Mechanisms underlying immobilization-induced muscle pain in rats.

INTRODUCTION: We investigated the mechanisms underlying immobilization-induced muscle pain in rats. METHODS: In rat skeletal muscle, pressure pain threshold (PPT) of the gastrocnemius muscle was measured, and nerve growth factor (NGF) level, peripheral nerve fiber density, macrophage number, and interleukin-1ß (IL-1ß) mRNA expression were examined. An NGF receptor inhibitor was injected intramuscularly to assess the relationship between PPT and NGF levels. RESULTS: Immobilization resulted in a decrease in PPT and increases in NGF level, C-fiber density, M1 macrophage number, and IL-1ß mRNA expression. Injection of NGF receptor inhibitor reversed the decrease in PPT. DISCUSSION: NGF upregulation may be a major contributor to immobilization-induced muscle pain. The increases in C-fiber density, M1 macrophage number, and IL-1ß mRNA expression may be related to immobilization-induced muscle pain.

ACBD3-mediated recruitment of PI4KB to picornavirus RNA replication sites.

Phosphatidylinositol 4-kinase IIIß (PI4KB) is a host factor required for genome RNA replication of enteroviruses, small non-enveloped viruses belonging to the family Picornaviridae. Here, we demonstrated that PI4KB is also essential for genome replication of another picornavirus, Aichi virus (AiV), but is recruited to the genome replication sites by a different strategy from that utilized by enteroviruses. AiV non-structural proteins, 2B, 2BC, 2C, 3A, and 3AB, interacted with a Golgi protein, acyl-coenzyme A binding domain containing 3 (ACBD3). Furthermore, we identified previously unknown interaction between ACBD3 and PI4KB, which provides a novel manner of Golgi recruitment of PI4KB. Knockdown of ACBD3 or PI4KB suppressed AiV RNA replication. The viral proteins, ACBD3, PI4KB, and phophatidylinositol-4-phosphate (PI4P) localized to the viral RNA replication sites. AiV replication and recruitment of PI4KB to the RNA replication sites were not affected by brefeldin A, in contrast to those in enterovirus infection. These results indicate that a viral protein/ACBD3/PI4KB complex is formed to synthesize PI4P at the AiV RNA replication sites and plays an essential role in viral RNA replication.

Reproducibility in the global indices for multifocal visual evoked potentials and Humphrey visual fields in controls and glaucomatous eyes within a 2-year period.

PURPOSE: In previous studies, we applied receiver operating characteristic curve analysis to the signal-to-noise ratio distributions in the signal and noise windows of multifocal VEP (mfVEP) response. The areas under the curve thus obtained (SNR-AUC) were found to quantitatively detect glaucomatous visual field damage. The present study evaluated the reproducibility of SNR-AUC and the Humphrey visual field (HVF) global indices in 37 eyes with primary open angle glaucoma (POAG; POAG group) and in 30 controls (control group) within a 2-year period. METHODS: The HVF SITA standard 24-2 and mfVEP were recorded at three separate sessions for each individual. The intersession variability for SNR-AUC, mean deviation (MD), and pattern standard deviation (PSD) was evaluated using the repeated measures of analysis of variance and Bland-Altman plots. The logarithmically converted coefficients of variation (CV) of PSD and SNR-AUC were compared between the control and POAG groups. Linear regression analyses were performed on the logarithmic CV of SNR-AUC against the average MD, PSD, and SNR-AUC. RESULTS: SNR-AUC in the POAG group was significantly lower and its CV was greater compared with the control group (P < 0.0001). MD value recorded at the third visit had significantly improved than that at the first visit in the control group (analysis of variance, P = 0.03), whereas PSD value was significantly worse in the POAG group (P = 0.024). In the POAG group, SNR-AUC CV increased as the glaucoma stage became more advanced when evaluated by any functional parameters tested (i.e., MD, PSD, or SNR-AUC). CONCLUSIONS: The SNR-AUC of mfVEP showed a high reproducibility in control group, whereas it fluctuated more in the POAG group according to the disease severity. MD in the control group and PSD in POAG group fluctuated among sessions during the 2-year period.

Effect of refractive errors on multifocal VEP responses and standard automated perimetry tests in a single population.

