Forebrain expression of serine racemase during postnatal development.

N-methyl-D-aspartate receptors (NMDARs) are important for synaptogenesis, synaptic maturation and refinement during the early postnatal weeks after birth. Defective synapse formation or refinement underlie cognitive and emotional abnormalities in various neurodevelopmental disorders (NDDs), including schizophrenia (Sz) and autism spectrum disorder (ASD). Serine racemase (SR) is a neuronal enzyme that produces D-serine, a co-agonist required for full NMDAR activation. NMDAR hypofunction as a result of genetic SR elimination and reduced synaptic availability of D-serine reduces neuronal dendritic arborization and spine density. In adult mouse brain, the expression of SR parallels that of NMDARs across forebrain regions including the striatum, amygdala, hippocampus, and medial prefrontal cortex (mPFC). However, there have yet to be studies providing a detailed characterization of the spatial and temporal expression of SR during early periods of synaptogenesis. Here, we examined the postnatal expression of SR in cortical and subcortical brain regions important for learning, memory and emotional regulation, during the first four weeks after birth. Using dual-antigen immunofluorescence, we demonstrate that the number of SR+ neurons steadily increases with postnatal age across the mPFC, amygdala, hippocampus and striatum. We also identified differences in the rate of SR protein induction both across and within brain regions. Analyzing existing human post-mortem brain in situ data, there was a similar developmental mRNA expression profile of SRR and GRIN1 (GluN1 subunit) from infancy through the first decade of life. Our findings further support a developmental role for D-serine mediated NMDAR activation regulating synaptogenesis and neural circuit refinement, which has important implications for the pathophysiology of Sz and other NDDs.

Bacterial production of maize and human serine racemases as partially active inclusion bodies for d-serine synthesis.

Recombinant protein overexpressed in Escherichia coli is often less folded, leading to form the insoluble aggregates also called as inclusion bodies (IBs). IBs are classified as active and inactive ones, and enzyme produced as active IBs is the novel carrier-free immobilized material. In this study, we determined that His6-tagged serine racemase (SR) from maize or human produced as partially active IBs maintained the activities for reversible racemization of l-serine to d-serine but lost the activities for irreversible ß-elimination of both enantiomers, in contrast to the soluble one displaying all activities. Fourier transform infrared spectroscopy analysis showed structural changes between the soluble and insoluble SR. Compared with the soluble SR with attachment of the N-terminal cellulose-binding module via the oriented immobilization of the regenerated amorphous cellulose, the insoluble SR with the fusion of the N-terminal aggregation-inducible tag GFIL8 displayed higher production and usage efficiency, lower leaky capacity, more stability at 4 °C storage with the prolonged time, less sensitivity to the limited proteolysis mediated by trypsin, and higher yield of the synthesized d-serine. These advantages allow the SRs as partially active IBs to synthesize d-serine for medical and agricultural applications.

RNA-based markers in biopsy cores with atypical small acinar proliferation: Predictive effect of T2E fusion positivity and MMP-2 upregulation for a subsequent prostate cancer diagnosis.

BACKGROUND: Atypical small acinar proliferation (ASAP) is a precursor lesion of prostate cancer (PC), and PC develops from this suspicious focus or an unsampled malignant gland nearby. However, PC-related molecular alterations that could guide the timing of repeat biopsies and help monitor PC risk in ASAP-diagnosed patients have not been investigated. The purpose of this study was to first investigate the expression of seven different PC-related RNAs that included serine 2 (TMPRSS2): erythroblastosis virus E26 oncogene homolog (ERG) gene (TMPRSS2-ERG, T2E) fusion, alpha-methylacyl-CoA racemase (AMACR), kallikrein related peptidase 3 (KLK3), androgen receptor (AR), prostate cancer specific antigen 3 (PCA3), and matrix metalloproteinases (MMP)-2 and 9. METHODS: PC-related RNAs were evaluated using a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) system in pathologically ASAP-diagnosed prostate biopsy cores from 55 patients presenting with a normal digital rectal examination and a PSA level of 4-10 ng/mL. RESULTS: We detected that positive T2E fusion status (P = 0.013) and the expression of AMACR (P = 0.016), AR (P = 0.016) and MMP-2 (P = 0.013) were independently and significantly associated with PC risk in ASAP patients. There were also several statistically significant correlations between expression levels. Additionally, we demonstrated that T2E fusion positive ASAP patients with higher MMP-2 expression were more likely to be diagnosed with PC at a subsequent biopsy during the follow-up period (P = 0.003). CONCLUSIONS: Although, more clinical validations are needed for the stratification of PC risk in ASAP-diagnosed biopsy cores, our current results indicate that the coexistence of T2E fusion positivity with MMP-2 upregulation may help clinicians adjust their biopsy timetable and/or assessment of PC risk in ASAP-diagnosed patients with a PSA level of 4-10 ng/mL.

