GABAA Receptors and Maternally Derived Taurine Regulate the Temporal Specification of Progenitors of Excitatory Glutamatergic Neurons in the Mouse Developing Cortex.

Temporal specification of the neural progenitors (NPs) producing excitatory glutamatergic neurons is essential for histogenesis of the cerebral cortex. Neuroepithelial cells, the primary NPs, transit to radial glia (RG). To coincide with the transition, NPs start to differentiate into neurons, undergoing a switch from symmetric to asymmetric cell division. After the onset of neurogenesis, NPs produce layer-specific neurons in a defined order with precise timing. Here, we show that GABAA receptors (GABAARs) and taurine are involved in this regulatory mechanism. Foetal exposure to GABAAR-antagonists suppressed the transition to RG, switch to asymmetric division, and differentiation into upper-layer neurons. Foetal exposure to GABAAR-agonists caused the opposite effects. Mammalian foetuses are dependent on taurine derived from the mothers. GABA and taurine function as endogenous ligands for GABAARs. Ca2+ imaging showed that NPs principally responded to taurine but not GABA before E13. The histological phenotypes of the taurine transporter knockout mice resembled those of the mice foetally exposed to GABAAR-antagonists. Foetal exposure to GABAAR-modulators resulted in considerable alterations in offspring behavior like core symptoms of autism. These results show that taurine regulates the temporal specification of NPs and that disrupting the taurine-receptor interaction possibly leads to neurodevelopmental disorders.

Two Revision Surgeries on Cemented Custom-made Tumor Prostheses.

We performed revision surgery in 2 patients for stem fracture of a cemented tumor prosthesis that occurred more than 25 years after the initial surgery. For revision, the global modular replacement system (GMRS) was used. However, as bone cement in the bone could not be adequately removed, stems with respective diameters of 11 and 12.5 mm were used. In revision surgery for cemented tumor prostheses, adequate removal of residual bone cement is optimal. However, when there is a risk of fracture, it may be appropriate to insert a thicker stem after reaming the femoral canal as much as possible, and then fix the stem using the cement-in-cement method.

Preventive Effects of Long-Term Caloric Restriction on Aging Related In Vivo Bladder Dysfunction and Molecular Biological Changes in the Bladder and Dorsal Root Ganglia in Rats.

PURPOSE: We evaluated aging related bladder dysfunctions and biological changes in the bladder and dorsal root ganglia in rats. We also investigated whether long-term caloric restriction may have preventive effects on these changes. MATERIALS AND METHODS: Male Fischer 344 rats were divided into a young group (age 6 months) and an old group (age 25 to 28 months), each with free access to normal food, and an old group (age 25 to 28 months) with food restricted to 3 days per week. Conscious cystometry, cDNA microarray analysis, immunohistochemistry and oxidative stress measurements of the bladder and dorsal root ganglia were performed. RESULTS: The old group with free access to normal food showed higher threshold pressure, more nonvoiding contractions and lower bladder compliance than the young group with free access to food. Old rats with free access showed greater post-void residual volume and lower voiding efficiency than old rats with caloric restriction and young rats. In the old group with free access 83 genes in the bladder and 48 in the L6 dorsal root ganglia were up-regulated compared with old rats with caloric restriction and young rats. These genes were mostly related to immune and inflammatory responses. Immunohistochemistry showed stronger expression of the immune response protease Gzm (granzyme) B and the collagenase Mmp13 (matrix metalloproteinase-13) in the bladder of old rats with free access vs old rats with caloric restriction and young rats. The level of malondialdehyde, an oxidative stress marker, was higher in the bladder of old rats with free access than in young rats but there was no difference between old rats with caloric restriction and young rats with free access to food. CONCLUSIONS: In rats aging leads to storage and voiding dysfunctions associated with immune and inflammatory related responses in the bladder and dorsal root ganglia, and with increased oxidative stress in the bladder. Caloric restriction reduced these aging related changes.

Growth differentiation factor 15 as a useful biomarker for mitochondrial disorders.

