Inhalation of hydrogen gas mitigates sevoflurane-induced neuronal apoptosis in the neonatal cortex and is associated with changes in protein phosphorylation.

Inhalation of hydrogen (H2) gas is therapeutically effective for cerebrovascular diseases, neurodegenerative disorders, and neonatal brain disorders including pathologies induced by anesthetic gases. To understand the mechanisms underlying the protective effects of H2 on the brain, we investigated the molecular signals affected by H2 in sevoflurane-induced neuronal cell death. We confirmed that neural progenitor cells are susceptible to sevoflurane and undergo apoptosis in the retrosplenial cortex of neonatal mice. Co-administration of 1-8% H2 gas for 3 h to sevoflurane-exposed pups suppressed elevated caspase-3-mediated apoptotic cell death and concomitantly decreased c-Jun phosphorylation and activation of the c-Jun pathway, all of which are induced by oxidative stress. Anesthesia-induced increases in lipid peroxidation and oxidative DNA damage were alleviated by H2 inhalation. Phosphoproteome analysis revealed enriched clusters of differentially phosphorylated proteins in the sevoflurane-exposed neonatal brain that included proteins involved in neuronal development and synaptic signaling. H2 inhalation modified cellular transport pathways that depend on hyperphosphorylated proteins including microtubule-associated protein family. These modifications may be involved in the protective mechanisms of H2 against sevoflurane-induced neuronal cell death.

Newer parameters of the octreotide test in patients with acromegaly.

PURPOSE: Predicting the therapeutic effects of first-generation somatostatin receptor ligands (fg-SRLs) is important when assessing or planning effective treatment strategies in patients with acromegaly. The oft-used maximum growth hormone (GH) suppression rate parameter of the octreotide test has a suboptimal predictive value. Therefore, this study explored newer parameters of the octreotide test for predicting the therapeutic effect of long-acting fg-SRLs. METHODS: In this single-center retrospective study, the octreotide test parameters and the therapeutic effects of fg-SRL at 3 months were investigated in 45 consecutive treatment-naïve patients with acromegaly between April 2008 and March 2023. Additionally, the relationship between the octreotide test parameters and the therapeutic effects of fg-SRLs was investigated. Tumor shrinkage was evaluated based on changes in the longitudinal diameter of the macroadenomas. The area GH suppression rate-time under the curve (AUC) and the time to nadir GH level were calculated and compared with the maximum GH suppression rate. RESULTS: The AUC estimated reductions in serum insulin-like growth factor I, and tumor shrinkage. The time to nadir GH level predicted tumor shrinkage more robustly than the maximum GH suppression rate in patients with macroadenoma. CONCLUSION: The AUC and time to nadir GH level may potentially be newer parameters of the octreotide test for estimating the therapeutic effect of fg-SRLs.

Serum Growth Differentiation Factor 15 Levels Predict the Incidence of Frailty among Patients with Cardiometabolic Diseases.

INTRODUCTION: Frailty is a crucial health issue among older adults. Growth differentiation factor 15 (GDF15) is associated with inflammation, oxidative stress, insulin resistance, and mitochondrial dysfunction, which are possible pathogeneses of frailty. However, few longitudinal studies have investigated the association between GDF15 and the incidence of frailty. Therefore, we investigated whether high serum GDF15 levels are associated with the incidence of frailty. METHODS: A total of 175 older adults (mean age: 77 ± 6 years; 63% women) with cardiometabolic diseases and no frailty out of the two criteria at baseline participated. Individuals with severe renal impairment or severe cognitive impairment were excluded. Serum GDF15 levels were measured at baseline. Patients were asked to assess frailty status at baseline and annually during follow-up using the modified version of the Cardiovascular Health Study (mCHS) and the Kihon Checklist (KCL). We examined the association between GDF15 tertiles and each frailty measure during follow-up (median 38-39 months). In the multivariate Cox regression analysis, with the GDF15 tertile groups as the explanatory variables, hazard ratios (HRs) and 95% confidence intervals (CIs) for incident frailty were calculated after adjusting for covariates and using the lowest tertile group as the reference. RESULTS: During the follow-up period, 25.6% and 34.0% of patients developed frailty, as defined by the mCHS and KCL, respectively. The highest GDF15 tertile group had a significantly higher incidence of mCHS- or KCL-defined frailty than the lowest GDF15 tertile group. Multivariate Cox regression analysis revealed that the adjusted HRs for incident mCHS- and KCL-defined frailty in the highest GDF15 tertile group were 3.9 (95% CI: 1.3-12.0) and 2.7 (95% CI: 1.1-6.9), respectively. CONCLUSION: High serum GDF15 levels predicted the incidence of frailty among older adults with cardiometabolic diseases and could be an effective marker of the risk for frailty in interventions aimed at preventing frailty, such as exercise and nutrition.