Same difference: A pilot study of cyclin D1, bcl-2, AMACR, and ALDH-1 identifies significant differences in expression between primary colon adenocarcinoma and its metastases.

Tumor heterogeneity implies the possibility of significantly different expression of key pathways between primary and metastatic clones. Colon adenocarcinoma is one of the few tumors where current practice includes resection of primary and isolated organ metastases simultaneously without neoadjuvant therapy. We performed a pilot study on 28 cases of colon adenocarcinoma resected simultaneously with metastases in patients with no history of neoadjuvant therapy. We assayed matched primary and metastatic tumors from each patient with common diagnostic antibodies to Bcl-2, Cyclin D1, AMACR, and ALDH-1 by immunohistochemistry with semi-quantitative interpretation on archived formalin fixed, paraffin embedded samples. We were powered for large, consistent differences between primary and metastatic expression, and found 21 of 28 had a significant difference in expression of at least one of the four proteins, accounting for multiplicity of testing. Cyclin D1 had significantly more cases with differential metastatic:primary expression than would be expected by chance alone (p-value 0.0043), favoring higher expression in the metastatic sample. Bcl-2 and ALDH-1 had trends in this direction (p-value 0.078 each). Proportionately more cases with significant differences were identified when a liver metastasis was tested. We conclude differences in expression between metastatic and primary colon adenocarcinoma within the same patient exist, and may have therapeutic and biomarker testing consequences.

High alpha-methylacyl-CoA racemase (AMACR) is associated with ERG expression and with adverse clinical outcome in patients with localized prostate cancer.

Alpha-methylacyl-CoA racemase (AMACR) is a well-characterized marker extensively utilized in prostate cancer (PCA) diagnosis. However, the prognostic value of AMACR expression and its relation to TMPRSS2-ERG gene rearrangement as one of the most common molecular alterations in PCA is not fully explored. AMACR expression was investigated in a cohort of 218 men with localized PCA treated by radical prostatectomy and correlated with ERG and various clinical and pathological parameters. In vitro studies assessed AMACR changes to ERG knockdown and other related genes. In addition, bioinformatics validated the significance of AMACR/ERG expression and assessed relevant genetic signatures in relation to AMACR/ERG expression. AMACR expression was significantly associated with disease progression and with ERG (p ∼0). Seventeen percent of cancer foci showed negative/weak AMACR expression while being ERG positive. High AMACR expression was significantly associated with positive surgical margins (p = 0.01), specifically in tumors with lower Gleason score <7, with ∼95 % exhibiting positive surgical margin (p = 0.008). High AMACR showed marginal association with PSA biochemical recurrence (BCR) (p = 0.06) which was slightly more pronounced in ERG-positive tumors (p = 0.04). This was validated in other public cohorts. However, in this cohort, the association with BCR was not statistically significant in multivariate analysis (p = 0.09). Using in vitro cellular models, AMACR messenger RNA (mRNA) expression, but not protein levels, showed an association with ERG expression. We report for the first time a significant association between AMACR and ERG with prognostic implication. Patients with high AMACR/ERG-positive PCA may be at higher risk for disease progression, and additional studies in larger cohorts are needed to confirm the above findings. Functional studies investigating the molecular pathways connecting AMACR and ERG may provide an additional insight into PCA progression pathways.

α-methylacyl-CoA racemase (AMACR) expression in chordomas differentiates them from chondrosarcomas.