OBJECTIVE: The diagnosis of mitochondrial disorders (MDs) is occasionally difficult because patients often present with solitary, or a combination of, symptoms caused by each organ insufficiency, which may be the result of respiratory chain enzyme deficiency. Growth differentiation factor 15 (GDF-15) has been reported to be elevated in serum of patients with MDs. In this study, we investigated whether GDF-15 is a more useful biomarker for MDs than several conventional biomarkers. METHODS: We measured the serum levels of GDF-15 and fibroblast growth factor 21 (FGF-21), as well as other biomarkers, in 48 MD patients and in 146 healthy controls in Japan. GDF-15 and FGF-21 concentrations were measured by enzyme-linked immunosorbant assay and compared with lactate, pyruvate, creatine kinase, and the lactate-to-pyruvate ratio. We calculated sensitivity and specificity and also evaluated the correlation based on two rating scales, including the Newcastle Mitochondrial Disease Rating Scale (NMDAS). RESULTS: Mean GDF-15 concentration was 6-fold higher in MD patients compared to healthy controls (2,711 ± 2,459 pg/ml vs 462.5 ± 141.0 pg/mL; p < 0.001). Using a receiver operating characteristic curve, the area under the curve was significantly higher for GDF-15 than FGF-21 and other conventional biomarkers. Our date suggest that GDF-15 is the most useful biomarker for MDs of the biomarkers examined, and it is associated with MD severity. INTERPRETATION: Our results suggest that measurement of GDF-15 is the most useful first-line test to indicate the patients who have the mitochondrial respiratory chain deficiency.

miR-130a activates apoptotic signaling through activation of caspase-8 in taxane-resistant prostate cancer cells.

BACKGROUND: The acquisition of drug resistance is one of the most malignant phenotypes of cancer and identification of its therapeutic target is a prerequisite for the development of novel therapy. MicroRNAs (miRNAs) have been implicated in various types of cancer and proposed as potential therapeutic targets for patients. In the present study, we aimed to identify miRNA that could serve as a therapeutic target for taxane-resistant prostate cancer. METHODS: In order to identify miRNAs related to taxane-resistance, miRNA profiling was performed using prostate cancer PC-3 cells and paclitaxel-resistant PC-3 cell lines established from PC-3 cells. Microarray analysis of mRNA expression was also conducted to search for potential target genes of miRNA. Luciferase reporter assay was performed to examine miRNA binding to the 3'-UTR of target genes. The effects of ectopic expression of miRNA on cell growth, tubulin polymerization, drug sensitivity, and apoptotic signaling pathway were investigated in a paclitaxel-resistant PC-3 cell line. RESULTS: The expression of miR-130a was down-regulated in all paclitaxel-resistant cell lines compared with parental PC-3 cells. Based on mRNA microarray analysis and luciferase reporter assay, we identified SLAIN1 as a direct target gene for miR-130a. Transfection of a miR-130a precursor into a paclitaxel-resistant cell line suppressed cell growth and increased the sensitivity to paclitaxel. Lastly, ectopic expression of miR-130a did not affect the polymerized tubulin level, but activated apoptotic signaling through activation of caspase-8. CONCLUSIONS: Our results suggested that reduced expression of miR-130a may be involved in the paclitaxel-resistance and that miR-130a could be a therapeutic target for taxane-resistant prostate cancer patients.

Long-term outcome following surgical treatment of sacral chordoma.

BACKGROUND: Sixteen sacral chordoma surgeries performed at a single institution during the 1983-2008 period were retrospectively studied. Our aim is to assess surgical treatment and long-term outcomes. METHODS: Fifteen patients underwent primary wide excision, and one intralesional excision using ethanol for local control and radiation therapy (RT). A combined anteroposterior approach for large tumors above S2, and wide excision was performed with the modified threadwire-saw (MT-saw) after 1997. RESULTS: Fourteen of the 15 patients had wide margins, one a wide margin with contamination. The MT-saw was facilitated sacral excision with wide margins. Eleven patients are alive for 5-28 years. Five patients died before 10 years, two patients experienced sepsis, and one of another disease. Two patients died of local recurrence (LR) and another of multiple metastases after intralesional excision and wide excision with contamination, respectively. LR and complications occurred 4 each of 11 patients with tumors ≥ 10 cm, neither with tumors < 10 cm. The overall 5- and 10-year survival rate with wide surgical margins was 13/16 (81.3%) and 8/13 (61.5%). CONCLUSIONS: A combined anteroposterior approach for large tumors, and the MT-saw facilitates sacral excision with wide margins. Wide excision is recommended for younger patients.