BACKGROUND: We have previously reported that the degree of signal-to-noise ratio (SNR) distribution overlaps between a signal window and a noise window in multifocal VEP (mfVEP) responses, which is determined by the area under the receiver-operating characteristic curve termed SNR-AUC, can quantitatively detect glaucomatous visual functional damage. However, the effect of high myopia on this parameter is not yet known. METHODS: SNR-AUC, total deviation, and retinal sensitivity on the Humphrey visual field (HVF) test were compared among 34 eyes>-6 diopters (control) and 21 eyes≤-6 diopters (high myopia), both of which were ophthalmoscopically normal and had a best-corrected visual acuity of 20/20. The mfVEP and HVF parameters were obtained from stimulus areas that corresponded to both HVF 24-2 and 10-2 programs. RESULTS: Both the HVF 24-2 total deviation and the SNR-AUC obtained from 60 sectors in high-myopia patients were significantly lower compared with controls (P=0.045 and P=0.003, respectively). The SNR-AUC obtained from the central 36 sectors that corresponded to the HVF 10-2 area in high-myopia patients was also significantly lower than that of the controls (P=0.01). Multiple regression analyses demonstrated that age and refractive error were significantly associated with retinal sensitivity on the HVF 24-2 and SNR-AUC for the whole field and central field, respectively. CONCLUSIONS: High myopia reduces the SNR-AUC of mfVEP responses, even with refractive correction. A normative database should be separately established for high myopes to evaluate the mfVEP responses obtained from highly myopic glaucoma patients.

Human protein factory for converting the transcriptome into an in vitro-expressed proteome,.

Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.

Receiver-operating characteristic analysis of multifocal VEPs to diagnose and quantify glaucomatous functional damage.

To test whether multifocal visual evoked potential (mfVEP) recording using two perpendicularly placed channels, as previously reported, to measure the degree of signal-to-noise ratio (SNR) distribution overlap between a signal window and a noise window would efficiently detect and quantify glaucomatous damage. Humphrey visual field (HVF) and mfVEP were recorded from 56 patients with primary open-angle glaucoma and mean deviation less than -15 dB and 62 age-matched ophthalmologically normal individuals. Areas under the receiver-operating characteristic curve (SNR-AUC) were calculated based on the proportion of mfVEP responses that exceeded a specific SNR criterion for both windows. Abnormal sectors with an SNR deviated from the previously established norm with P<5% and 1% were counted. Diagnostic accuracy of the SNR-AUC was similar to that of the average total deviation (TD) of the HVF. The hemifield agreement to detect a defect in mfVEP and HVF was 77.1-87.3%, which was similar to previous reports using multiple channels. Correlation coefficients between SNR-AUC and average TD (0.74 in the upper hemifield and 0.65 in the lower) were significantly higher than those between the sums of abnormal locations on the mfVEP and HVF probability plots (0.27 and 0.33, respectively). Two perpendicular channels can detect and quantify functional damage due to glaucoma. The SNR-AUC may be used as a global index to quantify diffuse glaucomatous functional loss.

Optimal conditions for multifocal VEP recording for normal Japanese population established by receiver operating characteristic analysis.

The purpose of this study was to establish optimal conditions for recording multifocal visual evoked potentials (mVEPs) in Japanese individuals, whose skull frame presumably differs from Caucasians. The scalp point that was extended from the calcarine fissure was identified using magnetic resonance imaging scans of 200 subjects. MVEPs were recorded from 56 individuals using three single channels and combinations of vertical and horizontal channels. Five electrodes were placed at the inion, 4 cm above the inion, 2.5 cm below the inion, 4 cm to the left or 4 cm to the right of the inion. The signal-to-noise ratio (SNR) was obtained by measuring the root-mean-square (RMS) amplitude of a signal window (45-150 ms) from each of 60-local responses that was divided by the average of the 60 RMS amplitudes of the noise window (325-430 ms). Receiver operating characteristic (ROC) analyses were performed based on the proportion of mVEP responses that exceeded a specific SNR criterion, calculated for both the signal window and the noise window. The position of the calcarine fissure relative to the inion was significantly lower than the value reported for Caucasians. The ROC analyses disclosed that bi-channel combinations (one vertical and one horizontal) had significantly better performance to discriminate signal from noise in 60-local mVEP responses compared to any single channel and performed similarly to the tri-channel combination. Two sets of perpendicular channels should be simultaneously used in recording mVEP responses from Japanese people, among whom skull frame characteristics differ from those observed in Caucasians.

Voluntary Forelimbs Exercise Reduces Immobilization-Induced Mechanical Hyperalgesia in the Rat Hind Paw.