AIMS: Chordomas and chondrosarcomas are malignant mesenchymal tumours with overlapping morphological and immunohistochemical (IHC) characteristics. Our aim was to evaluate the IHC expression of α-methylacyl-CoA racemase (AMACR/P504S), ß-catenin and E-cadherin in chordomas relative to chondrosarcomas and assess the utility of these markers for differential diagnosis. METHODS: Archival sections of 18 chordomas, 19 chondrosarcomas and 10 mature cartilage samples were immunostained and scored for AMACR, ß-catenin and E-cadherin and the relative differential capacity of each marker was calculated. In addition, AMACR mRNA level was assessed in 5 chordomas by RT-PCR and evaluated by comparative CT method. RESULTS: AMACR and ß-catenin stained 88.9% and 94.1% of the chordomas respectively, 21.1% and 10.5% of the chondrosarcomas correspondingly and none of the mature cartilage samples. E-cadherin stained positively 82.4% of the chordomas, 36.8% of the chondrosarcomas and 42.9% of the mature cartilage cases. Both AMACR and ß-catenin showed statistically significant difference between chordomas and chondrosarcomas (p < 0.001 for both), unlike E-cadherin. AMACR was detected at the mRNA level. CONCLUSIONS: AMACR is expressed in most of the chordomas but only in a minority of chondrosarcomas. AMACR may serve as IHC marker of chordoma with differentiating ability comparable to that of ß-catenin.

MiR-193a-3p and miR-193a-5p suppress the metastasis of human osteosarcoma cells by down-regulating Rab27B and SRR, respectively.

MicroRNAs have been identified as key players in the development and progression of osteosarcoma, which is the most common primary malignancy of bone. Sequencing-based miR-omic and quantitative real-time PCR analyses suggested that the expression of miR-193a-3p and miR-193a-5p was decreased by DNA methylation at their promoter region in a highly metastatic osteosarcoma cell line (MG63.2) relative to their expression in the less metastatic MG63 cell line. Further wound-healing and invasion assays demonstrated that both miR-193a-3p and miR-193a-5p suppressed osteosarcoma cell migration and invasion. Moreover, introducing miR-193a-3p and miR-193a-5p mimics into MG63.2 cells or antagomiRs into MG63 cells confirmed their critical roles in osteosarcoma metastasis. Additionally, bioinformatics prediction along with biochemical assay results clearly suggested that the secretory small GTPase Rab27B and serine racemase (SRR) were direct targets of miR-193a-3p and miR-193a-5p, respectively. These two targets are indeed involved in the miR-193a-3p- and miR-193a-5p-induced suppression of osteosarcoma cell migration and invasion. MiR-193a-3p and miR-193a-5p play important roles in osteosarcoma metastasis through down-regulation of the Rab27B and SRR genes and therefore may serve as useful biomarkers for the diagnosis of osteosarcoma and as potential candidates for the treatment of metastatic osteosarcoma.

Reduced serine racemase expression in aging rat cerebellum is associated with oxidative DNA stress and hypermethylation in the promoter.

Regulation of serine racemase (SR) occurs at transcriptional and translational levels; post-translational modification, cytosolic distribution as well as allosteric effect regulate SR activity. In this study, we report a new route of SR regulation, i.e. oxidative stress and hypermethylation of the srr (gene of SR) promoter correlate with its reduced transcription in aging rat cerebella. We first showed that the mRNA and protein level of srr were decreased in the homogenates of rat cerebellum at age 12 months compared with the counterparts from age 20 days. The reduction of SR protein level in aging cerebella was evidenced by decreased immunostaining observed in the cell body of granule cells or Purkinje cells. Staining for 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker for oxidative stress to DNA, was much stronger in granule cell or Purkinje cell nuclei from rat cerebella at 12 months compared with staining at 20 days. We further detected srr promoter hypermethylation at 12 months compared with that at 20 days by use of bisulfite sequencing PCR, coinciding with elevated protein levels of DNA methyltransferase 1 (DNMT1) in homogenates of aging cerebella. In vitro, we demonstrated that chronic treatment with the oxidant, menadione (VK3), reduced srr mRNA levels, which was reversed by the DNA demethylating agent 5-Aza-dC-2'-deoxycytidine (5-Aza-dC) in primary cerebellar granule cell cultures. Together, the in vivo and ex vivo results suggest that oxidative DNA stress and srr promoter hypermethylation are associated with reduced srr gene transcription and corresponding reduced protein expression in aging cerebella.