Inhibition of human osteosarcoma cell migration and invasion by a gene silencer, pyrrole-imidazole polyamide, targeted at the human MMP9 NF-κB binding site.

Osteosarcoma is one of the most prevalent bone tumors, occurring mostly in adolescence. However, no noticeable progress has been achieved in developing new therapeutic agents for this disease. Matrix metalloproteinase 9 (MMP9), a type IV collagenase, is a known anticancer target and is overexpressed in osteosarcomas. MMPs can degrade components of the extracellular matrix and are known to be involved in tumor invasion and metastasis. In the present study, we designed and synthesized a pyrrole-imidazole polyamide (HN.49), a gene-silencing agent that specifically targets the nuclear factor-kappa B (NF-κB) binding site of the human MMP9 promoter. We then examined the effect of HN.49 on the enzyme activity of MMP9 and the migration activity of osteosarcoma cells in vitro. It was clearly shown that HN.49 polyamide reduced the expression level of MMP9 mRNA and the enzymatic activity of MMP-9 in SaOS-2 cells. Moreover, HN.49 polyamide inhibited migration and invasion by SaOS-2 cells in in vitro wound-closure and matrigel-invasion assays. These results indicate that HN.49 may be a potential therapeutic agent for inhibiting the invasion and metastasis of osteosarcoma.

CCR1/CCL5 interaction promotes invasion of taxane-resistant PC3 prostate cancer cells by increasing secretion of MMPs 2/9 and by activating ERK and Rac signaling.

Castration-refractory prostate cancer (CRPC) is treated with taxane-based chemotherapy, but eventually becomes drug resistant. It is thus essential to identify novel therapeutic targets for taxane resistance in CRPC patients. We investigated the role of the chemokine (C-C motif) receptor 1 (CCR1) and its ligand, chemokine (C-C motif) ligand 5 (CCL5), in taxane-resistant CRPC using paclitaxel-resistant prostate cancer cells (PC3PR) established from PC3 cells. We found that the expression levels of CCR1 mRNA and protein were up-regulated in PC3PR cells compared to PC3 cells. In order to investigate the role of increased CCR1 in PC3PR cells, we stimulated cells with CCL5, one of the chemokine ligands of CCR1. In CCL5-stimulated PC3PR cells, siRNA-mediated knockdown of CCR1 expression reduced phosphorylation of ERK1/2 and Rac1/cdc42. Furthermore, CCR1 knockdown and MEK1/2 inhibition decreased CCL5-stimulated secretion of MMPs 2 and 9, which play important roles in cancer cell invasion and metastasis. In the Matrigel invasion assay, knockdown of CCR1 and inhibition of the ERK and Rac signaling pathways significantly decreased the number of invading cells. Finally, the serum CCL5 protein level as measured by ELISA was not different among the three groups of patients: those with negative prostate biopsy, those at initial diagnosis of prostate cancer, and those with taxane-resistant prostate cancer. These results demonstrated for the first time that the interaction of CCR1 with CCL5 caused by increased expression of CCR1 promotes invasion of PC3PR cells by increasing secretion of MMPs 2 and 9 and by activating ERK and Rac signaling. Our findings suggest that CCR1 could be a novel therapeutic target for taxane-resistant CRPC.

Hippocampal gene expression profiling in a rat model of posttraumatic epilepsy reveals temporal upregulation of lipid metabolism-related genes.

Traumatic brain injury occasionally causes posttraumatic epilepsy. To elucidate the molecular events responsible for posttraumatic epilepsy, we established a rodent model that involved the injection of microliter quantities of FeCl3 solution into the amygdalar nuclear complex. We previously compared hippocampal gene expression profiles in the traumatic epilepsy model and normal rats at 5 days after brain injury (acute phase) to determine the role of inflammation. In this study, we focused on later stages of epileptogenesis. We compared gene expression profiles at 5, 15 (sub-chronic phase), and 30 days (chronic phase) after brain injury to identify temporal changes in molecular networks involved in epileptogenesis. A total of 81 genes were significantly (at least twofold) up- or downregulated over the course of disease progression. We found that genes related to lipid metabolism, namely, Apoa1, Gh, Mc4r, Oprk1, and Pdk4, were temporarily upregulated in the sub-chronic phase. Changes in lipid metabolism regulation might be related to seizure propagation during epileptogenesis. This temporal description of hippocampal gene expression profiles throughout epileptogenesis provides clues to potential markers of disease phases and new therapeutic targets.