Responsiveness to DDAVP in Cushing's disease is associated with USP8 mutations through enhancing AVPR1B promoter activity.

PURPOSE: To clarify the characteristics of Cushing's disease (CD) patients who respond to the desmopressin (DDAVP) test and its underlying mechanisms. METHODS: Forty-seven patients with CD who underwent DDAVP testing were included. Patients were divided into two groups: DDAVP test (+) (adrenocorticotropic hormone [ACTH] levels increased by ≥ 1.5-fold during the DDAVP test) and DDAVP test (-) (ACTH levels increased by < 1.5-fold). AVP receptor expression levels in these tumors were quantified using quantitative RT-PCR and immunohistochemistry. AVP receptor promoter activity was analyzed using a dual-luciferase reporter assay system. RESULTS: Females (96.9%) and USP8 mutants (85.7%) were more prevalent in the DDAVP test (+) than in the DDAVP test (-). Indeed, the ACTH and cortisol responsiveness to DDAVP was greater in USP8 mutation positive tumors than that in USP8 wild type tumors (3.0-fold vs. 1.3-fold, 1.6-fold vs. 1.1-fold, respectively). Responsiveness to DDAVP was correlated with the expression levels of AVPR1B, but not with those of AVPR2. Comparably, Avpr1b promoter activity was enhanced by the overexpression of mutant USP8 compared to the wild type. CONCLUSIONS: We found that the responsiveness of ACTH to DDAVP in CD was greater in tumors with USP8 mutations. The present data suggest that USP8 mutations upregulate the AVPR1B promoter activity. Additionally, we showed that the DDAVP test can predict the presence of USP8 mutations.

Analysis of thyroid function in Japanese patients with coronavirus disease 2019.

Thyroid dysfunction that is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is becoming increasingly recognized. However, only a few reports in Japan have addressed this issue to date. In this study, we sought to clarify whether infection with SARS-CoV-2 affected thyroid hormone levels and whether these hormones could be better predictors of prognosis in patients with coronavirus disease 2019 (COVID-19). Accordingly, we retrospectively examined 147 cases wherein thyroid hormones were measured at the time of admission among 848 Japanese patients with COVID-19 admitted to the Hyogo Prefectural Kakogawa Medical Center. All patients underwent thyroid function testing upon hospital admission. More than half (59.1%) of the patients were euthyroid. Twenty-four percent of patients had serum thyroid-stimulating hormone (TSH) levels lower than the reference range with normal serum free thyroxine (fT4) levels, and 3.4% of the patients had low TSH with high fT4 levels. Over 70% of the patients with moderate and severe COVID-19 had low serum free triiodothyronine (fT3) levels. Serum TSH and fT3 levels were inversely correlated with disease severity. The mortality rate in patients with low serum fT3 levels was significantly higher than that in those with normal serum fT3 levels.

Proteomic analysis of extracellular vesicles identified PI3K pathway as a potential therapeutic target for cabazitaxel-resistant prostate cancer.