[Expression of P504S and matrix metalloproteinase-2 in circulating prostate cells disseminated as a result of transrectal ultrasound guided biopsy as determined by immunocytochemistry: Clinical implications]. / Condideraciones clínicas de la expressión de P504S y metaloproteinasa de matriz 2 en células prostáticas circulantes en sangre diseminadas como resultado de una biopsia prostática trans-rectal guiada por ecografía.

OBJECTIVES: Surgical manipulation of cancer has been shown to increase blood borne cancer cell dissemination and increase the risk of metastasis. We present the effect of prostate biopsy on prostate cell dissemination and the phenotypic characteristics of these cells. METHODS: 50 men undergoing initial prostate biopsy for suspicion of prostate cancer were studied. Blood samples were taken immediately before, and 1 and 24 hours after biopsy for circulating prostate cells (CPC) determination and phenotypic characterization. CPCs were detected and counted using standard immunocytochemistry using anti-PSA and then characterized using anti-P504S and anti-matrix metalloproteinase-2 (MMP-2). RESULTS: 14 (28%) men had cancer detected on biopsy. 13/14 had P504S (+) and MMP-2 (+) cells detected prior to biopsy. One hour after biopsy there was a mixture of P504S (+) and P504S (-) cells detected, as well as MMP-2 (+) and MMP-2 (-) cells detected. 24 hours after biopsy the same 13/14 men remained positive, although the number of CPCs increased 1 hour after biopsy and then the numbers decreased to pre-biopsy levels after 24 hours. In cancer negative men, P504S (-) and MMP-2 (-) cells were detected, some of these cells persisted 24 hours after biopsy. CONCLUSIONS: Prostate biopsy causes dissemination of prostate cells into the circulation, both malignant and benign; the majority of them are cleared within 24 hours. There was no conversion of negative to positive result in men with cancer, this suggests that the inherent capacity of malignant CPCs to disseminate is more important than the effect of dissemination caused by prostate biopsy.

Dietary influences on tissue concentrations of phytanic acid and AMACR expression in the benign human prostate.

BACKGROUND: Alpha-methylacyl-CoA racemase (AMACR) is an enzyme involved in fatty acid metabolism that is markedly over-expressed in virtually all prostate cancers (PCa), relative to benign tissue. One of AMACR's primary substrates, phytanic acid, is derived predominately from red meat and dairy product consumption. Epidemiological evidence suggests links between dairy/red meat intake, as well as phytanic acid levels, and elevated PCa risk. This study investigates the relationships among dietary intake, serum and tissue concentrations of phytanic acid, and AMACR expression (mRNA and protein) in the histologically benign human prostate. METHODS: Men undergoing radical prostatectomy for the treatment of localized disease provided a food frequency questionnaire (n = 68), fasting blood (n = 35), benign fresh frozen prostate tissue (n = 26), and formalin-fixed paraffin-embedded (FFPE) sections (n = 67). Serum and tissue phytanic acid concentrations were obtained by gas chromatography-mass spectrometry. We extracted RNA from epithelial cells using laser capture microdissection and quantified mRNA expression of AMACR and other genes involved in the peroxisomal phytanic acid metabolism pathway via qRT-PCR. Immunohistochemistry for AMACR was performed on FFPE sections and subsequently quantified via digital image analysis. Associations between diet, serum, and tissue phytanic acid levels, as well as AMACR and other gene expression levels were assessed by partial Spearman correlation coefficients. RESULTS: High-fat dairy intake was the strongest predictor of circulating phytanic acid concentrations (r = 0.35, P = 0.04). Tissue phytanic acid concentrations were not associated with any dietary sources and were only weakly correlated with serum levels (r = 0.29, P = 0.15). AMACR gene expression was not associated with serum phytanic acid (r = 0.13, P = 0.47), prostatic phytanic acid concentrations (r = 0.03, P = 0.88), or AMACR protein expression (r = -0.16, P = 0.20). CONCLUSIONS: Our data underscore the complexity of the relationship between AMACR and its substrates and do not support the unifying hypothesis that excess levels of dietary phytanic acid are responsible for both the overexpression of AMACR in prostate cancer and the potential association between PCa risk and intake of dairy foods and red meat.

D-serine and serine racemase are localized to neurons in the adult mouse and human forebrain.

