Yohei Mukai

Long-term effect of repeated lidocaine injections into the external oblique for upper camptocormia in Parkinson's disease

BACKGROUNDnParkinsons disease (PD) is occasionally complicated by camptocormia. In a previous study, we classified camptocormia into upper and lower types based on the inflection point, and reported that lidocaine injection into the external oblique muscle, but not into the internal oblique or rectus abdomen, improved upper camptocormia in PD. The effect of a single lidocaine injection disappeared over a period of few days. In this study, we used repeated lidocaine injections into the external oblique for 4-5 days and evaluated the effects of such treatment for up to 90 days.nnnMETHODSnThe study subjects were 12 patients with PD and upper camptocormia who were treated with repeated lidocaine injections into the bilateral external oblique followed by rehabilitation. The effect of treatment was evaluated by measuring the angle of truncal flexion before and after the injection. Patients who showed improvement with repeated injections were evaluated during a 90-day period.nnnRESULTSnEight out of 12 patients showed significant improvement in posture after a single lidocaine injection. However, the effect subsided several days after treatment. Repeated injections produced long-term improvement in 9 out of 12 patients, which was maintained during the 90-day observation period in eight of these patients.nnnCONCLUSIONSnOur results showed that repeated lidocaine injections into the external oblique improved upper camptocormia, and that the effect was maintained in the majority of patients during the 90-day observation period, indicating that repeated lidocaine injections into the external oblique have therapeutic effect on upper camptocormia in patients with Parkinsons disease.

Intestinal absorption and biological effects of orally administered amorphous silica particles

Although amorphous silica nanoparticles are widely used in the production of food products (e.g., as anticaking agents), there is little information available about their absorption and biological effects after oral exposure. Here, we examined the in vitro intestinal absorption and in vivo biological effects in mice of orally administered amorphous silica particles with diameters of 70, 300, and 1,000xa0nm (nSP70, mSP300, and mSP1000, respectively) and of nSP70 that had been surface-modified with carboxyl or amine groups (nSP70-C and nSP70-N, respectively). Analysis of intestinal absorption by means of the everted gut sac method combined with an inductively coupled plasma optical emission spectrometer showed that the intestinal absorption of nSP70-C was significantly greater than that of nSP70. The absorption of nSP70-N tended to be greater than that of nSP70; however, the results were not statistically significant. Our results indicate that silica nanoparticles can be absorbed through the intestine and that particle diameter and surface properties are major determinants of the degree of absorption. We also examined the biological effects of the silica particles after 28-day oral exposure in mice. Hematological, histopathological, and biochemical analyses showed no significant differences between control mice and mice treated with the silica particles, suggesting that the silica nanoparticles evaluated in this study are safe for use in food production.

Role of the external oblique muscle in upper camptocormia for patients with Parkinson's disease†‡