BACKGROUND: Cabazitaxel (CBZ) is now widely used for prostate cancer (PC) patients resistant to docetaxel (DOC), however, most patients eventually acquire resistance. It will, therefore, be of great benefit to discover novel therapeutic target for the resistance. We aimed to identify candidate therapeutic targets for CBZ-resistance by proteomic analysis of extracellular vesicles (EVs) isolated from serum of DOC-resistant PC patients who later developed CBZ-resistance as well as those harvested from culture medium of DOC- and CBZ-resistant PC cell lines. METHODS: Using T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4) conjugated to magnetic beads, EVs were purified from serum of PC patients with DOC-resistance that was collected before and after acquiring CBZ-resistance and conditioned medium of DOC-resistant (22Rv1DR) and CBZ-resistant (22Rv1CR) PC cell lines. Protein analysis of EVs was performed by nanoLC-MS/MS, followed by a comparative analysis of protein expression and network analysis. The cytotoxic effect of a phosphatidylinositol-3-kinase (PI3K) inhibitor, ZSTK474, was evaluated by WST-1 assay. The expression and phosphorylation of PI3K and PTEN were examined by western blot analysis. RESULTS: Among differentially regulated proteins, 77 and 61 proteins were significantly increased in EVs from CBZ-resistant PC cell line and patients, respectively. A comparison between the two datasets revealed that six proteins, fructose-bisphosphate aldolase, cytosolic nonspecific dipeptidase, CD63, CD151, myosin light chain 9, and peroxiredoxin-6 were elevated in EVs from both cell line and patients. Network analysis of the increased EV proteins identified pathways associated with CBZ-resistance including PI3K signaling pathway. ZSTK474 significantly inhibited growth of 22Rv1CR cells and improved their sensitivity to CBZ. In 22Rv1CR cells, PI3K was activated and PTEN that inhibits PI3K was deactivated. CONCLUSIONS: Proteomic analysis of serum EVs was successfully accomplished by using Tim-4 as a tool to isolate highly purified EVs. Our results suggest that the combination use of CBZ and PI3K inhibitor could be a promising treatment option for CBZ-resistant PC patients.

Muscle fiber type specific alterations of mitochondrial respiratory function and morphology in aged female mice.

Mitochondrial dysfunction is considered to be a major cause of sarcopenia, defined as age-related muscle fiber atrophy and muscle weakness, as reduced mitochondrial respiration and morphological changes such as ragged red fibers (RRFs) are observed in aging muscles. However, the role of mitochondrial dysfunction in sarcopenia is not fully elucidated. Although previous studies have suggested that aging has a fiber type-specific effect on mitochondrial function, little is known about mitochondrial changes in individual fiber types. Here, we used C57BL/6NCr female mice to identify fiber type-specific pathological changes, examine the significance of pathological changes in sarcopenia, and identify possible mechanisms behind mitochondrial changes in slow-twitch soleus muscle (SOL) and fast-twitch extensor digitorum longus muscle (EDL). We observed reduced type I fiber-specific mitochondrial respiratory enzyme activity, impaired respiration, and subsarcolemmal mitochondrial accumulation in aged SOL, which was different from RRFs. These pathological alterations were not directly associated with fiber atrophy. Additionally, we found increased oxidative stress markers in aged SOL, suggesting that oxidative stress is involved in the pathological and functional changes in mitochondria. Meanwhile, obvious mitochondrial changes were not seen in aged EDL. Thus, age-related mitochondrial dysfunction is specific to the fiber type and may correlate with the muscle quality rather than the muscle mass.

Mechanistic insights into immune checkpoint inhibitor-related hypophysitis: a form of paraneoplastic syndrome.