D-Serine, a co-agonist at the NMDA receptor (NMDAR), is synthesized from L-serine by the enzyme serine racemase (SR), which is heavily expressed in the forebrain. Although SR was originally reported to be localized exclusively to astrocytes, recent conditional knock out results demonstrate that little SR is expressed in forebrain astrocytes. As a consequence, the cellular location of its product, D-serine, in the brain is also uncertain. Immunocytochemistry now indicates that SR is expressed primarily in forebrain glutamatergic neurons with the remainder in GABAergic interneurons. We utilized SR deficient (SR-/-) mice, which have <15 % of normal D-serine levels, to validate and optimize a D-serine immunohistochemical method. Nearly all of the D-serine in neocortex and hippocampus (HP) is found in neurons, with virtually no D-serine co-localizing with two astrocyte markers. Interestingly, only a subset of the D-serine positive neurons contained SR in the neocortex and HP. Greater than half of the D-serine positive neurons were GABAergic interneurons, with a majority of these neurons containing parvalbumin and/or somatostatin. Only ~25-40 % of interneurons expressed SR in the neocortex and HP. Finally, we demonstrate in human post-mortem neocortex that SR is found in both excitatory and inhibitory neurons, but not in S100ß-containing astrocytes. In sum, these findings conclusively demonstrate that the majority of D-serine is both synthesized and stored in neurons. It will be important to determine the functional significance for the separation of synthesis and storage of D-serine in neurons, as well as the presence of this NMDAR co-agonist in GABAergic interneurons.

Utility of a triple antibody cocktail intraurothelial neoplasm-3 (IUN-3-CK20/CD44s/p53) and α-methylacyl-CoA racemase (AMACR) in the distinction of urothelial carcinoma in situ (CIS) and reactive urothelial atypia.

Urothelial carcinoma in situ (CIS) is a prognostically and therapeutically significant lesion with considerable morphologic overlap with reactive conditions especially in the setting of prior therapy. Various markers including CK20, CD44s, and p53 have been used as an adjunct in making this distinction; however, the utility of these markers in the posttreatment scenario is not fully established. α-Methylacyl-CoA racemase (AMACR) is a tumor-associated marker that is expressed in a subset of high-grade urothelial carcinomas but has not been studied in CIS. This study was undertaken to evaluate the immunoreactivity of CK20, CD44s, and p53 as a triple antibody cocktail intraurothelial neoplasm-3 (IUN-3) in distinguishing CIS from its mimics and to compare its utility with AMACR in the diagnosis of CIS. A total of 135 specimens (7 benign ureters and 128 bladder biopsies-28 reactive, 33 posttherapy reactive, 43 CIS, 24 CIS posttherapy) were included in this study. Immunostaining for p53 (brown, nuclear), CD44s (brown, membranous), and CK20 (red, cytoplasmic and membranous) was performed as a cocktail, and the staining pattern was further classified as: malignant (full-thickness CK20 and/or full-thickness p53 with CD44s negativity), reactive/benign (CK20 limited to the umbrella cell layer, p53 negative, and CD44s positivity ranging from basal to full thickness), and indeterminate (CK20 and p53 positive but not full thickness and/or CD44s positive). AMACR staining was performed in 50 cases. Cytoplasmic staining for AMACR was graded as negative (absent to weak focal staining [<5% cells]) and positive (≥5%). The "IUN-3 malignant" pattern was observed in 84% of cases of CIS without a history of prior therapy and in 71% of the cases of CIS with a history of prior therapy. Cases with posttherapy reactive atypia showed an "IUN-3 reactive" pattern in 84% cases and "IUN-3 indeterminate" pattern in 16% of the cases; the IUN-3 malignant pattern was not identified in any of the cases. Benign and reactive urothelium (with and without a history of therapy) showed an IUN-3 reactive pattern and negative AMACR staining in all the cases (100%). AMACR positivity was observed in 78% of nontreated CIS cases and 50% of CIS posttherapy cases. In these cases, the IUN-3 cocktail showed an IUN-3 malignant pattern in 83% of untreated CIS cases and 88% of CIS posttherapy cases. AMACR positivity is a potentially useful marker of CIS. However, the IUN-3 malignant pattern is a more reliable indicator of CIS compared with AMACR, especially in the posttreatment setting. The simultaneous evaluation of all 3 markers (p53, CD44s, and CK20) in a single slide in the form of a cocktail is advantageous, especially in small biopsy specimens.