Patients with Parkinson’s disease (PD) often experience camptocormia, a postural disorder with unclear pathophysiology and unestablished treatments. We clinically categorized camptocormia as upper and lower types based on the location of inflection points. We defined upper camptocormia as abnormal truncal flexion at a point between the lower thoracic and upper lumbar vertebrae whose flexion angle exceeded 40 degrees, whereas lower camptocormia was defined as abnormal truncal flexion at the hip joint. This study focused on upper camptocormia. We performed lidocaine injections into the abdominal muscles of PD patients with upper camptocormia and evaluated their effects on posture to investigate its pathophysiology. Patients with fixed posture because of spinal disease or truncal muscle weakness were excluded. We included 5 patients (4 women and 1 man; mean age, 70.8 6 4.4 years; PD duration, 8.2 6 3.9 years; Hoehn & Yahr stage, 2.6 6 0.8) treated with antiparkinson drugs in our hospital. Camptocormia did not respond to these drugs in any of the patients. Ultrasound guidance was used for lidocaine injections into the abdominal muscle (rectus abdomen [RA] and external oblique [EO] in all patients; internal oblique [IO] in 2 patients; 50 mg in each muscle bilaterally). Although the order of each injection was different in each patient, the following injection was performed on confirming that improvement diminished or if no improvement was observed after several days. Flexion angles were measured before and after each injection. The angle formed between a line perpendicular to the ground and a line linking the C7 vertebra with the inflection point of the trunk was defined as the flexion angle. This study was approved by the NCNP ethics committee. Informed consent was obtained from all patients. The posture of all patients improved following injection into the EO. The average flexion angle decreased from 49 6 6.0 degrees to 37 6 10 degrees (truncal angle of agematched PD patients without camptocormia was 29.4 6 3.7 degrees; Fig. 1), Only 1 patient showed mild improvement after injection into the RA. No improvements were observed following injection into the IO. The RA performs truncal anteflexion, whereas the EO and IO work bilaterally for truncal flexion. Azher et al previously reported improvement of camptocormia by botulinum toxin injection into the RA, although they did not classify camptocormia by type. In the present study, forward flexion was reduced in all patients after injection into the EO, not RA nor IO. Our results may suggest that the EO is primarily associated with upper camptocormia pathogenesis. Considering that previous studies have speculated that lidocaine could suppress dystonic excitation, dystonia of the EO may be a cause of upper camptocormia. Although this study has some limitations, such as the small number of patients and not adjusting lidocaine dose for muscle size, this is the first report to investigate camptocormia pathophysiology by classification, and these findings may contribute to the treatment of upper camptocormia in patients with PD. To confirm our results, we have been carrying out a larger study.

Ephrin receptor A10 is a promising drug target potentially useful for breast cancers including triple negative breast cancers.

Ephrin receptor A10 (EphA10) is a relatively uncharacterized protein which is expressed in many breast cancers but not expressed in normal breast tissues. Here, we examined the potential of EphA10 as a drug target in breast cancer. Immunohistochemical staining of clinical tissue sections revealed that EphA10 was expressed in various breast cancer subtypes, including triple negative breast cancers (TNBCs), with no expression observed in normal tissues apart from testis. Ligand-dependent proliferation was observed in EphA10-transfected MDA-MB-435 cells (MDA-MB-435(EphA10)) and native TNBC cells (MDA-MB-436). However, this phenomenon was not observed in parental MDA-MB-435 cells which express a low level of EphA10. Finally, tumor growth was significantly suppressed by administration of an anti-EphA10 monoclonal antibody in a xenograft mouse model. These results suggest that inhibition of EphA10 signaling may be a novel therapeutic option for management of breast cancer, including TNBCs which are currently not treated with molecularly targeted agents.

Identification and evaluation of metastasis-related proteins, oxysterol binding protein-like 5 and calumenin, in lung tumors

Metastasis is an important prognosis factor in lung cancer, therefore, it is imperative to identify target molecules and elucidate molecular mechanism of metastasis for developing new therapeutics and diagnosis methods. We searched for metastasis-related proteins by utilizing a novel antibody proteome technology developed in our laboratory that facilitated efficient screening of useful target proteins. Two-dimensional differential in-gel electrophoresis (2D-DIGE) analysis identified sixteen proteins, which were highly expressed in metastatic lung cancer cells, as protein candidates. Monoclonal single-chain variable fragments (scFvs) binding to candidates were isolated from a scFv-displaying phage library by affinity selection. Tissue microarray analysis of scFvs binding to candidates revealed that oxysterol binding protein-like 5 (OSBPL5) and calumenin (CALU) were expressed at a significantly higher levels in the lung tissues of metastasis-positive cases than that in the metastasis-negative cases (OSBPL5; p=0.0156, CALU; p=0.0055). Furthermore, 80% of OSBPL5 and CALU double-positive cases were positive for lymph node metastasis. Consistent with these observations, overexpression of OSBPL5 and CALU promoted invasiveness of lung cancer cells. Conversely, knockdown of these proteins using respective siRNAs reversed the invasiveness of the lung cancer cells. Moreover, these proteins were expressed in lung tumor tissues, but not in normal lung tissues. In conclusion, OSBPL5 and CALU are related to metastatic potential of lung cancer cells, and they could be useful targets for cancer diagnosis and also for development of drugs against metastasis.