BACKGROUND: Immune checkpoint inhibitors (ICIs) as a cancer immunotherapy have emerged as a treatment for multiple advanced cancer types. Because of enhanced immune responses, immune-related adverse events (irAEs), including endocrinopathies such as hypophysitis, have been associated with the use of ICIs. Most underlying mechanisms of ICI-related hypophysitis remain unclear, especially for programmed cell death-1 (PD-1)/PD-1 ligand 1 (PD-L1) inhibitors. We hypothesized that ICI-related hypophysitis is associated with paraneoplastic syndrome caused by ectopic expression of pituitary-specific antigens. METHODS: Twenty consecutive patients with ICI-related hypophysitis between 2017 and 2019 at Kobe University Hospital were retrospectively analyzed. Circulating anti-pituitary antibodies were detected using immunofluorescence staining and immunoblotting. Ectopic expression of pituitary autoantigens in tumor specimens was also examined. RESULTS: Eighteen patients were treated with PD-1/PD-L1 inhibitors, and two were treated with a combination of cytotoxic T-lymphocyte antigen-4 (CTLA-4) and PD-1 inhibitors. All patients showed adrenocorticotropic hormone (ACTH) deficiency and additionally, three showed thyroid-stimulating hormone (TSH) deficiency, and one showed gonadotropin-releasing hormone (GnRH) deficiency. Among these patients, three exhibited anti-pituitary antibodies, two with anti-corticotroph antibody and one with anti-somatotroph antibody. Interestingly, the anti-corticotroph antibody recognized proopiomelanocortin (POMC) and those two patients exhibited ectopic ACTH expression in the tumor, while the patients without anti-corticotroph antibody did not. CONCLUSIONS: We demonstrated 10% of PD-1/PD-L1 inhibitors-related hypophysitis were associated with the autoimmunity against corticotrophs and maybe caused as a form of paraneoplastic syndrome, in which ectopic expression of ACTH in the tumor was observed. It is also suggested that the pathophysiology is heterogenous in ICI-related hypophysitis.

Serum Exosomal Gamma-Glutamyltransferase Activity Increased in Patients with Renal Cell Carcinoma with Advanced Clinicopathological Features.

BACKGROUND: There has been no clinically useful diagnostic or prognostic biomarker for renal cell carcinoma (RCC). Serum γ-glutamyltransferase (GGT) activity has been reported to be a prognostic marker for several types of cancer including RCC. Exosomes or small extracellular vesicles present in body fluids have potential as a biomarker. We have recently demonstrated that GGT activity on exosomes isolated from serum is useful for the differential diagnosis of prostate cancer and benign prostate hyperplasia. In this study, we aimed to examine if serum exosomal GGT activity could be a marker for RCC. METHODS: We examined GGT1 expression and GGT activity in cell lysates and exosomes from culture medium of HK-2 proximal tubule epithelial and RCC cell lines. GGT activity was measured using a fluorescent probe for GGT, γ-glutamyl hydroxymethyl rhodamine green. Serum and serum exosomal GGT activities were measured in patients with RCC. GGT1 expression in RCC tissues was evaluated by immunohistochemical staining. RESULTS: GGT1 levels in exosomes from KMRC-1, OS-RC-2 and 786-O cells were elevated compared with those from HK-2 cells. In exosomes, GGT1 expression correlated with GGT activity determined using a fluorescent probe for GGT. In RCC patients, serum exosomal GGT activity was elevated in those with advanced stages (III/IV vs. I/II, p = 0.037) and those with microvascular invasion (with vs. without, p = 0.034). Immunohistochemical analysis showed that membranous GGT1 expression was increased in RCC with microvascular invasion. Notably, preoperative serum exosomal GGT activity could predict the likelihood of having microvascular invasion diagnosed by pathological examination of surgically resected specimens. CONCLUSIONS: Our results suggest that serum exosomal GGT activity could be a clinically useful marker for advanced clinicopathological features of RCC patients, and its combined use with conventional diagnostic modalities may improve the diagnosis and treatment of patients.

Mitochondrial stress and GDF15 in the pathophysiology of sepsis.

Mitochondria are multifunctional organelles that regulate diverse cellular processes. Mitochondrial stress, including stress generated by electron transport chain defects and impaired mitochondrial proteostasis, is intimately involved in various diseases and pathological conditions. Sepsis is a life-threatening condition that occurs when an imbalanced host response to infection leads to organ dysfunction. Metabolic disturbances and impaired immune responses are implicated in the pathogenesis and development of sepsis. Given that mitochondria play central roles in cellular metabolism, mitochondrial stress is predicted to be involved in the pathological mechanism of sepsis. Under mitochondrial stress, cells activate stress response systems to maintain homeostasis. This mitochondrial stress response transcriptionally activates genes involved in cell survival and death. Mitochondrial stress also induces the release of distinctive secretory proteins from cells. Recently, we showed that growth differentiation factor 15 (GDF15) is a major secretory protein induced by mitochondrial dysfunction. In this article, we provide a brief overview of mitochondrial stress response and GDF15, and discuss the potential role of GDF15 in the pathophysiology of sepsis.