Clinical significance of the expression of alpha-methylacyl-CoA racemase in squamous cell carcinoma and adenocarcinoma of the lung.

BACKGROUND: The role of alpha-methylacyl-CoA racemase (AMACR) in pathogenesis and diagnosis of different types of cancer has been investigated during last decade. This study is conducted to investigate AMACR expression in adenocarcinoma and squamous cell carcinoma (SCC) of lung and its correlation with clinical characteristics and survival. METHODS: The clinicopathologic characteristics of 146 patients who underwent a potentially curative surgical resection between June 2000-2009 in our clinic were reviewed retrospectively. The patients who were given adjuvant chemotherapy and/or radiotherapy, with an evidence of residual tumor at resection margin and who died due to postoperative mortality and due to reasons not related to lung cancer were excluded. Data from remaining 67 patients were analyzed for survival. For the correlation between progression and AMACR immunoreactivity, data from 62 patients who had postoperative follow up in our center were analyzed. RESULTS: AMACR immunoreactivity was observed more frequently in adenocarcinoma group than SCC group (p = 0,046). The samples with invasive adenocarcinoma--lepidic predominant pattern also showed high rates of positive staining (73%). We could not show a statistically significant correlation between AMACR immunostaining and degree of differentiation, age, gender, pathologic T status, N status, or stage. We failed to show a statistically meaningful effect of AMACR on overall and progression-free survival. CONCLUSION: Adenocarcinoma had higher rates of positive immunostaining for AMACR than SCC (p = 0,046). But the presence of AMACR expression did not have a statistically meaningful effect on overall and progression-free survival in adenocarcinoma and SCC of lung.

High-level extracellular production of D-Psicose-3-epimerase with recombinant Escherichia coli by a two-stage glycerol feeding approach.

The aim of this study is to achieve high-level extracellular production of D-Psicose-3-epimerase (DPE) with recombinant Escherichia coli. High-level production of DPE is one of the key factors in D-Psicose production. In the present study, the gene AAL45544.1 from Agrobacterium tumefaciens str. C58 was modified by artificial synthesis for overexpression in E. coli. The total DPE activity reached 3.96 U mL(-1) after optimization of the media composition, induction temperature, and concentration of inducer. Furthermore, it was found that addition of glycine had a positive effect on the extracellular production of DPE, which reached 3.5 U mL(-1). Finally, a two-stage glycerol feeding strategy based on both the specific growth rate before induction and the amount of glycerol residues after induction was applied in a 3-L fermenter. After a series of optimal strategies in the 3-L fermenter, the total and extracellular DPE activity were 5.08- and 3.11-fold higher than that noted in the shake flask. The extracellular and intracellular DPE activity reached 10.9 and 13.2 U mL(-1), achieving 25.5 and 31.1 % conversion of D-fructose to D-psicose, respectively. The systemic strategies presented in this study provide valuable novel information for the industrial application of DPE.

Hyposmolality differentially and spatiotemporally modulates levels of glutamine synthetase and serine racemase in rat supraoptic nucleus.

Prolonged hyposmotic challenge (HOC) has a dual effect on vasopressin (VP) secretion [Yagil and Sladek (1990) Am J Physiol 258(2 Pt 2):R492-R500]. We describe an electrophysiological correlate of this phenomenon, whereby in vitro HOC transiently reduced the firing activity of VP neurons within the supraoptic nucleus of brain slices, which was followed by a rebound increase of their activity; this was paralleled by changes in the level of proteins relevant to astroglia-neuronal interactions. Hence, in vitro HOC transiently (at 5 min) increased the level of astrocyte-specific glial fibrillary acidic protein (GFAP), which then declined to control or base level (at 20 min); this was blocked by the gliotoxin L-aminoadipic acid, but not by tetanus toxin, which was used to inhibit neurotransmission. Similarly, in vivo HOC led to changes in GFAP level, which after an early increase (10 min) returned to normal (30 min). Immunoassays revealed that neuronal, but not astrocytic, expression of serine racemase (SR) was increased at the late stage of HOC in vivo, whereas at an early stage there was a transient increase in level of the astrocyte-specific glutamine synthetase (GS). Furthermore, there was an increased molecular association between GFAP and GS at 10 min, whereas SR increased its association with the neuronal nuclear antigen NeuN at 30 min. These results suggest that the dual effect of HOC on VP neuronal secretion/activity could be related to metabolic/signaling changes in astrocytes (glutamate-glutamine conversion) and neurons (D-serine synthesis/ammonia production), which may account for the rebound in VP neuronal activity, presumably by promoting the activation of neuronal glutamate receptors.

