Structural and Functional Basis of GenB2 Isomerase Activity from Gentamicin Biosynthesis.

Aminoglycosides are essential antibiotics used to treat severe infections caused mainly by Gram-negative bacteria. Gentamicin is an aminoglycoside and, despite its toxicity, is clinically used to treat several pulmonary and urinary infections. The commercial form of gentamicin is a mixture of five compounds with minor differences in the methylation of one of their aminosugars. In the case of two compounds, gentamicin C2 and C2a, the only difference is the stereochemistry of the methyl group attached to C-6'. GenB2 is the enzyme responsible for this epimerization and is one of the four PLP-dependent enzymes encoded by the gentamicin biosynthetic gene cluster. Herein, we have determined the structure of GenB2 in its holo form in complex with PMP and also in the ternary complex with gentamicin X2 and G418, two substrate analogues. Based on the structural analysis, we were able to identify the structural basis for the catalytic mechanism of this enzyme, which was also studied by site-directed mutagenesis. Unprecedently, GenB2 is a PLP-dependent enzyme from fold I, which is able to catalyze an epimerization but with a mechanism distinct from that of fold III PLP-dependent epimerases using a cysteine residue near the N-terminus. The substitution of this cysteine residue for serine or alanine completely abolished the epimerase function of the enzyme, confirming its involvement. This study not only contributes to the understanding of the enzymology of gentamicin biosynthesis but also provides valuable details for exploring the enzymatic production of new aminoglycoside derivatives.

Molecular genetic analysis of AKR1C2-4 and HSD17B6 genes in subjects 46,XY with hypospadias.

BACKGROUND: The formation of the male urethra depends to enzyme-mediated testosterone (T) conversion into 5α-dihydrotestosterone (DHT). Two metabolic pathways could be operating in the fetal testis to synthesize androgens: 1) the "classic" route (T→DHT) mediated by SRD5A2 and 2) a "backdoor" pathway in which DHT is synthesized by aldo-keto reductase family 1, member C2 (AKR1C2), AKR1C3, and AKR1C4 enzymes without formation of a T intermediate. OBJECTIVE: We studied four genes of the "backdoor" pathway in karyotypic males with hypospadias to ascertain whether gene defects in AKRs impair urethral DHT formation that result in hypospadias. DESIGN AND PATIENTS: The coding regions of the AKR1C2-4 and HSD17B6 genes were analyzed by PCR-SSCP and sequencing in a cohort of 25 Mexican patients (0.3-9 year-old-children) with 46,XY-hypospadias. Chi-squared tests was performed to evaluate the distribution of genotypes, alleles, and the Hardy-Weinberg (H-W) equilibrium. The effect of the genetic variants was investigated by in silico studies. RESULTS: Screening studies revealed distinct genotypic patterns at different exons of AKR1C2-4 whereas HSD17B6 presented a wild-type sequence. The DNA analyses detected two synonymous variants (c.327C>T, c.666T>C/unreported) in AKR1C2. The AKR1C3 had two variants (c.15C>G, c.230A>G), two unreported variants (c.538T>C, c.596G>A), and one silent variant (c.312G>A). Two variants (c.434C>G, c.931C>G) were identified in AKR1C4. All variants were in H-W equilibrium without structural changes. DISCUSSION: Hypospadias have been associated with defects that alter androgen biosynthesis in the human fetal testis, specifically 5α-DHT. We selected four candidate genes involved in the "backdoor" pathway for the formation of 5α-DHT. Molecular assays of the AKR1C2, AKR1C3, and AKR1C4 genes revealed a total of nine genetic single nucleotide variants. Several variants in the AKR1C genes have been associated with a variety of human pathologies. However, our studies suggest that active steroid biosynthesis via AKR1C might not be involved in hypospadias. Additionally, genetic research suggests a low involvement in the "backdoor" 5α-DHT pathway during human sexual development, specifically, the differentiation of male external genitalia. CONCLUSION: These results indicate that substitutions in AKR1C2-4 are polymorphisms and all genetic variants lacks deleterious significant association with hypospadias. The data suggest that inactivating mutations in the AKR1C2-4 and HSD17B6 genes are an infrequent cause of hypospadias, which might weaken the contribution of the "backdoor" pathway to embryonic urethral masculinization.

Auditory cognitive training improves prepulse inhibition in serine racemase mutant mice.