Urinary exosome as a potential biomarker for urinary tract infection.

Unlike urinary tract infection (UTI), asymptomatic bacteriuria (ABU) should not be treated, with some exceptions such as pregnant women and patients who will undergo traumatic urologic interventions. However, there has been no clinically available marker for their differential diagnosis. Exosomes or small extracellular vesicles carry proteins contained in cells from which they are derived, thus having the potential as a biomarker of several diseases. On the basis of the hypothesis that the molecular signature of exosomes in urine may differ between UTI and ABU patients, we examined if urinary exosomes could serve as a marker for their differential diagnosis. Exosomes were isolated by ultracentrifugation or affinity-based method from cell culture medium of monocytic THP-1 and uroepithelial SV-HUC-1 cells and human urine. Protein expression was examined by Western blot analysis, ELISA, and CLEIA. The results showed that the levels of intracellular signalling molecules Akt and ERK and transcription factor NF-κB increased in exosomes isolated from THP-1 and SV-HUC-1 cells cocultured with Escherichia coli and/or treated with lipopolysaccharide. In urinary exosomes of UTI patients, Akt significantly diminished, and an exosomal marker CD9 showed a trend to decrease after treatment with antimicrobial agents. More importantly, Akt and CD9 levels in urinary exosomes were higher in UTI patients than in ABU patients, which was also observed after correction by urine creatinine. Collectively, these results suggest that Akt and CD9 in urinary exosomes could be useful markers for differential diagnosis of UTI and ABU.

IgG4-related hypophysitis in patients with autoimmune pancreatitis.

PURPOSE: IgG4-related disease involves various organs including the pituitary and pancreas. The prevalence of IgG4-related hypophysitis is relatively rare compared with IgG4-related pancreatitis (autoimmune pancreatitis). Although several cases demonstrating both autoimmune pancreatitis and hypophysitis have been reported, the prevalence of IgG4-related hypophysitis in patients with autoimmune pancreatitis remains unknown. This study aimed at screening for IgG4-related hypophysitis to accurately determine its prevalence in patients with autoimmune pancreatitis. METHODS: In this cohort study, we screened IgG4-related hypophysitis via pituitary magnetic resonance imaging (MRI) and endocrinological examination in 27 patients who were undergoing follow-up for autoimmune pancreatitis at Kobe University Hospital between 2014 and 2018. RESULTS: Among 27 patients with autoimmune pancreatitis, 5 patients exhibited morphological abnormalities in the pituitary (18.5%). Among them, one patient (3.7%) met the criteria for hypophysitis with an enlarged pituitary and stalk concomitant with hypopituitarism. After glucocorticoid treatment, the enlarged pituitary shrank and became empty sella during the clinical course. Four patients (14.8%) revealed empty sella without obvious pituitary dysfunction. Four of 5 patients with morphological pituitary abnormalities showed multiple organ involvement in addition to pancreatic and pituitary involvement. Accordingly, multiple organ involvement was more prevalent in patients with morphological pituitary abnormalities (80%) compared to those without (48%). CONCLUSIONS: Although a large-scale study is necessary to validate these results, these data suggest that the prevalence of hypophysitis in patients with autoimmune pancreatitis may be underestimated. Based on our findings, we recommend screening for hypophysitis, especially in patients with multiple organ involvement.

Isolated adrenocorticotropic hormone deficiency as a form of paraneoplastic syndrome.