Regulation of NADPH oxidase gene expression with PKA and cytokine IL-4 in neurons and microglia.

Neuronal excitation is mediated by the activation of NMDA receptor and associated with the formation of reactive oxygen species due to the activation of NADPH oxidase complex proteins. The activation of Gs protein coupled receptors (GPCRs) induces neuronal activation in the cAMP-dependent protein kinase A (PKA)-mediated signal cascade and regulates NADPH oxidase activity. However, it is unknown whether PKA regulates NADPH oxidase gene expression in neurons and microglia. In the present research, the NADPH oxidase gene expression was studied in rat cortical neurons and microglia in vitro. Purified microglial cells were identified with OX-42 antibody and they also expressed apolipoprotein E (ApoE). The time-dependent effect of cytokine interleukin-4 (IL-4) (20 ng/ml) in NADPH oxidase gene expression was studied in microglial cells. The levels of mRNA were determined by quantitative RT-PCR. The expression of NOX1, NOX2, and NCF2 was upregulated after IL-4 treatment for 4 h, but it was downregulated after 8-24 h. The expression of NCF1 was suppressed during any time of cytokine effect. IL-4 upregulated arginase1 (Arg1) and serine racemase1 (SRR1) gene expressions in microglia. Amyloid beta (Ab) suppressed NOX2, NCF1, and NCF2 gene expressions and upregulated glutamate cystine transporter (xCT), although IL-4 attenuated the effect of Ab (500 µM) in the upregulation of xCT gene expression. The activation of PKA with agonist dibutyryl cAMP (dbcAMP) (100 µM) induced the upregulation of Arg1 gene expression in microglia involving in the process of microglial activation. The transcription of NOX1, NOX2, and NCF1 was suppressed in microglial cells after dbcAMP treatment within 24 h. Neurons were identified with the microtubule-associated protein tau. The uniform distribution of tau along axons was established in normal neurons. Tau protein was redistributed after PKA agonist dbcAMP treatment for 24 h. L-glutamate (50 µM) caused the apoptotic processes and the accumulation of tau in the soma of neurons and along axons. The activation of PKA for 24 h induced the transcriptional upregulation of NOX1 and NCF1 in cortical neurons. However, L-glutamate suppressed NOX1 gene expression in neurons. These data demonstrate that the effects of IL-4 and dbcAMP are similar in the regulation of SRR1, Arg1, and NADPH oxidase complex gene expressions in neurons and microglia. IL-4 prevents glutamate release from microglia suppressing xCT expression induced by Ab. These findings suggest that the activation of GPCR in PKA-mediated pathway leads to transcriptional regulation of NADPH oxidase complex. The modulation of GPCR activation may inhibit the oxidative stress in neurons.

Prognostic value of alpha-methyl CoA racemase (AMACR) expression in renal cell carcinoma.

PURPOSE: Alpha-methyl CoA racemase (AMACR) is used as an immunohistochemical marker for renal cell carcinoma (RCC) subtyping to distinguish papillary (pap) RCC. Expression of AMACR in other renal tumor subtypes is inhomogeneous, and the clinical and prognostic value of AMACR is unknown. The aim of this study was to asses AMACR protein expression in different RCC subtypes and to investigate its prognostic significance. METHODS: Protein expression of AMACR was analyzed in 1,088 renal tumor samples, among them 809 clear cell RCC and 151 papRCC, by immunohistochemistry using tissue microarry (TMA) technique. Results were correlated with clinicopathological data and to follow-up data [overall (OS)/cancer-specific survival (CSS)]. RESULTS: Frequency of AMACR expression was significantly higher in papRCC compared to other tumor subtypes (83% vs. 15-35%, p < 0.0001). Presence of AMACR did not correlate with stage or nodal metastases in papRCC. In a dichotomized scoring (negative vs. positive expression), an inverse correlation between higher grade (p = 0.03) and presence of distant metastasis (p = 0.014) was observed in papRCC. AMACR expression correlated with the presence of nodal metastasis in ccRCC (p = 0.02). Both in ccRCC and in papRCC, OS and CSS did not correlate with the AMACR expression status. CONCLUSIONS: The high expression in papRCC confirms AMACR to be a marker for subtype differentiation in RCC, while a missing expression in this subtype seems to be associated with negative pathological features. However, in contrast to other tumor entities, AMACR expression seems to have a limited prognostic impact in renal carcinoma, especially with regard to survival.