Evidence indicates that neuroplasticity-based cognitive training can improve cognition in patients with schizophrenia, but the individual response to training varies greatly between subjects. Hence, there is a need to understand the neurological underpinnings of cognitive training to reveal predictors of treatment response. D-serine is a crucial modulator of neuroplasticity, and decreased levels of D-serine may contribute to deficits in neuroplasticity in schizophrenia. Interestingly, we observed that training mice to identify auditory oddballs increased extracellular levels of D-serine in the hippocampus during training. Serine racemase (Srr) is the only source of brain D-serine; thus, it is possible that Srr may mediate the response to training. To test this hypothesis, we trained mice that have a mutated version of Srr (SrrY269*/SrrY269*) and reduced levels of D-serine in the same auditory training. SrrY269*/SrrY269* mice showed decreased performance during auditory training (defined as the capacity to discriminate an oddball during a sequence of tones). Importantly, auditory training improved prepulse inhibition (PPI) in SrrY269*/SrrY269* but not in wild-type mice. Finally, D-serine (100 mg/kg i.p.) given 30 min before training sessions to SrrY269*/SrrY269* mice improved training performance, but it did not enhance PPI. Taken together, our results show that D-serine is involved in the response to neuroplasticity-based auditory training and that PPI deficits can be improved by auditory oddball training even in the presence of neuroplasticity deficits.

Netrin 1 and Alpha-Methyl Acylcoenzim-A Racemase in diagnosis of prostate cancer.

OBJECTIVES: To investigate serum and urine levels of Alpha-methylacyl-CoA-racemase (AMACR) and Netrin 1 in patients with and without prostate cancer and to determine whether these markers could be used as alternatives in diagnosis of prostate cancer instead of serum prostate specific antigen (PSA) levels. METHODS: One hundred and seventy five patients between 45-75 years to whom transrectal ultrasound guided biopsies were performed for abnormal serum PSA levels or digital rectal examinations were included. The levels of AMACR and Netrin 1 levels of blood and urine samples of 5 mL those were taken prior to biopsies were measured. . RESULTS: The mean age of the patients was 62.7 ±6.4 years. Prostate cancer was detected in 40 patients (22.8%) while 135 of them (77.2%) were diagnosed as benign prostate hyperplasia (BPH). In BPH group, serum and urine levels of AMACR and Netrin 1 were 13.4 ±16.9 ng/mL; 7.1 ±3.4 ng/mL; 164.1±46 pg/mL and 19.5 ±5.0 pg/mL respectively. The levels of serum and urine levels of AMACR and Netrin 1 were 10.2 ±9.8 ng/mL; 6.8 ±2.5 ng/mL; 159.1 ±44.1 pg/mL and 20.1 ±5.3 pg/mL respectively in prostate cancer group. There was no statistically significant difference or correlation between these two groups serum and urine AMACR and Netrin 1 results. CONCLUSIONS: Serum and urine levels of AMACR and Netrin 1 were not found to be alternatives for serum PSA levels in the diagnosis of prostate cancer in this study.

OBJETIVOS: Investigar los niveles de alfa-metil acilcoenzima-A y Netrina 1 en pacientes con y sin cáncer de próstata y determinar si estos marcadores pueden ser usados como una alternativa en el diagnóstico de cáncer de próstata en lugar del antígeno prostático específico en suero (PSA). MÉTODOS: Fueron incluidos 175 pacientes entre 45-75 años, a quienes se les realizó una biopsia de próstata guiada por ultrasonido por presentar un nivel anormal de PSA en el suero o un tacto rectal. Se tomó una muestra de 5 mL de sangre y orina para medir los niveles de alfa-metil acilcoenzima-A y Netrina 1. Estos niveles se midieron antes del análisis de la biopsia. RESULTADOS: La edad media de los pacientes fue de 62.7±6.4 años. Se detectó cander en 40 pacientes (22.8%), mientras que a 135 de ellos (77.2%) se les diagnóstico una hiperplasia benigna de próstata (HBP). En el grupo HBP los niveles en suero y orina de alfa-metil acilcoenzima-A y Netrina 1 fueron 13.4 ±16.9 ng/mL; 7.1 ±3.4 ng/mL; 164.1 ±46 pg/mL y 19.5 ±5.0 pg/mL respectivamente. En el grupo con cáncer de próstata los niveles en suero y orina de alfa-metil acilcoenzima-A y Netrina 1 fueron 10.2 ±9.8 ng/mL; 6.8 ±2.5 ng/mL; 159.1 ±44.1 pg/mL y 20.1 ±5.3 pg/mL respectivamente. No hubo una diferencia significativa o una correlación entre los niveles de alfa-metil acilcoenzima-A y Netrina 1 en suero y orina al comparar estos dos grupos de pacientes. CONCLUSIONES: Los niveles de alfa-metil acilcoenzima-A y Netrina 1 en suero y orina no son una alternativa para reemplazar el PSA en suero para el diagnóstico de cáncer de próstata.