PURPOSE: In isolated adrenocorticoropic hormone (ACTH) deficiency (IAD), autoimmunity against corticotrophs has been suggested; however, the pathogenesis remains largely unknown. Large cell neuroendocrine carcinoma (LCNEC) of the lung is a pulmonary tumor of high-grade malignant neuroendocrine tumor and it reportedly caused paraneoplastic syndrome by autoimmunity in several cases. METHODS: A 42-year-old woman with isolated adrenocorticotropic (ACTH) hormone deficiency (IAD) was diagnosed with large cell neuroendocrine carcinoma (LCNEC) 3 years after being diagnosed with IAD. We hypothesized that the LCNEC played a causal role in the development of IAD as a paraneoplastic syndrome and analyzed the autoimmunity. We also analyzed another case of ectopic ACTH syndrome to prove this hypothesis. RESULTS: The LCNEC tissue revealed an ectopic ACTH expression and lymphocyte infiltration. Interestingly, autoantibody against the proopiomelanocortin (POMC) protein was detected in the peripheral blood. Although, patient's serum did not show any effects on cell viability, proliferation, nor pomc expression in a corticotroph cell line, AtT20 cells, patient's lymphocytes in the peripheral blood specifically reacted toward POMC protein, indicating a presence of cytotoxic T lymphocytes (CTLs). In addition, the analysis of another case of ectopic ACTH syndrome showed lymphocyte infiltration not only in the metastatic liver tumors but also in the pituitary. Moreover, most CD8-positive cells resided adjacent to corticotrophs. CONCLUSIONS: These data indicate that the ectopic ACTH expression in the tumor evoked the autoimmunity to corticotrophs and caused IAD as a form of paraneoplastic syndrome.

'Haemophilus quentini' in the urethra of men complaining of urethritis symptoms.

We isolated a cryptic genospecies of Haemophilus influenzae referred to as 'Haemophilus quentini' in the urethra of 3 men complaining of urethritis symptoms. H. influenzae strains, which had been isolated from the urethra in 77 of 1518 men complaining of urethritis symptoms, identified by the conventional test, and stored, were re-cultured for this study. Sixty-seven strains surviving storage were screened by a PCR-based assay specific for the cryptic genital Haemophilus genospecies. Three strains (HI09003, HI11006, and HI14016) were screened by PCR and identified as 'H. quentini' by 16S rRNA sequencing. The men positive for HI09003 and HI11006 were diagnosed as having non-chlamydial non-gonococcal urethritis (NGU), and their demographic and clinical features were similar to those of NGU caused by other pathogens. The man positive for HI14016 was ultimately diagnosed as having condyloma acuminatum on the glans. The 3 strains of 'H. quentini' produced no ß-lactamase and were susceptible to ampicillin and other antimicrobial agents, including cephalosporins, fluoroquinolones, tetracyclines, and macrolides, recommended for treatment for urethritis. 'H. quentini' would be an uncommon pathogen in men with urogenital infections. Based on the clinical features of the two patients with 'H. quentini'-positive NGU, it would be difficult to predict the presence of 'H. quentini' in the urethra. The 3 strains of 'H. quentini' were susceptible to a variety of antimicrobial agents. Further accumulation of data regarding 'H. quentini' infections is needed to characterize the pathogenic roles of this genospecies in urogenital infections and to establish appropriate management of 'H. quentini' infections.

Exosomes expressing carbonic anhydrase 9 promote angiogenesis.

Exosomes or microvesicles that are secreted from cells are considered to play important roles in tumor microenvironment. Carbonic anhydrase 9 (CA9), which is induced by hypoxia-inducible factor 1 (HIF1) in response to hypoxia, is overexpressed in many types of cancer including renal cell carcinoma (RCC). We examined the expression level of CA9 in several RCC cell lines and found that the basal level of CA9 was much higher in OSRC-2 cells than in Caki-1, KMRC-1 and 786-O cells. Consistent with the intracellular expression levels, CA9 was abundantly detected in exosomes isolated by ultracentrifugation from OSRC-2 cells. Density gradient centrifugation of OSRC-2 and 786-O exosomes confirmed the co-presence of CA9 with exosomal markers. Upon hypoxia and treatment with CoCl2, a hypoxia mimic agent, the CA9 level in exosomes was increased for all cell lines. In order to examine the effects of CA9 exosomes on angiogenesis, we generated stably transfected HEK293 cells expressing CA9. Immunocytochemical staining demonstrated the uptake of CA9 exosomes by human umbilical vein endothelial cells (HUVEC). In vitro angiogenesis assays using HUVEC revealed that CA9 exosomes promoted migration and tube formation. Lastly, MMP2 expression was increased by treatment with CA9 exosomes in HUVEC. Taken together, our results suggest the possibility that CA9 exosomes released from hypoxic RCC may enhance angiogenesis in microenvironment, thereby contributing to cancer progression.

