A Novel Mutation of Human Liver Alanine:Glyoxylate Aminotransferase Causes Primary Hyperoxaluria Type 1: Immunohistochemical Quantification and Subcellular Distribution

A novel alanine:glyoxylate aminotransferase (AGT) mutation involved in primary hyperoxaluria type 1 (PH1) was studied in Japanese patients. Two mutations in exon 7, c.751T>A and c.752G>A, lead to a W251K amino acid substitution. Proband 1 (patient 1) was homozygous for the W251K mutation allele (DDBJ Accession No. AB292648), and AGT-specific activity in the patient’s liver was very low. To reveal the cause of the low enzymatic activity, the intracellular localization of AGT (W251K) was studied using immunohistochemistry and immunoelectron microscopy. The latter analysis showed that patient 2 had only one-fifth of the normal AGT expression per catalase, suggesting impairment of AGT (W251K) dependent transport into peroxisomes. Peroxisomal transport of human AGT is believed to be dependent on the presence of the type 1 peroxisomal targeting sequence. The C-terminal tripeptide of AGT, KKL is necessary for peroxisomal targeting. In cultured cells, EGFP-AGT (W251K) localized both in the peroxisome and cytosol. These results were consistent with the data obtained from liver analysis of patient 2. The subcellular distribution of AGT (W251K) and the results from a random mutagenesis study suggest that KKL is necessary for peroxisomal targeting of human AGT, but additional signal other than KKL may be necessary

Sorafenib-induced apoptosis in colonic neuroendocrine carcinoma cells

　Background: Neuroendocrine carcinoma (NEC) is a rare disease, and therapy for this malignant tumor is controversial. Conventionally, platinum doublet chemotherapy has been used for advanced gastroentero-pancreatic (GEP) neuroendocrine carcinoma (GEP- NEC), but the efficacy of molecular-targeted drugs for GEP-NEC is unknown. In this study, we investigated the antitumor effect of molecular-targeted drugs on colorectal neuroendocrine carcinoma cells.　Materials and methods: A colonic neuroendocrine carcinoma cell line COLO320 was treated with molecular-targeted drugs, and cell growth suppression and apoptosis induction were evaluated.　Results: The cytostatic effects of molecular-targeted drugs against COLO320 were higher in the order of sorafenib, sunitinib, rapamycin, and imatinib. Flow cytometry analysis showed that sorafenib induced G1 cell cycle arrest and a high rate of apoptosis. Sunitinib showed condensation and fragmentation of nuclear chromatin, but also necrosis with cell swelling. In contrast, sorafenib strongly induced apoptosis via condensation and fragmentation of nuclear chromatin. Sorafenib-induced apoptosis was due to caspase-3 activation, and this apoptosis was inhibited with a caspase inhibitor.　Conclusion: Sorafenib induces apoptosis in COLO320 cells and is a potential therapeutic agent for colonic neuroendocrine carcinomas

The actin family member Arp6 and the histone variant H2A.Z are required for spatial positioning of chromatin in chicken cell nuclei

The spatial organization of chromatin in the nucleus contributes to genome function and is altered during the differentiation of normal and tumorigenic cells. Although nuclear actin-related proteins (Arps) have roles in the local alteration of chromatin structure, it is unclear whether they are involved in the spatial positioning of chromatin. In the interphase nucleus of vertebrate cells, gene-dense and gene-poor chromosome territories (CTs) are located in the center and periphery, respectively. We analyzed chicken DT40 cells in which Arp6 had been knocked out conditionally, and showed that the radial distribution of CTs was impaired in these knockout cells. Arp6 is an essential component of the SRCAP chromatin remodeling complex, which deposits the histone variant H2A.Z into chromatin. The redistribution of CTs was also observed in H2A.Z-deficient cells for gene-rich microchromosomes, but to lesser extent for gene-poor macrochromosomes. These results indicate that Arp6 and H2A.Z contribute to the radial distribution of CTs through different mechanisms. Microarray analysis suggested that the localization of chromatin to the nuclear periphery per se is insufficient for the repression of most genes

The actin family member Arp6 and the histone variant H2A.Z are required for spatial positioning of chromatin in chicken cell nuclei