Functional annotation of genes differentially expressed between primary motor and prefrontal association cortices of macaque brain.

DNA microarray-based genome-wide transcriptional profiling and gene network analyses were used to characterize the molecular underpinnings of the neocortical organization in rhesus macaque, with particular focus on the differences in the functional annotation of genes in the primary motor cortex (M1) and the prefrontal association cortex (area 46 of Brodmann). Functional annotation of the differentially expressed genes showed that the list of genes selectively expressed in M1 was enriched with genes involved in oligodendrocyte function, and energy consumption. The annotation appears to have successfully extracted the characteristics of the molecular structure of M1.

Effect of zonisamide co-administration with levodopa on global gene expression in the striata of rats with Parkinson's disease.

The anti-epileptic drug zonisamide is reported to exert beneficial effects in patients with Parkinson's disease. To elucidate the pathophysiological mechanisms underlying the anti-parkinsonism effects of zonisamide, we examined the effect of zonisamide co-administered with levodopa in the striata of rats with 6-hydoroxydopamine hemiparkinsonism by using a DNA microarray for genome-wide gene expression profiling. We found that the expression of some genes related to metabolism and nervous system development and function were upregulated by zonisamide; expression of these genes was downregulated by levodopa. Furthermore, many genes related to the immune system and inflammation were downregulated by zonisamide, and their expression was upregulated by levodopa. These results indicate that zonisamide has a protective effect when co-administered with levodopa.

ETS1 promotes chemoresistance and invasion of paclitaxel-resistant, hormone-refractory PC3 prostate cancer cells by up-regulating MDR1 and MMP9 expression.

ETS1, which belongs to the ETS transcription factor family, plays important roles in diverse aspects of cancer such as drug resistance and metastasis. In the present study, we examined the functional roles of ETS1 in paclitaxel resistance and invasion using human prostate cancer PC3 cells and paclitaxel-resistant PC3PR cells established from PC3 cells. Our results showed that ETS1mRNA and protein expression was markedly up-regulated in paclitaxel-resistant PC3PR cells compared with paclitaxel-sensitive PC3 cells. The mRNA levels of MDR1 as well as MMP1, MMP3, MMP9 and uPA were positively correlated with that of ETS1. In PC3PR cells, silencing of ETS1 expression by siRNAs inhibited the activity of the MDR1 promoter containing ETS binding sites, reduced the mRNA and protein levels of MDR1 and suppressed paclitaxel resistance. Furthermore, ETS1 knockdown decreased secretion of MMP9 as well as its intracellular mRNA level, and dramatically inhibited invasion of PC3PR cells. Our results suggest that ETS1 promotes paclitaxel resistance and invasion in part by up-regulating MDR1 and MMP9 expression. Taken together, a novel therapeutic strategy targeting the ETS1 gene could be designed to overcome chemoresistance and metastasis of taxane-resistant, hormone-refractory prostate cancer.

CKIepsilon/delta-dependent phosphorylation is a temperature-insensitive, period-determining process in the mammalian circadian clock.

A striking feature of the circadian clock is its flexible yet robust response to various environmental conditions. To analyze the biochemical processes underlying this flexible-yet-robust characteristic, we examined the effects of 1,260 pharmacologically active compounds in mouse and human clock cell lines. Compounds that markedly (>10 s.d.) lengthened the period in both cell lines, also lengthened it in central clock tissues and peripheral clock cells. Most compounds inhibited casein kinase Iepsilon (CKIepsilon) or CKIdelta phosphorylation of the PER2 protein. Manipulation of CKIepsilon/delta-dependent phosphorylation by these compounds lengthened the period of the mammalian clock from circadian (24 h) to circabidian (48 h), revealing its high sensitivity to chemical perturbation. The degradation rate of PER2, which is regulated by CKIepsilon/delta-dependent phosphorylation, was temperature-insensitive in living clock cells, yet sensitive to chemical perturbations. This temperature-insensitivity was preserved in the CKIepsilon/delta-dependent phosphorylation of a synthetic peptide in vitro. Thus, CKIepsilon/delta-dependent phosphorylation is likely a temperature-insensitive period-determining process in the mammalian circadian clock.