Voluntary exercise is sufficient to protect against neuropathic pain. However, it is unclear whether voluntary exercise reduces immobilization-induced hyperalgesia. We examined the effect of voluntary forelimb exercise on immobilized-induced hyperalgesia in hind paws of rats. Wistar rats were randomly divided into the (1) both hind limbs immobilized group (IM group), (2) immobilization and exercise with nonimmobilized fore limbs group (EX group), and (3) control group. In the IM and EX groups, the bilateral ankle joints of each rat were immobilized in full plantar flexion with a plaster cast for eight weeks. In the EX group, voluntary exercise using nonimmobilized forelimbs in the running wheel was administered during the immobilization period, while hind limbs were kept immobilized (60 min/day, 5 days/week). Mechanical hyperalgesia in the hind paw was measured using a digital von Frey device every week. To investigate the abnormality of primary sensory neurons and central sensitization, the number of calcitonin gene-related peptide-positive cells in the dorsal root ganglion and the expression level of calcitonin gene-related peptide in the spinal dorsal horn were analyzed by immunohistochemical staining. Immobilization-induced mechanical hyperalgesia was inhibited in the EX group compared to the IM group at three weeks after immobilization. In the EX group, the number of calcitonin gene-related peptide-positive cells in the dorsal root ganglion and the expression level of calcitonin gene-related peptide were significantly decreased compared to those in the IM group. Our results therefore suggest that voluntary forelimb exercise during hind limb immobilization partially reduces immobilization-induced hyperalgesia by suppressing that the plastic changes of the primary sensory nerves that excessively transmit pain and increased responsiveness of nociceptive neurons in the spinal dorsal horn.

[Influence of commercial soft drinks or green tea intake to occult blood and sugar tests with urinalysis reagent strips].

Consumption of commercial soft drinks or tea has increased in over recent years. In many case, these drinks contains large amount of ascorbic acid as an antioxidant. We examined the influence of ascorbic acid to the result of urine occult blood and sugar tests with urinalysis reagent strips. Three subjects took 3 brands of soft drinks (P, C, B) in 3 different days once in the morning. Other 3 subjects took 4 brands of green tea in 4 different days 4 times in a day. After 2 hours of first administration, urine specimen were collected every hour for 8-10 hours. Constant amount of hemoglobin or sugar was added in urine specimen and the reflectance was measured with AX-4280. As a results, urine ascorbic acid induced false negative results in both occult blood and sugar tests in all groups. These results indicate the necessity of patient education about commercial soft drinks or green tea consumption on the previous night and the day of the urinalysis.

Knockdown of XAB2 enhances all-trans retinoic acid-induced cellular differentiation in all-trans retinoic acid-sensitive and -resistant cancer cells.

Xeroderma pigmentosum group A (XPA)-binding protein 2 (XAB2) is composed of 855 amino acids, contains 15 tetratricopeptide repeat motifs, and associates with Cockayne syndrome group A and B proteins and RNA polymerase II, as well as XPA. In vitro and in vivo studies showed that XAB2 is involved in pre-mRNA splicing, transcription, and transcription-coupled DNA repair, leading to preimplantation lethality, and is essential for mouse embryogenesis. Retinoids are effective for the treatment of preneoplastic diseases including xeroderma pigmentosum and other dermatologic diseases such as photoaging. We therefore focused on defining the effect of XAB2 on cellular differentiation in the presence of ATRA treatment. In the present study, we showed that overexpression of XAB2 inhibited ATRA-induced cellular differentiation in human rhabdomyosarcoma cell line, and that knockdown of XAB2 by small interfering RNA (siRNA) increased ATRA-sensitive cellular differentiation in the human promyelocytic leukemia cell line HL60 at both physiologic (10(-9)-10(-8) mol/L) and therapeutic (10(-7) mol/L) concentrations of ATRA. Moreover, we found that XAB2 was associated with retinoic acid receptor alpha (RARalpha) and histone deacetylase 3 in the nuclei. Finally, using siRNA against XAB2, we showed that the ATRA-resistant neuroblastoma cell line IMR-32 underwent cellular differentiation induced by ATRA at a therapeutic concentration (10(-6) mol/L). These results strongly suggest that XAB2 is a component of the RAR corepressor complex with an inhibitory effect on ATRA-induced cellular differentiation and that XAB2 plays a role in ATRA-mediated cellular differentiation as an important aspect of cancer therapy.