Gamma-glutamyltransferase activity in exosomes as a potential marker for prostate cancer.

BACKGROUND: Exosomes or extracellular vesicles have the potential as a diagnostic marker for various diseases including cancer. In order to identify novel exosomal markers for prostate cancer (PC), we performed proteomic analysis of exosomes isolated from PC cell lines and examined the usefulness of the marker in patients. METHODS: Exosomes isolated by differential centrifugation from the culture medium of androgen-dependent LNCaP prostate cancer cell line and its sublines of partially androgen-independent C4, androgen-independent C4-2 and bone metastatic C4-2B were subjected to iTRAQ-based proteomic analysis. Exosomes were also isolated by immunocapture and separated by size exclusion chromatography and density gradient centrifugation. Protein expression was determined by Western blot analysis. GGT activity was measured using a fluorescent probe, γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG). Immunohistochemical analysis of tissues was performed using anti-GGT1 antibody. RESULTS: Among proteins upregulated in C4-2 and C4-2B cells than in LNCaP cells, we focused on gamma-glutamyltransferase 1 (GGT1), a cell-surface enzyme that regulates the catabolism of extracellular glutathione. The levels of both GGT1 large and small subunits were elevated in exosomes isolated from C4-2 and C4-2B cells by differential centrifugation and by immunocapture with anti-CD9 or -prostate-specific membrane antigen (PSMA) antibody. In cell lysates and exosomes, GGT1 expression correlated with GGT activity. Size exclusion chromatography of human serum demonstrated the presence of GGT activity and GGT1 subunits in fractions positive for CD9. Density gradient centrifugation revealed the co-presence of GGT1 subunits with CD9 in exosomes isolated by differential centrifugation from human serum. Since GGT activity correlated with GGT1 expression in serum exosomes isolated by differential centrifugation, we measured serum exosomal GGT activity in patients. Unexpectedly, we found that serum exosomal GGT activity was significantly higher in PC patients than in benign prostatic hyperplasia (BPH) patients. In support of this finding, immunohistochemical analysis showed increased GGT1 expression in PC tissues compared with BPH tissues. CONCLUSIONS: Our results suggest that serum exosomal GGT activity could be a useful biomarker for PC.

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.

Ascorbic acid deficiency affects genes for oxidation-reduction and lipid metabolism in livers from SMP30/GNL knockout mice.

BACKGROUND: We sought to elucidate the effect of an ascorbic acid (AA) deficiency on gene expression, because the water soluble antioxidant AA is an important bioactive substance in vivo. METHODS: We performed microarray analyses of the transcriptome in the liver from senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which are unable to synthesize AA in vivo. RESULTS: Our microarray analysis revealed that the AA deficiency increased gene expression related to the oxidation-reduction process, i.e., the nuclear factor, erythroid derived 2, like 2 (Nrf2) gene, which is a reactive oxygen species-sensitive transcriptional factor. Moreover, this AA deficiency increased the expression of genes for lipid metabolism including the cytochrome P450, family 7, subfamily a, polypeptide 1 (Cyp7a1), which is a late-limiting enzyme of the primary bile acid biosynthesis pathway. Although an AA deficiency increased the Cyp7a1 protein level, bile acid levels in the liver and gallbladder decreased. Since Cyp7a1 has a heme iron at the active site, AA must function as a reductant of the iron required for the continuous activation of Cyp7a1. CONCLUSIONS: This experimental evidence strongly supports a role for AA in the physiologic oxidation-reduction process and lipid metabolism including bile acid biosynthesis. GENERAL SIGNIFICANCE: Although many effects of AA supplementation have been reported, no microarray analysis of AA deficiency in vivo is available. Results from using this unique model of AA deficiency, the SMP30/GNL-KO mouse, now provide new information about formerly unknown AA functions that will implement further study of AA in vivo.

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.