Giant-cell tumor of the patella.

We report a 38-year old man with a giant-cell tumor in a rare site, the patella. Primary patellar neoplasms are highly unusual. According to a survey by the Bone and Soft Tissue Tumor Committee of the Japanese Orthopaedic Association, of more than 2,126 giant-cell tumors of bone reported since 1972, only 22 were primary patellar neoplasms. We present a case of this rare entity along with its clinical and radiographic features. The first clinical symptom was anterior knee pain. Though anterior knee pain has numerous and varied causes, it is necessary to consider patellar bone tumors in the differential diagnosis.

Revision of tumor prosthesis of the knee joint.

BACKGROUND: Among 40 patients with primary malignant tumors of the knee joint who underwent reconstruction of the affected limb with tumor prosthesis, revision was required in 7 due to stem breakage or loosening. SUBJECTS AND METHODS: In the 7 cases undergoing revision, conditions and background factors at the time of breakage, the breakage site, time of revision, models of previous and new prostheses, stem diameters before and after revision, details of the revision (blood loss, operative time), and the presence or absence of adjuvant therapy were determined. RESULTS: The replacement site was the distal femur in 5 and proximal tibia in 2. Revision was performed 6 years and 2 months after the previous prosthesis placement on average. The broken prosthesis model was KMFTR in 4 and HMRS and the physio-hinge type in one each. Revision due to loosening was performed in a case requiring replacement with Growing Kotz prosthesis. The model was switched to HMRS in 3, and the stem diameter was changed to 12 mm in 3 KMFTR breakage cases. The mean stem diameters were 11.2 and 10.2 mm in the non-revision and revision groups. The respective resection rates were 36 and 45%. The mean functional evaluation was 70.1% before and 76.2% after revision. CONCLUSION: To reduce the risk of tumor prosthesis breakage, the amount of bone resection should be limited to 30% or less in the affected bone, the stem diameter should be at least 12 mm, and the stem shape should be fitted to the anatomical shape of the femur.

MiR-148a attenuates paclitaxel resistance of hormone-refractory, drug-resistant prostate cancer PC3 cells by regulating MSK1 expression.

MicroRNAs are involved in cancer pathogenesis and act as tumor suppressors or oncogenes. It has been recently reported that miR-148a expression is down-regulated in several types of cancer. The functional roles and target genes of miR-148a in prostate cancer, however, remain unknown. In this report, we showed that miR-148a expression levels were lower in PC3 and DU145 hormone-refractory prostate cancer cells in comparison to PrEC normal human prostate epithelial cells and LNCaP hormone-sensitive prostate cancer cells. Transfection with miR-148a precursor inhibited cell growth, and cell migration and invasion, and increased the sensitivity to anti-cancer drug paclitaxel in PC3 cells. Computer-aided algorithms predicted mitogen- and stress-activated protein kinase, MSK1, as a potential target of miR-148a. Indeed, miR-148a overexpression decreased expression of MSK1. Using luciferase reporter assays, we identified MSK1 as a direct target of miR-148a. Suppression of MSK1 expression by siRNA, however, showed little or no effects on malignant phenotypes of PC3 cells. In PC3PR cells, a paclitaxel-resistant cell line established from PC3 cells, miR-148a inhibited cell growth, and cell migration and invasion, and also attenuated the resistance to paclitaxel. MiR-148a reduced MSK1 expression by directly targeting its 3'-UTR in PC3PR cells. Furthermore, MSK1 knockdown reduced paclitaxel-resistance of PC3PR cells, indicating that miR-148a attenuates paclitaxel-resistance of hormone-refractory, drug-resistant PC3PR cells in part by regulating MSK1 expression. Our findings suggest that miR-148a plays multiple roles as a tumor suppressor and can be a promising therapeutic target for hormone-refractory prostate cancer especially for drug-resistant prostate cancer.

Hippocampal gene network analysis in an experimental model of posttraumatic epilepsy.