Netrin 1 and Alpha-Methyl Acylcoenzim-A Racemase in diagnosis of prostate cancer / Netrina 1 y Alfa Metil-Acil coenzima-A Racemasa para el diagnóstico de cáncer de próstata

Abstract Objectives: To investigate serum and urine levels of Alpha-methylacyl-CoA-racemase (AMACR) and Netrin 1 in patients with and without prostate cancer and to determine whether these markers could be used as alternatives in diagnosis of prostate cancer instead of serum prostate specific antigen (PSA) levels. Methods: One hundred and seventy five patients between 45-75 years to whom transrectal ultrasound guided biopsies were performed for abnormal serum PSA levels or digital rectal examinations were included. The levels of AMACR and Netrin 1 levels of blood and urine samples of 5 mL those were taken prior to biopsies were measured. . Results: The mean age of the patients was 62.7 ±6.4 years. Prostate cancer was detected in 40 patients (22.8%) while 135 of them (77.2%) were diagnosed as benign prostate hyperplasia (BPH). In BPH group, serum and urine levels of AMACR and Netrin 1 were 13.4 ±16.9 ng/mL; 7.1 ±3.4 ng/mL; 164.1±46 pg/mL and 19.5 ±5.0 pg/mL respectively. The levels of serum and urine levels of AMACR and Netrin 1 were 10.2 ±9.8 ng/mL; 6.8 ±2.5 ng/mL; 159.1 ±44.1 pg/mL and 20.1 ±5.3 pg/mL respectively in prostate cancer group. There was no statistically significant difference or correlation between these two groups serum and urine AMACR and Netrin 1 results Conclusions: Serum and urine levels of AMACR and Netrin 1 were not found to be alternatives for serum PSA levels in the diagnosis of prostate cancer in this study.

Resumen Objetivos: Investigar los niveles de alfa-metil acilcoenzima-A y Netrina 1 en pacientes con y sin cáncer de próstata y determinar si estos marcadores pueden ser usados como una alternativa en el diagnóstico de cáncer de próstata en lugar del antígeno prostático específico en suero (PSA). Métodos: Fueron incluidos 175 pacientes entre 45-75 años, a quienes se les realizó una biopsia de próstata guiada por ultrasonido por presentar un nivel anormal de PSA en el suero o un tacto rectal. Se tomó una muestra de 5 mL de sangre y orina para medir los niveles de alfa-metil acilcoenzima-A y Netrina 1. Estos niveles se midieron antes del análisis de la biopsia. Resultados: La edad media de los pacientes fue de 62.7±6.4 años. Se detectó cander en 40 pacientes (22.8%), mientras que a 135 de ellos (77.2%) se les diagnóstico una hiperplasia benigna de próstata (HBP). En el grupo HBP los niveles en suero y orina de alfa-metil acilcoenzima-A y Netrina 1 fueron 13.4 ±16.9 ng/mL; 7.1 ±3.4 ng/mL; 164.1 ±46 pg/mL y 19.5 ±5.0 pg/mL respectivamente. En el grupo con cáncer de próstata los niveles en suero y orina de alfa-metil acilcoenzima-A y Netrina 1 fueron 10.2 ±9.8 ng/mL; 6.8 ±2.5 ng/mL; 159.1 ±44.1 pg/mL y 20.1 ±5.3 pg/mL respectivamente. No hubo una diferencia significativa o una correlación entre los niveles de alfa-metil acilcoenzima-A y Netrina 1 en suero y orina al comparar estos dos grupos de pacientes. Conclusiones: Los niveles de alfa-metil acilcoenzima-A y Netrina 1 en suero y orina no son una alternativa para reemplazar el PSA en suero para el diagnóstico de cáncer de próstata.

Extended use of P504S positive primary circulating prostate cell detection to determine the need for initial prostate biopsy in a prostate cancer screening program in Chile.

