve action potential(SNAP) in the evaluation of diabetic polyneuropathy, we studied measurements of SNAPs in the median, ulnar and sural nerves. Subjects were 253 patients with non-insulin dependent diabetes mellitus; 167 men and 86 women, aged 58.2 +/- 12.8(mean +/- SD) years old. Their diabetic history was 10.2 +/- 8.6 years. SNAPs were recorded antidromically from index finger, little finger and lateral to the Achilles tendon, respectively. Twenty-eight patients, in whom any one of the SNAPs couldn't be obtained, were already excluded from this study. The polyneuropathy index (PNI) was calculated from 12 indices concerning to the velocity or long distance latency in motor nerve conduction studies of 4 nerves. The PNI is known to be an excellent index to express the degree of diabetic polyneuropathy. Amplitude and conduction velocity in each nerve was 28.6 +/- 15.6 microV and 46.2 +/- 7.4 m/sec in the median nerve, 26.7 +/- 15.8 microV and 47.0 +/- 6.5 m/sec in the ulnar nerve, 13.1 +/- 6.5 microV and 43.1 +/- 6.0 m/sec in the sural nerve, respectively. The coefficient of correlation of the measurements between median and ulnar nerves was larger than other assortment of nerves. The coefficient of correlation of each measurement with PNI was around 0.40 in the amplitude and around 0.55 in the conduction velocity. Nevertheless, the mean value of the 3 nerves had a higher coefficient of correlation with PNI; 0.48 in the amplitude and 0.60 in the conduction velocity. SNAP measurements of a single nerve are often largely affected by the inter-individual differences, inter-nerve differences or measuring errors. But the mean value of the 3 nerves will be better in exploring the degree of diabetic polyneuropathy. Evaluation of diabetic polyneuropathy by SNAPs will be best achieved by using the mean value of these 3 nerves.

A combination of vermiculite and paper pulp supporting material for the photoautotrophic micropropagation of sweet potato.

A mixture of vermiculite (hydrous silicates) and paper pulp (waste product of paper industry) was used as a supporting material for the in vitro photoautotrophic micropropagation of plantlets. Sweet potato was used as a model plant to find out the appropriate proportion of vermiculite and paper pulp for the optimum growth of the plantlets. The plantlets grown in the conventional supporting material, agar, were used as the control. The study revealed that in all aspects, the plantlets grown in vermiculite mixed with 30% (w/w) paper pulp exhibited the highest growth performance. The shoot and root fresh mass were x2.7 greater than those in agar (control); the leaf, stem and root dry mass were also greater and at least two fold in this treatment compared with those in the control. The net photosynthetic rate per plantlet was highest in this treatment, and on day 20 it was 15.3 µmol CO(2) h(-1) as compared with 9.8 µmol CO(2) h(-1) in the control. The growth of both shoots and roots decreased gradually with the increase or decrease of percentage of paper pulp in the supporting material. In general, the growth was significantly poorer in the plantlets grown in 100% vermiculite than that in vermiculite mixed with 30% paper pulp but still greater than in the control. The porosity of the supporting materials increased with the increase in the percentage of paper pulp in the supporting material. After transplanting to the ex vitro condition the survival percentage did not vary significantly (90-100%) among the treatments, except in control where it was only 73%. The number of unfolded leaves and the stem height were similar among the treatments except those in the control.

Visual event-related potentials in progressive supranuclear palsy, corticobasal degeneration, striatonigral degeneration, and Parkinson's disease.

To determine whether there are characteristic changes in event-related potentials (ERPs) in parkinsonian syndromes we studied 8 patients with progressive supranuclear palsy (PSP), 10 patients with corticobasal degeneration (CBD), 9 patients with striatonigral degeneration (SND), and 16 patients with idiopathic Parkinson's disease (PD) with a mean duration of illness shorter than 5 years in each group. A visual oddball paradigm was employed to elicit P300. P300 to the rare target and rare nontarget stimuli and reaction time (RT) to rare target stimuli in each group were compared with those in the corresponding age-matched normal control group and to each other after age correction. The correlation of P300 and RT to motor disability score was also studied. In PSP P300 amplitude was markedly reduced while in CBD P300 latency was prolonged. P300 amplitude to rare nontargets in SND and PD was attenuated. The mean RT in the PSP and the CBD group was significantly longer than in the other two groups. The mean RT in PD and P300 amplitude to rare nontargets in both CBD and PD showed significant correlation with the severity of motor disability. Simultaneous measurement of P300 and RT may yield useful supplementary information in facilitating diagnosis of parkinsonian syndromes in addition to clinical criteria.