The effects of nucleotide usage in key nucleotide positions +4 and -3 flanking start codon on translation levels mediated by IRES of hepatitis C virus.

Internal ribosomal entry site (IRES) functions as a cis-acting RNA element, which drives an alternative and cap-independent translation initiation pathway. Currently, there are few studies on effects of nucleotide usages at key nucleotide positions +4 and -3 flanking start codon mediated by IRES of hepatitis C virus (HCV). Herein, we focus on the effect of nucleotide usages at -3 and +4 positions mediated by HCV IRES. The nucleotide contexts flanking AUG start codon employed by HCV IRES is firstly analyzed. We found that each position in the six nucleotide positions (-4 to +6) flanking start codon of HCV has a strong tendency to select the specific nucleotide. A set of bicistronic expression vectors containing CAT gene, HCV IRES and EGFP gene were constructed, including 16 different nucleotide combinations at position -3 and +4. Each set, in which nucleotide at the -3 and +4 position has been changed into different nucleotides, included 16 types of bicistronic expression vectors. It was found that the purine nucleotide at the position -3 or +4 obviously impacts on HCV IRES-related expression, and IRES-driven translation is potentially influenced by the Kozak rule. Our results suggest that optimization of nucleotides at positions -3 and +4 is a convenient and efficient way to enhance the level of IRES-mediated translation. Keywords: Cap-independent translation; internal ribosomal entry site; hepatitis C virus; bicistronic expression vector; translation efficiency.

Effect of manual acupuncture and transcutaneous electrical nerve stimulation on the H-reflex.

Several studies find that manual acupuncture and transcutaneous electrical nerve stimulation (TENS) are via different mechanisms and generate different effect on the central nervous system, therefore, the aim of the present study was to compare the effect of manual acupuncture (MA) and TENS on the spinal cord using H-reflex recordings. A total of 13 healthy adult volunteers were studied. The electrical stimuli were delivered to the posterior tibial nerve transcutaneously at the left popliteal fossa to evoke the soleus H-reflex. MA, 2Hz TENS, 100 Hz TENS, respectively, was applied to the surface of the right first dorsal interosseous muscle exactly at the Hegu acupoint (LI.4). Four assessments were performed randomly, separated by an interval of at least three days in all the subjects as follows: 1) Control assessment: a pair of electrodes placed on the surface of the right Hegu acupoint (LI.4), but no electrical stimulation was delivered throughout the test; 2) MA assessment: MA was done at the right Hegu acupoint (LI.4) for 15 minutes; 3) TENS assessment at 2Hz: electrical stimulation (40 mA in intensity) at 2Hz was applied to the surface of the right Hegu acupoint (LI.4) for 15 minutes; 4) TENS assessment at 100 Hz: electrical stimulation (20 mA in intensity) at 100 Hz was applied to the surface of the right Hegu acupoint (LI.4) for 15 minutes. Each assessment was divided into three periods as follows: 1) Baseline period: H-reflexes recorded prior to MA or TENS; 2) TENS period: six H-reflex recordings after MA or TENS for a duration of 4-5 min, 9-10 min and 14-15 min, respectively; 3) Post TENS period: H-reflex recordings of 6 after TENS period 4-5 min and 9-10 min, respectively. Our results indicate that both 2Hz TENS and 100 Hz TENS increased the amplitude of the H-reflex, and that these increases may be retained longer with 100 Hz TENS than with 2 Hz TENS, whereas MA could not increase the amplitude of the H-reflex. MA, TENS at 2 Hz or 100 Hz didn't change the latencies of the H-reflexes. We conclude that both 2Hz and 100 Hz TENS increased the amplitude of the H-reflex, suggesting that TENS enhances the excitability of the motoneuron pool in the spinal cord, and 100 Hz TENS has a greater effect than 2Hz TENS, whereas MA was not similar effect to TENS on spinal cord.

Changes of pulse rate and skin temperature evoked by electroacupuncture stimulation with different frequency on both Zusanli acupoints in humans.

The purpose of this study was to investigate the effect of electroacupuncture stimulation (EA) of different frequency on pulse rate and skin temperature. Sixteen healthy male medical student volunteers received EA of 2 Hz, and 100 Hz, respectively on the both Zusanli acupoints (St-36) while resting. Their pulse rates were measured on the middle finger, and skin temperature was taken between the thumb and index finger before, during, and after EA stimulation. Each test took 35 minutes. The initial 10 min were defined as baseline period (no EA), the following 15 min as the EA period and the last 10 min as the post-EA period. Three assessments were performed on each subject as follows: A) control assessment: no EA was done throughout the test; B) 2 Hz EA assessment: 2 Hz EA was applied to both Zusanli acupoints during the EA period; and C) 100 Hz EA assessment: 100 Hz EA was applied to both Zusanli acupoints during the EA period. Our results indicate that both 2 Hz EA and 100 Hz EA decreased pulse rates during the EA period, and these changes remained throughout the post-EA period in 2 Hz EA assessment, but not in 100 Hz EA assessment. Both 2 Hz and 100 Hz EA resulted in decreases of skin temperature during the EA period. Our conclusions are that 2 Hz EA and 100 Hz EA applied to both Zusanli acupoints resulted in the decrease of pulse rate, which possibly evoked greater parasympathetic nerve activity on heart beats. 2 Hz EA had a more sustained effect on heart beats than 100 Hz EA. Decreased skin temperatures in the EA period may have resulted from cutaneous vasoconstriction caused by EA induced sympathetic stress response, suggesting EA at least remains for 15 min in clinical application.

Cerebral cortex participation in the physiological mechanisms of acupuncture stimulation: a study by auditory endogenous potentials (P300).

Although acupuncture has traditionally used the acupoints formula to treat diseases, the physiological mechanisms involved and the effectiveness of therapy remain unclear. This study investigated the physiological mechanism(s) and response to acupuncture stimulation using the acupoints formula. Scalp-recorded potentials P300 were evoked by auditory stimulation of non-target and target in 13 normal adult volunteers. Latencies and amplitudes were measured. Three assessments were performed in each subject over a period of at least one week. Each assessment was divided into a control period with no acupuncture stimulation, followed by an acupuncture period and then a post-acupuncture period. Acupuncture needles were inserted into the body as follows: 1) non-acupoint: acupuncture needles were inserted 2 cm lateral to both Zusanli acupoints; 2) acupoint: acupuncture needles were inserted into both Zusanli acupoints; 3) acupoints formula: acupuncture needles were inserted into both Zusanli and Shousanli acupoints. Our results showed that both acupoint and acupoints formula assessments resulted in a significant decrease of P300 amplitudes during the acupuncture and post-acupuncture periods. However, there was significant difference in P300 amplitudes in the non-acupoint assessment during these periods. P300 changes in latencies and amplitudes were not significantly different between the acupoint assessment and the acupoints formula assessment. We concluded that acupuncture stimulation of both Zusanli acupoints resulted in a decrease of P300 amplitudes, suggesting the involvement of the cerebral cortex in sensory interaction when simultaneous sensations of the two types are received. No similar changes were observed in the non-acupoint assessment, which have been suggested to be related to so-called acupoint specificity. Results obtained using the acupoints formula were not significantly different from those using acupoints alone. These findings suggested that neuropsychological effects from stimulation of Zusanli acupoints and Shousanli acupoints are different.