BACKGROUND: To determine the frequency of primary circulating prostate cells (CPC) detection according to age and serum PSA levels in a cohort of men undergoing screening for prostate cancer and to determine the diagnostic yield in those men complying with the criteria for prostate biopsy. MATERIALS AND METHODS: A prospective study was carried out to analyze all men evaluated in a hospital prostate cancer screening program. Primary CPCs were obtained by differential gel centrifugation and detected using standard immunocytochemistry using anti-PSA, positive samples undergoing a second process with anti-P504S. A malignant primary CPC was defined as PSA+ P504S+, and a test positive if 1 cell/4ml was detected. The frequency of primary CPC detection was compared with age and serum PSA levels. Men with a PSA >4.0ng/ml and/or abnormal rectal examination underwent 12 core prostate biopsy, and the results were registered as cancer/no-cancer and compared with the presence/absence of primary CPCs to calculate the diagnostic yield. RESULTS: A total of 1,117 men participated; there was an association of primary CPC detection with increasing age and increasing serum PSA. Some 559 men underwent initial prostate biopsy of whom 207/559 (37.0%) were positive for primary CPCs and 183/559 (32.0%) had prostate cancer detected. The diagnostic yield of primary CPCs had a sensitivity of 88.5%, a specificity of 88.0%, and positive and negative predictive values of 78.3% and 94.9%, respectively. CONCLUSIONS: The use of primary CPCs for testing is recommended, since its high negative predictive value could be used to avoid prostate biopsy in men with an elevated PSA and/or abnormal DRE. Men positive for primary CPCs should undergo prostate biopsy. It is a test that could be implemented in the routine immunocytochemical laboratory.

Antinociceptive effect of rat D-serine racemase inhibitors, L-serine-O-sulfate, and L-erythro-3-hydroxyaspartate in an arthritic pain model.

N-methyl-D-aspartic acid receptor (NMDAr) activation requires the presence of D-serine, synthesized from L-serine by a pyridoxal 5'-phosphate-dependent serine racemase (SR). D-serine levels can be lowered by inhibiting the racemization of L-serine. L-serine-O-sulfate (LSOS) and L-erythro-3-hydroxyaspartate (LEHA), among others, have proven to be effective in reducing the D-serine levels in culture cells. It is tempting then to try these compounds in their effectiveness to decrease nociceptive levels in rat arthritic pain. We measured the C-reflex paradigm and wind-up potentiation in the presence of intrathecally injected LSOS (100 µg/10 µL) and LEHA (100 µg/10 µL) in normal and monoarthritic rats. Both compounds decreased the wind-up activity in normal and monoarthritic rats. Accordingly, all the antinociceptive effects were abolished when 300 µg/10 µL of D-serine were injected intrathecally. Since no in vivo results have been presented so far, this constitutes the first evidence that SR inhibitions lower the D-serine levels, thus decreasing the NMDAr activity and the consequent development and maintenance of chronic pain.

Pseudohyperplastic prostatic carcinoma in simple prostatectomy.

Pseudohyperplastic carcinoma (PHPC) is a prostatic neoplasm that can be easily mistaken for nodular hyperplasia or atypical adenomatous hyperplasia. To determine the frequency and clinicopathologic characteristics of PHPC, we reviewed 200 simple prostatectomy specimens. We found 3 cases (1.5%) of PHPC. The tumors were small and ranged in size from 4 to 6 mm. Two of them were erroneously diagnosed as benign glandular proliferations in the original interpretation. Their histologic aspect at low magnification showed nodules of well-differentiated medium-sized glands with cystic dilation in a tight arrangement that imparted a benign appearance. Corpora amylacea were found in 2 cases. However, the lining cells showed nucleomegaly and prominent nuclei in most of the neoplastic glands, and the high-molecular-weight keratin (34BE12) immunostain revealed absence of basal cells. α-Methylacyl-CoA-racemase was positive in 2 cases. In one case, a small focus of moderated acinar adenocarcinoma was found adjacent to the pseudohyperplastic glands facilitating the diagnosis. The 3 patients are disease-free 3 and 4 years after surgery probably because of the small size of the tumors; however, it must be emphasized that most PHPC are considered moderately differentiated and potentially aggressive neoplasms.

Protein kinase C activity regulates D-serine availability in the brain.