Aminopeptidase P3, a new member of the TNF-TNFR2 signaling complex, induces phosphorylation of JNK1 and JNK2.

ABSTRACT Tumor necrosis factor (TNF) is an important mediator that triggers onset of autoimmune diseases and exerts its biological effects by interacting through two receptors, TNFR1 (also known as TNFRSF1A) and TNFR2 (also known as TNFRSF1B). TNFR2 signaling has significant potential to exert pro-survival and protective roles in several diseases. Unlike TNFR1 signaling, however, the mechanism of TNFR2 signal transduction is poorly understood, and few of its adaptor molecules are known. The present study utilized a proteomics approach to search for adaptor molecules in the TNFR2 signaling complex and identified aminopeptidase P3 (APP3, also known as XPNPEP3) to be a key molecule. One of its two isoforms, mitochondrial APP3 (APP3m) but not cytosolic APP3 (APP3c), was recruited to TNFR2 and shown to regulate TNF–TNFR2-dependent phosphorylation of JNK1 (also known as MAPK8) and JNK2 (also known as MAPK9). Furthermore, APP3m was released from mitochondria upon TNF stimulation in the absence of mitochondrial outer membrane permeabilization (MOMP). The observation of increased cell death upon downregulation of APP3m also suggested that APP3m exerts an anti-apoptotic function. These findings reveal that APP3m is a new member of the TNF–TNFR2 signaling complex and characterize an APP3-mediated TNFR2 signal transduction mechanism that induces activation of JNK1 and JNK2.

Mechanism of camptocormia in Parkinson's disease analyzed by tilt table-EMG recording

BACKGROUNDnWe previously classified camptocormia of Parkinsons disease (PD) into upper and lower types based on the inflection point, and reported improvement of upper camptocormia after lidocaine injection into the external oblique. However, the exact pathophysiology of this phenomenon remains obscure.nnnMETHODSnSurface electromyography (sEMG) was recorded in 11 PD patients with upper camptocormia, 11 PD patients with lower camptocormia, and 10 age-matched PD patients without postural deformity. Electrodes were positioned above the external oblique, hip flexors and paraspinal muscles at Th11 level bilaterally. Recording commenced with the patient in supine position on a tilt table, and continued when the table was tilted up to vertical position. Lidocaine was injected into the external oblique in patients with upper camptocormia and the psoas major in patients with lower camptocormia.nnnRESULTSnAll patients with upper and lower camptocormia developed the corresponding camptocormic posture during tilt up. The onset of camptocormic posture was preceded by the appearance of sEMG activity in the external oblique in 10 out of 11 patients with upper camptocormia, but less frequently in patients with lower camptocormia and the controls. Hip flexors sEMG activity was recorded in almost all patients. Posture was improved in 8 out of 9 patients with upper camptocormia, and 9 out of 11 patients with lower camptocormia following injections of lidocaine.nnnCONCLUSIONSnThe results suggest the external oblique is involved, at least in part, in the development of upper camptocormia. Although EMG findings cannot differentiate pathogenicity, the psoas major is probably involved in lower camptocormia.