Alpha-methylacyl-coenzyme A racemase expression in neuroendocrine neoplasms of the stomach.

The enzyme alpha-methylacyl-coenzyme A racemase plays an important role in the beta-oxidation of branched-chain fatty acid and its derivatives. It has been used to detect prostatic adenocarcinoma and high-grade intraepithelial neoplasia, and recently also as a marker for other neoplasms, including those of the genitourinary system, breast, upper and lower gastrointestinal tract and their precursor lesions. We assessed expression of alpha-methylacyl-coenzyme A racemase by immunohistochemistry in neuroendocrine tumours of the stomach to determine differences in the incidence and pattern of expression among different types of gastric neuroendocrine tumours. While none of the grade 1 neuroendocrine tumours were immunoreactive, 67 % of grade 2 neuroendocrine tumours and 90 % of neuroendocrine carcinomas were positive for alpha-methylacyl-coenzyme A racemase. Furthermore, an adenocarcinoma component was found in 72.5 % (37 of 51) of neuroendocrine carcinomas, whereas none of the grade 1 and 2 neuroendocrine tumours contained an adenocarcinoma component. In 83 % of neuroendocrine carcinomas, the adenocarcinoma component was positive for alpha-methylacyl-coenzyme A racemase, and both adenocarcinoma and neuroendocrine carcinoma components stained positively in 78 % of these cases. Our results indicate that alpha-methylacyl-coenzyme A racemase is a useful marker for distinguishing between grade 1 (negative) and grade 2 neuroendocrine tumours, and neuroendocrine carcinoma of the stomach (frequently positive). Different patterns of alpha-methylacyl-coenzyme A racemase expression between gastric neuroendocrine tumours and neuroendocrine carcinoma suggest that these might develop via different tumourigenic pathways.

Relationship between alpha-methylacyl-coenzyme A racemase expression and mucin phenotype in gastric cancer.

Alpha-methylacyl-coenzyme A racemase controls ß-oxidation of branched-chain fatty acid and their derivatives. Many investigators have described alpha-methylacyl-coenzyme A racemase expression in various neoplasias and their precursor lesions. Although there have been a few reports regarding alpha-methylacyl-coenzyme A racemase expression in gastric neoplasia, these reports did not discuss the relationship between alpha-methylacyl-coenzyme A racemase expression and mucin phenotype. This study analyzed alpha-methylacyl-coenzyme A racemase expression of gastric carcinomas with regard to mucin phenotype. Alpha-methylacyl-coenzyme A racemase expression was evaluated in 85 cases of gastric biopsies including gastric epithelial neoplasia and nonneoplasia and in 108 cases of surgically resected early gastric cancer. In biopsy cases, alpha-methylacyl-coenzyme A racemase was more highly expressed in neoplasia (69.7%, 23/33) than in nonneoplasia (0%, 0/42) (P = .001). Alpha-methylacyl-coenzyme A racemase was overexpressed in 20.0% (2/10) of cases that were indefinite for neoplasia, and the 2 positive cases were ultimately diagnosed as adenocarcinoma. In resected cases of early gastric adenocarcinoma, alpha-methylacyl-coenzyme A racemase expression significantly correlated with mucin phenotype (P = .003), but not with tumor progression, histologic classification, or clinicopathologic features. Alpha-methylacyl-coenzyme A racemase expression was significantly higher in intestinal-phenotype carcinoma (90.2%, 37/40) than in gastric-phenotype carcinoma (56.3%, 18/31) (P = .006) and also correlated with an increase in CDX2 expression (P = .018) and a decrease in MUC5AC expression (P = .048). This tendency was observed in all histologic types. Our results indicate that alpha-methylacyl-coenzyme A racemase is a useful marker for distinguishing gastric neoplasia from nonneoplasia even at an early stage. Alpha-methylacyl-coenzyme A racemase expression is associated with mucin phenotypes of gastric neoplasia, particularly with the expression of CDX2 and MUC5AC.