D-serine is a co-agonist of NMDA receptor (NMDAR) and plays important roles in synaptic plasticity mechanisms. Serine racemase (SR) is a brain-enriched enzyme that converts L-serine to D-serine. SR interacts with the protein interacting with C-kinase 1 (PICK1), which is known to direct protein kinase C (PKC) to its targets in cells. Here, we investigated whether PKC activity regulates SR activity and D-serine availability in the brain. In vitro, PKC phosphorylated SR and decreased its activity. PKC activation increased SR phosphorylation in serine residues and reduced D-serine levels in astrocyte and neuronal cultures. Conversely, PKC inhibition decreased basal SR phosphorylation and increased cellular D-serine levels. In vivo modulation of PKC activity regulated both SR phosphorylation and D-serine levels in rat frontal cortex. Finally, rats that completed an object recognition task showed decreased SR phosphorylation and increased D-serine/total serine ratios, which was markedly correlated with decreased PKC activity in both cortex and hippocampus. Results indicate that PKC phosphorylates SR in serine residues and regulates D-serine availability in the brain. This interaction may be relevant for the regulation of physiological and pathological mechanisms linked to NMDAR function.

Expresion de la proteína P504S en las células prostáticas en la circulación sanguínea (CPCS) en pacientes con cáncer prostático y el efecto de la supresión adrogénica: estudio inmunocitoquímico / Circulating prostate cells(CPCS)in patient with prostate cancer express P504S and androgen blockage effect: an immunocytochemical based study

Determinar la presencia de CPCS en pacientes con cáncer prostático, la expresión de p504 S yel efecto de la supresión androgénica. Pacientes, materiales y método: en muestras de sangre venosa de 92 pacientes portadores de cáncer a la prostáta se separaron las células mononucleares por centrifugación diferencial. Las cpcs fueron identificadas utilizando anticuerpos monoclonales contra APE y P504S. Muestras de sangre de 10 mujeres fueron usadas como controles. Resultados: En ninguna de las muestras utilizadas como control y en el 68 por ciento de los hombres estudiados se detectaron CPCS. Todas las células detectadas fueron positivas para la expresión de P504S. Los pacientes con supresión androgénica, DES o después de una orquidectomía, tuvieron un nivel de P504S promedio menor que aquellos sin terapia sistémica p menor que 0,03. Conclusiones: la detección de CPCS P504S positivas en biopsias de prostáta es utilizada para el diagnóstico de cáncer, las celulas benignas no expresan este antígeno. Este estudio pionero demuestra que la expresión de P504S en CPCS es menor eb hombres con tratamiento hormonal sistémico.

Objective To determine the effect of androgen blockage on the expression of P504S en circulating prostate cells (CPCs) in men with prostate cancer. Patients, material and method: mononuclear cells were separated from venous blood using differential centrifugation and identification fied using monoclonal antibodies against PSA and P504S. 10 women were used as controls and 92 men with prostate cancer formesd the study group. Results: 64,8 percent of men were positive for CPCs, all the CPCs detected expressed the antigen P504S. No controls were positive. Conclusions. The detection of P504S postive cells in prostate biopsies is used to determine whether they are malignant or not, benign cells P504S negative. This is pioner study to show that CPCs are P504S positive, with the implication that they are malignant cells.

Non-synonymous variants in the AMACR gene are associated with schizophrenia.

BACKGROUND: The AMACR gene is located in the schizophrenia susceptibility locus on chromosome 5p13, previously identified in a large Puerto Rican pedigree of Spanish origin. The AMACR-encoded protein is an enzyme involved in the metabolism of branched-chain fatty and bile acids. The enzyme deficiency causes structural and functional brain changes, and disturbances in fatty acid and oxidative phosphorylation pathways observed in individuals with schizophrenia. Therefore, AMACR is both a positional and functional candidate gene for susceptibility to schizophrenia. METHODS: The study had a two-step design: we performed mutation analysis of the coding and flanking regions of AMACR in affected members of the pedigree, and tested the detected sequence variants for association with schizophrenia in a Puerto Rican case-control sample (n=383) of Spanish descent. RESULTS AND CONCLUSION: We identified three missense variants segregating with the disorder in the family, rs2278008, rs2287939 and rs10941112. Two of them, rs2278008 and rs2287939, demonstrated significant differences in genotype (P = 4 × 10-4, P = 4 × 10-4) and allele (P = 1 × 10-4, P = 9.5 × 10-5) frequencies in unrelated male patients compare to controls, with the odds ratios (OR) 2.24 (95% CI: 1.48-3.40) and 2.25 (95% CI: 1.49-3.38), respectively. The G-C-G haplotype of rs2278008-rs2287939-rs10941112 revealed the most significant association with schizophrenia (P = 4.25 × 10-6, OR = 2.96; 95% CI: 1.85-4.76) in male subjects. There were no statistically significant differences in genotype, allele, and haplotype frequencies between female schizophrenia subjects and controls. Our results suggest that AMACR may play a significant role in susceptibility to schizophrenia in male patients.