Endothelial Cell–Specific Expression of Roundabout 4 Is Regulated by Differential DNA Methylation of the Proximal Promoter

Objective—The molecular basis of endothelial cell (EC)–specific gene expression is poorly understood. Roundabout 4 (Robo4) is expressed exclusively in ECs. We previously reported that the 3-kb 5′-flanking region of the human Robo4 gene contains information for lineage-specific expression in the ECs. Our studies implicated a critical role for GA-binding protein and specificity protein 1 (SP1) in mediating overall expression levels. However, these transcription factors are also expressed in non-ECs. In this study, we tested the hypothesis that epigenetic mechanisms contribute to EC-specific Robo4 gene expression. Methods and Results—Bisulfite sequencing analysis indicated that the proximal promoter of Robo4 is methylated in non-ECs but not in ECs. Treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine increased Robo4 gene expression in non-ECs but not in ECs. Proximal promoter methylation significantly decreased the promoter activity in ECs. Electrophoretic mobility shift assays showed that DNA methylation of the proximal promoter inhibited SP1 binding to the −42 SP1 site. In DNase hypersensitivity assays, chromatin condensation of the Robo4 promoter was observed in some but not all nonexpressing cell types. In Hprt (hypoxanthine phosphoribosyltransferase)-targeted mice, a 0.3-kb proximal promoter directed cell-type–specific expression in the endothelium. Bisulfite sequencing analysis using embryonic stem cell–derived mesodermal cells and ECs indicated that the EC-specific methylation pattern of the promoter is determined by demethylation during differentiation and that binding of GA-binding protein and SP1 to the proximal promoter is not essential for demethylation. Conclusions—The EC-specific DNA methylation pattern of the Robo4 proximal promoter is determined during cell differentiation and contributes to regulation of EC-specific Robo4 gene expression.

Size and surface modification of amorphous silica particles determine their effects on the activity of human CYP3A4 in vitro

Because of their useful chemical and physical properties, nanomaterials are widely used around the world - for example, as additives in food and medicines - and such uses are expected to become more prevalent in the future. Therefore, collecting information about the effects of nanomaterials on metabolic enzymes is important. Here, we examined the effects of amorphous silica particles with various sizes and surface modifications on cytochrome P450 3A4 (CYP3A4) activity by means of two different in vitro assays. Silica nanoparticles with diameters of 30 and 70xa0nm (nSP30 and nSP70, respectively) tended to inhibit CYP3A4 activity in human liver microsomes (HLMs), but the inhibitory activity of both types of nanoparticles was decreased by carboxyl modification. In contrast, amine-modified nSP70 activated CYP3A4 activity. In HepG2 cells, nSP30 inhibited CYP3A4 activity more strongly than the larger silica particles did. Taken together, these results suggest that the size and surface characteristics of the silica particles determined their effects on CYP3A4 activity and that it may be possible to develop silica particles that do not have undesirable effects on metabolic enzymes by altering their size and surface characteristics.

Eph receptor A10 has a potential as a target for a prostate cancer therapy

We recently identified Eph receptor A10 (EphA10) as a novel breast cancer-specific protein. Moreover, we also showed that an in-house developed anti-EphA10 monoclonal antibody (mAb) significantly inhibited proliferation of breast cancer cells, suggesting EphA10 as a promising target for breast cancer therapy. However, the only other known report for EphA10 was its expression in the testis at the mRNA level. Therefore, the potency of EphA10 as a drug target against cancers other than the breast is not known. The expression of EphA10 in a wide variety of cancer cells was studied and the potential of EphA10 as a drug target was evaluated. Screening of EphA10 mRNA expression showed that EphA10 was overexpressed in breast cancer cell lines as well as in prostate and colon cancer cell lines. Thus, we focused on prostate cancers in which EphA10 expression was equivalent to that in breast cancers. As a result, EphA10 expression was clearly shown in clinical prostate tumor tissues as well as in cell lines at the mRNA and protein levels. In order to evaluate the potential of EphA10 as a drug target, we analyzed complement-dependent cytotoxicity effects of anti-EphA10 mAb and found that significant cytotoxicity was mediated by the expression of EphA10. Therefore, the idea was conceived that the overexpression of EphA10 in prostate cancers might have a potential as a target for prostate cancer therapy, and formed the basis for the studies reported here.