Low-intensity muscle contraction exercise following the onset of arthritis improves hyperalgesia via reduction of joint inflammation and central sensitization in the spinal cord in a rat model.

We examined the effect of immobilization, low-intensity muscle contraction exercise, and transcutaneous electrical nerve stimulation (TENS) on tissue inflammation and acute pain following the onset of arthritis in a rat model. Sixty Wistar rats were divided into five groups: (1) Arthritis group, (2) arthritis and immobilization (Immobilization group), (3) arthritis and low intensity muscle contraction (Exercise group), (4) arthritis and TENS (TENS group), and (5) sham arthritis (Sham group). Arthritis was induced in the right knee joints by single injection of 3% kaolin and carrageenan. Immobilization of the right hindlimb was conducted by full extension of the right knee joints and full plantar flexion of the ankle joints using a plaster cast for 7 days after injection. The right quadriceps muscles were subjected to electrical stimulation (frequency: 50 Hz; intensity: 2-3 mA) for 20 min/day as contraction exercise for one week. TENS was delivered at 20 min/day for one week (frequency: 50 Hz; intensity: 1 mA). The pressure pain threshold (PPT) and paw withdrawal response (PWR) were evaluated at 1 and 7 days after injection. We also analyzed the number of CD68-positive cells in the synovium by immunohistochemistry and determined the expression level of calcitonin gene-related peptide (CGRP) in the spinal dorsal horn with immunofluorescence. Improvements of both PPT and PWR were observed in the Exercise group at 7 days after injection compared to those of the Arthritis and Immobilization groups, although only improvement of PPT was observed in the TENS group. The number of CD68-positive cells in the synovium and CGRP expression in the dorsal horn decreased only in the Exercise group. These results suggested that low-intensity muscle contraction exercise might be a better treatment for reduction of arthritis-induced inflammation and acute pain compared to immobilization and TENS.

The effect of donor leukocyte infusion on refractory pure red blood cell aplasia after allogeneic stem cell transplantation in a patient with myelodysplastic syndrome developing from Kostmann syndrome.

We describe the clinical course of a patient who experienced refractory pure red cell aplasia (PRCA) after undergoing HLA-matched allogeneic peripheral blood stem cell transplantation (allo-PBSCT) for refractory anemia with an excess of blasts in transformation that had evolved from Kostmann syndrome. The treatment for patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) developing from Kostmann syndrome has not been standardized. We treated this patient with allo-PBSCT using a regimen combining high-dose cytosine arabinoside with granulocyte colony-stimulating factor, in addition to total body irradiation and cyclophosphamide without preceding intensive chemotherapy. The donor was ABO incompatible. Myeloid and platelet recoveries were achieved rapidly. Erythroid engraftment was not evident, however, and the patient was given a diagnosis of PRCA. Regimen-related toxicity and graft-versus-host disease (GVHD) were limited. The PRCA did not respond to various therapies, including the discontinuation of immunosuppressants for the induction of chronic GVHD, human recombinant erythropoietin, immunosuppressive treatment with steroids, cyclosporin A, and human anti-CD20 antibody (rituximab). The patient received transfusions 48 times until the resolution of his anemia by donor leukocyte infusion (DLI) at 25 months after PBSCT. He is now clinically well (performance status, 100%) with normal blood cell counts at 5 years after SCT. An in vitro study demonstrated that serum from the recipient blocked the differentiation of erythroid cells in the bone marrow. The results indicate that the conditioning regimen we describe seems safe and effective for those who have MDS/AML and that DLI might be a valuable approach for refractory PRCA after ABO-incompatible SCT.

A novel high-throughput (HTP) cloning strategy for site-directed designed chimeragenesis and mutation using the Gateway cloning system.

There is an increasing demand for easy, high-throughput (HTP) methods for protein engineering to support advances in the development of structural biology, bioinformatics and drug design. Here, we describe an N- and C-terminal cloning method utilizing Gateway cloning technology that we have adopted for chimeric and mutant genes production as well as domain shuffling. This method involves only three steps: PCR, in vitro recombination and transformation. All three processes consist of simple handling, mixing and incubation steps. We have characterized this novel HTP method on 96 targets with >90% success. Here, we also discuss an N- and C-terminal cloning method for domain shuffling and a combination of mutation and chimeragenesis with two types of plasmid vectors.

Novel ENU-Induced Mutation in Tbx6 Causes Dominant Spondylocostal Dysostosis-Like Vertebral Malformations in the Rat.