P504S expressing circulating prostate cells as a marker for prostate cancer.

Prostate cancer is the most commonly diagnosed cancer in men and the second leading cause of cancer deaths. The serum prostate specific antigen (PSA) is the only biomarker routinely used in screening. The aim of this study was to develop a system to test the presence of circulating prostate cells in men without a diagnosis of prostate cancer in relation with age, serum PSA levels and prostate biopsy by determining the co-expression of several markers such as CD82, HER-2 and matrix metalloproteinase 2 (MMP-2). For this purpose mononuclear cells were separated from blood using differential centrifugation and then prostate cells were identified by using standard immunocytochemical method. Results indicated that among 409 men screened for prostate cancer 16.6% were positive for circulating prostate cells. Cells were positive for MMP-2 and HER-2 in 100 and 14.3% of cases, respectively, without an association with age or PSA levels. However, CD82 protein expression was associated with older age and low grade tumors. It can be concluded that the study of circulating prostate cells with various markers could be a useful complementary screening test for prostate cancer in men with increased PSA level.

Prostate carcinogenesis induced by N-methyl-N-nitrosourea (mnu) in gerbils: histopathological diagnosis and potential invasiveness mediated by extracellular matrix components.

In the present study prostate lesions were induced in gerbils (Meriones unguiculatus) treated with a single N-methyl-N-nitrosourea (MNU) dose; thus, the incidence, latency and histology of these lesions were evaluated. Fibrillar elements of the extracellular matrix associated with microinvasive sites were also investigated. Animals were divided into 5 groups, including 2 control groups: (1) remained untreated; (2) received the corn oil vehicle (vehicle, 0.1 ml/application) and three different tumor induction regimens: (1) received MNU (30 mg/kg) and weekly testosterone (2 mg/kg) (MNU+testosterone); (2) received only MNU (30 mg/kg); (3) received weekly testosterone doses (2 mg/kg). After 3 and 6 months the animals were dissected and the prostates were evaluated morphologically, immunohistochemically and quantitatively. MNU plus androgen contributed to the development of prostatic intraepithelial neoplasia, microinvasive carcinoma and adenocarcinoma in gerbil prostate. However, these lesions occurred earlier in time in groups that received MNU and androgen compared to control animals as they over time also developed to a high extent microinvasive lesions. Cytochemistry and immunohistochemistry showed that these injuries were commonly associated with inflammatory cells whereas the epithelial cells presented proliferative activity. The alpha-methylacyl-CoA racemase (AMACR) expression in prostate cancer cells facilitated diagnosis of gerbil lesions. Testosterone, MNU and MNU+testosterone showed an increased epithelial volume, although the secretory activity was significantly suppressed mainly at neoplastic foci. In the prostatic stroma, reticular fibers increased significantly in MNU, MNU+testosterone and among the lesions found in these groups, while collagen fibers decreased at neoplastic sites. The disruption of the basement membrane was proven at malignant sites by ultrastructural analysis and type IV collagen and laminin degradation. The prostate carcinogenesis mediated by MNU and androgen stimulated the emergence of proliferative lesions in gerbils after short periods and showed the importance of a dynamic remodeling of stromal components for cellular invasiveness.

Expression of alpha-methylacyl coenzyme A racemase in partial and complete focal atrophy on prostate needle biopsies.