The spatial organization of chromatin in the nucleus contributes to genome function and is altered during the differentiation of normal and tumorigenic cells. Although nuclear actin-related proteins (Arps) have roles in the local alteration of chromatin structure, it is unclear whether they are involved in the spatial positioning of chromatin. In the interphase nucleus of vertebrate cells, gene-dense and gene-poor chromosome territories (CTs) are located in the center and periphery, respectively. We analyzed chicken DT40 cells in which Arp6 had been knocked out conditionally, and showed that the radial distribution of CTs was impaired in these knockout cells. Arp6 is an essential component of the SRCAP chromatin remodeling complex, which deposits the histone variant H2A.Z into chromatin. The redistribution of CTs was also observed in H2A.Z-deficient cells for gene-rich microchromosomes, but to lesser extent for gene-poor macrochromosomes. These results indicate that Arp6 and H2A.Z contribute to the radial distribution of CTs through different mechanisms. Microarray analysis suggested that the localization of chromatin to the nuclear periphery per se is insufficient for the repression of most genes

Pathophysiological analysis of detrusor overactivity following partial bladder outlet obstruction

　Introduction: Detrusor overactivity (DO) following partial bladder outlet obstruction (PBOO) is a common urological condition in humans, with 50-70% patients with PBOO complicated with DO. The pathological mechanisms of DO following PBOO are largely unknown, but inflammatory changes may play a key role. We hypothesized that inflammation is important in the earlier pathophysiological phase before overproduction of oxidative stress in DO following PBOO. Therefore, we investigated the relationships among bladder function, ischemia, oxidative stress and inflammation in DO following PBOO in PBOO model mice.　Materials and Methods: C57BL/6J male mice aged 10 to 15 weeks were used in the study. PBOO model mice were created surgically by ligation of the proximal urethra with 5-0 nylon suture under inhalation anesthesia. Sham-operated mice were used as controls. Pathophysiological changes in the bladder at 1, 3 and 5 weeks after creation of the PBOO model mice were compared with those in sham-operated mice using functional, histological, biochemical and immunohistochemical analyses.　Results: Functional analysis using a pressure flow study showed increased maximum detrusor pressure at 1 week and DO from 3 to 5 weeks after creation of the PBOO model. Histological analysis using hematoxylin-eosin and Masson-Trichrome staining showed greater invasion of inflammatory cells and fibrosis in PBOO model mice compared with sham-operated mice at 3 and 5 weeks. Inflammatory cells were mainly present in interstitial tissue, and fibrosis gradually infiltrated from interstitial tissue to the muscular layer. Ischemia analysis showed significantlyincreased HIF-1α in PBOO model mice at all time points. Oxidative stress analysis indicated significantly increased levels of ROS from 1 week and 8-OHdG from 3weeks in PBOO model mice. An inflammation-related proteome assay showed high levels of colony stimulating factor (CSF) family proteins at 1 week and IL-2, IL-3, IL-17A, IL-23, MMP-3, MMP-9 and periostin from 3 to 5 weeks in PBOO model mice.　Conclusions: Oxidative stress and inflammatory changes showed contemporaneous increase in pathophysiology of detrusor overactivity following partial bladder outlet obstruction. Especially, CSF family and ROS changes are showed in the early stage, and might be a predict marker in the pathophysiology of DO following PBOO at the early stage

Pathological analysis of spermatic dysfunction following testicular ischemia-reperfusion injury\n