Congenital vertebral malformations caused by embryonic segmentation defects are relatively common in humans and domestic animals. Although reverse genetics approaches in mice have provided information on the molecular mechanisms of embryonic somite segmentation, hypothesis-driven approaches cannot adequately reflect human dysmorphology within the population. In a N-ethyl-N-nitrosourea (ENU) mutagenesis project in Kyoto, the Oune mutant rat strain was isolated due to a short and kinked caudal vertebra phenotype. Skeletal staining of heterozygous rats showed partial loss of the cervical vertebrae as well as hemivertebrae and fused vertebral blocks in lumbar and sacral vertebrae. In homozygous embryos, severe displacement of the whole vertebrae was observed. The Oune locus was genetically mapped to rat chromosome 1 using 202 backcross animals and 50 genome-wide microsatellite markers. Subsequently, a miss-sense mutation in the Tbx6 gene was identified in the critical region. Although the mutation is located within the T-box domain near a predicted dimmer-interface, in vitro experiments revealed that the Tbx6 variant retains normal DNA binding ability and translational efficiency. However, the variant has decreased transcriptional activation potential in response to Notch-mediated signaling. Recently, it was reported that a dominant type of familial spondylocostal dysostosis is caused by a stoploss mutation in TBX6. Thus, we propose that partial dysfunction of Tbx6 leads to similar congenital vertebral malformations in both humans and rats. The Oune strain could be a unique animal model for dominant spondylocostal dysostosis and is useful for molecular dissection of the pathology of congenital vertebral malformations in humans.

Early pathogenesis of cardiac amyloid deposition in senile systemic amyloidosis: close relationship between amyloid deposits and the basement membranes of myocardial cells.

Despite a number of in vitro studies of transthyretin (TTR) amyloidogenesis the early stage of in vivo amyloidogenesis in the human heart is largely unknown. A heart with a mild degree of cardiac amyloidosis removed from a 90-year old woman at autopsy was selected for analysis. The genotype of the TTR was the wild type. An immunohistochemical study with anti-TTR antibody was performed on serial paraffin sections, and 17 TTR-positive lesions less than 50 micro m in diameter consisting of 13 interstitial and 4 vascular lesions were identified. The early interstitial lesions start as thick membranous deposits between interfacing myocardial cells. They are Congophilic with green birefringence and positive for apolipoprotein E but negative for amyloid P component. The TTR-positive amyloid extends along intercellular spaces and becomes larger, involving several myocardial fibers. The media is the initial site of arteriolar involvement. According to the in vitro studies of amyloid fibrillogenesis, the most critical step is formation of the nucleus under supersaturated conditions. The supersaturated conditions are speculated to be achieved by binding to proteoglycans or lipid membranes. Our results indicate that the basement membrane of myocardial cells is the initial site of amyloid deposition, providing a suitable place for concentration of TTR.

Viral Infections in Juvenile Myelomonocytic Leukemia: Prevalence and Clinical Implications.

OBJECTIVES: Viral infections may complicate the diagnosis of juvenile myelomonocytic leukemia (JMML) in a substantial proportion of patients, but this possibility has not been tested in a prospective study. The authors therefore measured the cellular expression of the MxA protein, a reliable marker of viral infection, at diagnosis in children with JMML to estimate the prevalence of such infections. METHODS: Eighteen children, aged 1 to 69 months, who met the diagnostic criteria of the International JMML Working Group were prospectively studied. MxA expression was assessed by flow cytometric analysis of peripheral blood mononuclear cells stained with an antihuman MxA antibody. All data were obtained through the MDS Committee of the Japanese Society of Pediatric Hematology. RESULTS: Twelve patients (67%) had elevated levels of the MxA protein, with rotavirus, RS virus, or CMV infection documented in three of these patients. Although none of the patients had primary Epstein-Barr virus (EBV) infection, reactivation of the virus was strongly suspected in four children, including two with monosomy 7, each having increased levels of MxA. Southern blot analysis revealed monoclonal integration of the EBV genome into bone marrow mononuclear cells from one of these patients. There was no discernible correlation between increases in the marker protein and the presenting features or course of the disease. CONCLUSIONS: Viral infection may be present in two thirds of children with newly diagnosed JMML, but it does not constitute a basis for revising clinical management. The possibility that EBV or other viruses contribute to JMML pathogenesis by stimulating pre-exiting malignant clones warrants further investigation.