OBJECTIVE: To describe the behavior of alpha-methylacyl coenzyme A racemase (AMACR) in focal atrophy including all subtypes. Focal prostatic atrophy is the benign lesion that most frequently mimicks adenocarcinoma particularly the partial variant. STUDY DESIGN: We analyzed the immunohistochemical expression of AMACR in normal glands and acini of adenocarcinoma, partial atrophy and all variants of complete atrophy of a total of 1,198 acini on needle prostatic biopsies. RESULTS: Partial atrophy showed negative and weak expression of AMACR in 143/190 (75.3%), and 47/190 (24.7%) acini, respectively. The secretory (luminal) compartment in all variants of complete atrophy showed aberrant immunohistochemical expression: AMACR negative, prostate-specific antigen negative and 34betaE12 positive, suggesting an intermediate phenotype for these cells. CONCLUSION: No strong positivity was seen in partial atrophy; however, the absence of basal cells in 23.2% acini of partial atrophy and the weak positivity seen in 24.7% acini that overlaps with 22.5% acini with weak expression in adenocarcinoma may be a pitfall for the correct interpretation in the differential diagnosis of cancer. Recognizing the hematoxylin-eosin features of partial atrophy is still the most critical aspect in preventing a misdiagnosis of adenocarcinoma.

Las células prostáticas en la circulación sanguínea en pacientes con cáncer prostático expresan la proteína P504S: un estudio utilizando inmunocitoquímica / Circulating prostate cells in patients with prostate cancer express P504S: an immunocytochem

Determinar la presencia de células prostáticas en la circulación sanguínea (CPCs) en pacientes con cáncer prostático y la expresión de P504S. Método: Las células mononucleares fueron separadas de la sangre venosa por centrifugación diferencial, e identificadas utilizando anticuerpos monoclonales contra APE y P504S. Diez mujeres fueron usadas como controles. 66 hombres con cáncer prostático formaron el grupo de estudio. Resultados: 69,7 por ciento tuvieron células prostáticas en la sangre venosa, todas las células detectadas fueron positivas para la expresión de P504S. Conclusiones: La detección de células prostáticas P504S positivas en biopsias de la próstata esutilizando para el diagnóstico de cáncer, células benignas no se expresan el antígeno. Este es el primer estudio que demuestra la expresión de P504S en CPCs, con la inferencia que estas células son malignas.

To determine the expression of P504S en circulating prostate cells (CPCs) in men with prostate cancer. Method: Mononuclear cells were separated from venous blood using differential centrifugation andidentified using monoclonal antibodies against PSA and P504S. 10 women were used as controls and 66 men with prostate cancer formed the study group. Results: 69.7 percent of men were positive for CPCs, all the CPCs detected expressed the antigen P504S. Conclusions: The detection of P504S positive cells in prostate biopsies is used to determine whether they are malignant or not, benign cells are P504S negative. This is the first study to show that CPCsare P504S with the implication that they are malignant cells.

Cytotoxic epimeric withaphysalins from leaves of Acnistus arborescens.

Phytochemical analysis of the leaves of Acnistus arborescens (Solanaceae) resulted in the isolation of two new epimeric withaphysalins (17S,20R,22R)-5beta,6beta: 18,20-diepoxy-4beta,18-dihydroxy-1-oxowitha-24-enolide (2, 18R and 18S), together with the known withaphysalin F (1, 18R and 18S). Their structures were established by spectroscopic methods, including 2D NMR data and comparison with literature data. Compounds 1 and 2 dis-played potent cytotoxic activities against several cancer cell lines with IC50 values in the range of 0.20 to 1.46 microg/mL for 1 and 0.89 to 8.08 microg/mL for 2. The strong cytotoxicity presented by 1 suggests that in this series of compounds, the 2,3-unsaturated ketone moiety is an important pharmacophoric unit. The cytotoxic activity seemed to be related to DNA synthesis inhibition, as revealed by the reduction of 5-bromo-2'-deoxyuridine incorporation after 24 hours of incubation on leukemic cells.

Role of the transmembrane domain of the VanT serine racemase in resistance to vancomycin in Enterococcus gallinarum BM4174.