In the present study, we performed comprehensive gene expression and gene network analyses using a DNA microarray to elucidate the molecular events responsible for the pathology of posttraumatic epilepsy at the partial seizure stage. We used an experimental posttraumatic epilepsy model of amygdalar focal FeCl(3)-injected rats and compared gene expression profiles in the hippocampus at the partial seizure stage (less than stage 3 on Racine's convulsion scale) and that of sham-operated animals. At the partial seizure stage, upregulation of phospholipase A2 (PLA2) and lipid metabolism were observed, which have been reported to be caused by brain injury and seizures in previous studies. Furthermore, significant upregulation of genes related to inflammation and the immune system was observed. These molecular changes in PLA2 and lipid metabolism may be related to seizure propagation.

Mutational analysis of GABRG2 in a Japanese cohort with childhood epilepsies.

A few mutations in the gene encoding the gamma 2 subunit of the gamma-aminobutyric acid receptor type A (GABRG2) have been reported in various types of epilepsy. The aim of this study is to investigate the role of GABRG2 in the pathogenesis of childhood epilepsy in a large Japanese cohort. Genetic analysis of GABRG2 was performed on 140 Japanese patients with various childhood epilepsies largely including Dravet syndrome and genetic epilepsy with febrile seizures plus. The mutational analysis identified one novel missense mutation of GABRG2 (c.236A>G: p.N40S) in a patient with generalized tonic-clonic seizures (GTCS). The mutation was heterozygous and replacing a highly conserved Asn residue with a Ser. The affected amino acid was located at residue 40 of the mature GABRG2 protein, which was near the first one of two high-affinity benzodiazepine-binding domains of the gamma2 subunit (Lys-41-Trp-82). This mutation in such an important position may hamper the function of the channel and contribute to the case's pathogenesis of GTCS.

A bent needle tip during irrigation for enchondroma of the distal phalanx: a new curettage tool.

BACKGROUND: We employed a novel curettage tool, a bent needle tip, during irrigation for enchondroma of the distal phalanx. This study aimed to evaluate our new curettage tool for treating enchondroma of the distal phalanx. METHODS: Seven distal phalanx enchondromas were pathologically diagnosed at our institute. We evaluated age, gender, tumor location, affected side, clinical symptoms, Takigawa classification, size, recurrence, complications, residual pain, Tordai score, and follow-up period. We bent an 18G needle tip connected to an extension tube and syringe. The bent needle was inserted through the small hole, and the cavity for bone grafting was adequately filled with injectable calcium phosphate cement through the small hole. RESULTS: There were five centric-type and two giant-type tumors, with a mean size of 52.7%. All patients had clinical symptoms at the initial presentation. All patients showed complete bone healing within 3 months on post-radiological examinations and were Grade 1 according to the Tordai score. CONCLUSIONS: This tool is extremely simple, and both the incision and the cortical window can be small. We recommend a bent needle tip, easily devised in any hospital, as a curettage tool for treating enchondroma in small bones, especially of the distal phalanx.

Rats harboring S284L Chrna4 mutation show attenuation of synaptic and extrasynaptic GABAergic transmission and exhibit the nocturnal frontal lobe epilepsy phenotype.

Mutations of genes encoding alpha4, beta2, or alpha2 subunits (CHRNA4, CHRNB2, or CHRNA2, respectively) of nAChR [neuronal nicotinic ACh (acetylcholine) receptor] cause nocturnal frontal lobe epilepsy (NFLE) in human. NFLE-related seizures are seen exclusively during sleep and are characterized by three distinct seizure phenotypes: "paroxysmal arousals," "paroxysmal dystonia," and "episodic wandering." We generated transgenic rat strains that harbor a missense mutation S284L, which had been identified in CHRNA4 in NFLE. The transgenic rats were free of biological abnormalities, such as dysmorphology in the CNS, and behavioral abnormalities. The mRNA level of the transgene (mutant Chrna4) was similar to the wild type, and no distorted expression was detected in the brain. However, the transgenic rats showed epileptic seizure phenotypes during slow-wave sleep (SWS) similar to those in NFLE exhibiting three characteristic seizure phenotypes and thus fulfilled the diagnostic criteria of human NFLE. The therapeutic response of these rats to conventional antiepileptic drugs also resembled that of NFLE patients with the S284L mutation. The rats exhibited two major abnormalities in neurotransmission: (1) attenuation of synaptic and extrasynaptic GABAergic transmission and (2) abnormal glutamate release during SWS. The currently available genetically engineered animal models of epilepsy are limited to mice; thus, our transgenic rats offer another dimension to the epilepsy research field.