　Introduction & Objectives: Torsion, which may result in testicular ischemia, requires emergency surgery to restore testicular blood flow. However, the risk of spermatic dysfunction remains even if surgery is performed. The pathology of spermatic dysfunction in testicular ischemia-reperfusion injury (TIRI) remains unclear. A previous study showed the relevance of inflammation and oxidative stress in the other organs of ischemia-reperfusion injury. We hypothesized that inflammation and oxidative stress play key roles in causing spermatic dysfunction following TIRI. We investigated the pathophysiology of spermatic dysfunction in TIRI focusing on inflammatory changes using TIRI model mice.　Materials and Methods: The study used C57BL/6J male mice aged 10 to 15 weeks. To create TIRI model mice, the unilateral (left side) testicular vessels were clamped using Dieffenbach clamps (Bulldog clamps) for 1 hour and de-clamped. The bilateral testes were removed at 0 (ischemic state), 1, 3, and 5 weeks after creating the TIRI model mice. Spermatic changes following TIRI were investigated by analyzing the histology of the testes and semen and assessing levels of inflammation and oxidative stress. Semen was collected from the bilateral cauda epididymites and investigated using the sperm motility analysis system (SMAS).　Results: Histological analysis after hematoxylin-eosin staining showed tissue thickening in interstitial tissues at week 1 and 3 on the left (affected) testis, and week 1, 3 and 5 on the right (unaffected) testis. The infiltration of lymphocytes-predominant inflammatory cells were observed at week 1 and week 3 on the left (affected) testis. The destruction of ductal structures and giant cells were observed at weeks 3 and 5 on the left (affected) testis and week 5 on the right (unaffected) testis. SMAS showed significantly decreased spermatic concentration and motility in both testes of TIRI model mice compared with those of sham-operated mice at weeks 1, 3 and 5. Inflammation analysis using an inflammation-related proteome assay showed significantly increased levels of cytokines (IL-2, IL-3, IL-17A, and IL-23) and chemokines (CCL2, CCL5, CXCL1, and CX3CL1) at weeks 1, 3, and 5 in both testes of TIRI model mice. For the assessment of oxidative stress, enzyme-linked immuno-sorbent assay (ELISA) for 8-hydroxy-2’-deoxyguanosine (8-OHdG) was performed, which showed that levels of 8-OHdG were significantly increased in the left (affected) testis of TIRI model mice compared with that of sham-operated mice at all observation periods. Meanwhile, ELISA showed that levels of 8-OHdG in the right (unaffected) testis were significantly increased in TIRI model mice at weeks 3 and 5 compared with that of sham-operated mice.　Conclusions: Spermatic dysfunction following TIRI is induced by inflammation and oxidative stress. Inflammation and oxidative stress may be novel regulatory factors to prevent spermatic dysfunction following TIRI

Preventive effect of indoleamine 2,3-dioxygenase 1 inhibition on lipopolysaccharide-induced prostatitis

　Introduction and Objectives: Bacterial infections are the main cause of acute prostatitis and are treated with appropriate antimicrobial therapy. However, approximately 5% of patients continue to have inflammatory symptoms even after receiving antibacterial therapy, leading to refractory conditions. Bacterial prostatitis requires additional therapy, focusing on inflammatory changes. Indoleamine 2,3-dioxygenase 1 (IDO1) catalysis is the first rate-limiting step of tryptophan metabolism. IDO1 is expressed in the prostate and plays a key role in the immune response. As the first step in investigating the relationship between acute prostatitis and IDO1, we investigated the preventive effect of IDO1 inhibition on lipopolysaccharide (LPS)- induced prostatitis using IDO knockout (Ido1 −/−) mice in this study.　Materials and Methods: The study used Ido1 −/− and wild-type (Ido1 +/+) C57BL/6J malemice aged 10–15 weeks. LPS Escherichia coli O26 (100μg/PBS, 100μL) was administered transurethrally into the lower urinary tract to create a mouse model of LPS-induced prostatitis. The prostates were removed 1, 3, 5, and 7 days after creating the model mice. Histological, immunohistochemical, and biochemical analyses were used to compare the preventive effect in Ido1 −/− mice compared with that in Ido1+/+ mice.　Results: HE staining showed suppression of ductal destruction following infiltration of inflammatory cells in Ido1 −/− mice compared with Ido1 +/+ mice. The enzyme-linked immunosorbent assay (ELISA) method was used for kynurenine pathway analysis, which showed significantly maintained tryptophan levels and decreased L-kynurenine levels in Ido1 −/− mice compared to Ido1 +/+ mice. The IDO1 assay in Ido1 +/+ mice showed significantly increased levels during all observation periods after creating the model compared with that under normal conditions. Immunofluorescent staining using five types of cytokines and chemokines (IL-2, IL-4, IL-17, CCL2, and CCL3) related to the pathophysiology of acute prostatitis showed decreased expression of these cytokines and chemokines in Ido1 -/- mice compared with Ido1 +/+ mice. Inflammation-related proteome assays showed decreased levels of IL-1β, IL-4, IL-5, IL-6, IL-17, CCL2, CCL3, CXCL1, CXCL11, and tissue inhibitor of matrix metalloproteinases (TIMP)-1 in Ido1 −/− mice compared with Ido1 −/− mice during all observation periods after model creation.　Conclusions: IDO1 is involved in LPS-induced prostatitis through cytokines and chemokines. IDO1 inhibition contributes to the prevention of LPS-induced prostatitis. IDO1 inhibition has the potential to serve as an additional therapy for acute prostatitis