Photocontrol of mitotic kinesin Eg5 facilitated by thiol-reactive photochromic molecules incorporated into the loop L5 functional loop.

Kinesin Eg5 is a plus-end-directed microtubule-based motor that is essential for bipolar spindle formation during eukaryotic cell division. Loop L5 of mitotic kinesin Eg5 is a key region determining ATPase activity and motor function. Photochromic molecules undergo reversible isomerization in response to ultraviolet and visible light irradiation. We introduced three kinds of photochromic molecules, 4-phenylazomaleinanil (PAM), 4-(N-(2-iodoacetyl)amino)-4'-(N-(2-(N-(triphenylmethyl)amino)acetyl)amino)azobenzene (IATAB) and 3,3-dimethyl-1-(2-(2-iodoacetoxy)ethyl)-3H-1,2-dihydroindole-2-spiro-2'-(2H)-6'-nitrochromene (IASP) into L5 to control the Eg5 ATPase activity using light irradiation. We prepared five kinesin Eg5 motor domain mutants, E116C, E118C, Y125C, W127C and D130C, which contained a single reactive cysteine residue in loop L5. The ability of S-trityl-l-cysteine (STLC), a specific Eg5 inhibitor, to inhibit E116C, W127C and D130C was significantly reduced. The photochromic molecules were stoichiometrically incorporated into the cysteine residues in L5 of mutants. W127C and D130C modified with IASP exhibited reversible ATPase activity alterations when subjected to light irradiation-induced photoisomerization. The two IASP modified mutants also demonstrated photocontrolled alterations following treatment with STLC. Additionally, the ATPase activity of the mutant D130C modified with PAM could be photocontrolled. Our findings demonstrate that incorporation of photochromic molecules into the key region of loop L5 facilitates the photocontrol of the function of kinesin Eg5.

Photocontrol of the mitotic kinesin Eg5 using a novel S-trityl-L-cysteine analogue as a photochromic inhibitor.

Because the mitotic kinesin Eg5 is essential for the formation of bipolar spindles during eukaryotic cell division, it has been considered as a potential target for cancer treatment. A number of specific and potent inhibitors of Eg5 are known. S-trityl-L-cysteine is one of the inhibitors of Eg5 whose molecular mechanism of inhibition was well studied. The trityl group of S-trityl-L-cysteine was shown to be a key moiety required for potent inhibition. In this study, we synthesized a novel photochromic S-trityl-L-cysteine analogue, 4-(N-(2-(N-acetylcysteine-S-yl) acetyl) amino)-4'- (N-(2-(N-(triphenylmethyl)amino)acetyl)amino)azobenzene (ACTAB), composed of a trityl group, azobenzene and N-acetyl-L-cysteine, which exhibits cis-trans photoisomerization in order to photocontrol the function of Eg5. ACTAB exhibited cis-trans photoisomerization upon alternating irradiation at two different wavelengths in the visible range, 400 and 480 nm. ACTAB induced reversible changes in the inhibitory activity of ATPase and motor activities correlating with the cis-trans photoisomerization. Compared with cis-ACTAB, trans-ACTAB reduced ATPase activity and microtubule gliding velocity more significantly. These results suggest that ACTAB could be used as photochromic inhibitor of Eg5 to achieve photocontrol of living cells.

3CD, but not 3C, cleaves the VP1/2A site efficiently during Aichi virus polyprotein processing through interaction with 2A.

Picornavirus genomes are translated into a single large polyprotein, which is processed by virus-encoded proteases into individual functional proteins. 3C of all picornaviruses is a protease, and the leader (L) and 2A proteins of some picornaviruses are also involved in polyprotein processing. Aichi virus (AiV), which is associated with acute gastroenteritis in humans, is a member of the genus Kobuvirus of the family Picornaviridae. The AiV L and 2A proteins have already been shown to exhibit no protease activity. In this study, we investigated AiV polyprotein processing by 3C and 3CD using a cell-free translation system. 3C and 3CD were capable of processing the polyprotein in trans; 3C, however, cleaved the VP1/2A site inefficiently, while 3CD cleaved this site almost completely. Mammalian two-hybrid and coimmunoprecipitation assays showed an interaction between 2A and 3CD. Using a 3CD mutant and various 2A mutants of substrate proteins, we showed a clear correlation between the 2A-3CD interaction and the VP1/2A cleavage by 3CD. Thus, this study suggests that tight interaction of 3CD with the 2A region of a precursor protein is required for efficient cleavage at the VP1/2A site.