Enterococcus gallinarum BM4175 (a vancomycin-susceptible derivative of BM4174 obtained by insertional inactivation of vanC-1) was transformed with plasmid constructs pCA10 (containing the genes necessary for resistance, vanC-1-XYc-T), pJP1 (with a fragment lacking the DNA encoding the transmembrane region of VanT, -vanC-1-XYc-T((Delta))(2-322)-) and with plasmids containing fragments encoding either the transmembrane (mvanT(1-322)) or racemase (svanT(323-698)) domains of VanT under the control of a constitutive promoter. Accumulated peptidoglycan precursors were measured in all strains in the presence of L-Ser, D-Ser (50 mM) or in the absence of any growth supplement. Uptake of 0.1 mM L-[(14)C]serine was also determined in BM4174, BM4175 and BM4175/pCA10. Vancomycin resistance was restored in BM4175 transformed with pCA10(C-1-XYc-T), and the profile of peptidoglycan precursors was similar to wild-type E. gallinarum BM4174. Transformation of E. gallinarum BM4175 with plasmid pJP1(vanC-1-XYc-T((Delta))(2-322)) resulted in: (i) vancomycin MICs remaining within susceptible levels (< or =4 mg/L) in the absence of any growth supplement, but increasing to 8 mg/L when either L-Ser or D-Ser was added to the medium; and (ii) the relative amounts of accumulated UDP-MurNAc-pentapeptide[D-Ser] and tetrapeptide precursors decreasing substantially compared with BM4175/pCA10 and BM4174. The effect on the appearance of tetrapeptide appeared to be host dependent, since a substantial amount was present when the same plasmid construct pJP1(vanC-1-XYc-T((Delta))(2-322)) was electroporated into Enterococcus faecalis JH2-2. The uptake of L-[(14)C]Ser at 240 s was decreased by approximately 40% in BM4175 compared with BM4174. Plasmid pCA10(C-1-XY(C)-T) restored uptake of L-[(14)C]Ser at 180 and 240 s in BM4175. The results suggest that the transmembrane domain of VanT is likely to be involved in the transport of L-Ser, and that in its absence the resistance phenotype is compromised.

Cofactors of serine racemase that physiologically stimulate the synthesis of the N-methyl-D-aspartate (NMDA) receptor coagonist D-serine.

High levels of d-serine occur in the brain, challenging the notion that d-amino acids would not be present or play a role in mammals. d-serine levels in the brain are even higher than many l-amino acids, such as asparagine, valine, isoleucine, and tryptophan, among others. d-serine is synthesized by a serine racemase (SR) enzyme, which directly converts l- to d-serine. We now report that SR is a bifunctional enzyme, producing both d-serine and pyruvate in cultured cells and in vitro. Transfection of SR into HEK 293 cells elicits synthesis of d-serine and augmented release of pyruvate to culture media. We identified substances present in HEK 293 and astrocyte cell extracts that strongly stimulate d-serine production by SR and elicit production of pyruvate. Experiments with recombinant enzyme reveal that Mg(2+) and ATP present in the cell extracts are physiological cofactors and increase 5- to 10-fold the rates of racemization and production of pyruvate. As much as three molecules of pyruvate are synthesized for each molecule of d-serine produced by SR. This finding constitutes a previously undescribed mechanism underlying d-amino acid synthesis in mammals, different from classical amino acid racemases present in bacteria. Our data link the production of d-serine to the energy metabolism, with implications for the metabolic and transmitter crosstalk between glia and neurons.

Hemangioendotelioma hepatico associado a galactosemia em um recem-nascido: relato de caso / Hepatic hemangioendothelioma associated to galactosemia in a newborn: case report

Objetivo: Descrever um recem-nascido portador de galactosemia e de hemangioendotelioma hepatico (HH). Descricao: um RN pre-termo nascido de parto normal apresentou ao nascimento hidropisia, hepato-esplenomegalia e ictericia. Os exames laboratoriais...

A new strategy to decrease N-methyl-D-aspartate (NMDA) receptor coactivation: inhibition of D-serine synthesis by converting serine racemase into an eliminase.

Serine racemase is a brain-enriched enzyme that synthesizes d-serine, an endogenous modulator of the glycine site of N-methyl-d-aspartate (NMDA) receptors. We now report that serine racemase catalyzes an elimination reaction toward a nonphysiological substrate that provides a powerful tool to study its neurobiological role and will be useful to develop selective enzyme inhibitors. Serine racemase catalyzes robust elimination of l-serine O-sulfate that is 500 times faster than the physiological racemization reaction, generating sulfate, ammonia, and pyruvate. This reaction provides the most simple and sensitive assay to detect the enzyme activity so far. We establish stable cell lines expressing serine racemase and show that serine racemase can also be converted into a powerful eliminase in cultured cells, while the racemization of l-serine is inhibited. Likewise, l-serine O-sulfate inhibits the synthesis of d-serine in primary astrocyte cultures. We conclude that the synthetic compound l-serine O-sulfate is a better substrate than l-serine as well as an inhibitor of d-serine synthesis. Inhibition of serine racemase provides a new strategy to selectively decrease NMDA receptor coactivation and may be useful in conditions in which overstimulation of NMDA receptors plays a pathological role.