D-allulose-3-epimerase (DPEase) is the key enzyme for isomerization of D-fructose to D-allulose. In order to improve its thermal stability, short amphiphilic peptides (SAP) were fused to the N-terminal of DPEase. SDS-PAGE analysis showed that the heterologously expressed DPEase folded correctly in Bacillus subtilis, and the protein size was 33 kDa. After incubation at 40 °C for 48 h, the residual enzyme activity of SAP1-DSDPEase was 58%. To make the recombinant B. subtilis strain reusable, cells were immobilized with a composite carrier of sodium alginate (SA) and titanium dioxide (TiO2). The results showed that 2% SA, 2% CaCl2, 0.03% glutaraldehyde solution and a ratio of TiO2 to SA of 1:4 were optimal for immobilization. Under these conditions, up to 82% of the activity of immobilized cells could be retained. Compared with free cells, the optimal reaction temperature of immobilized cells remained unchanged at 80 °C but the thermal stability improved. After 10 consecutive cycles, the mechanical strength remained unchanged, while 58% of the enzyme activity could be retained, with a conversion rate of 28.8% achieved. This study demonstrated a simple approach for using SAPs to improve the thermal stability of recombinant enzymes. Moreover, addition of TiO2 into SA during immobilization was demonstrated to increase the mechanical strength and reduce cell leakage.
Bacillus subtilis/metabolism* ; Carbohydrate Epimerases/genetics* ; Enzyme Stability ; Enzymes, Immobilized/metabolism* ; Fructose ; Hydrogen-Ion Concentration ; Racemases and Epimerases ; Temperature

BACKGROUND: The histologic classification of renal cell carcinoma (RCC) is based on the cytoarchitectural features, yet sometimes this requires correlation with the immunophenotype. Alpha-methylacyl-CoA racemase (AMACR) and claudin-7 have recently been introduced as useful markers that are frequently expressed in papillary RCC (PRCC) and chromophobe RCC (ChRCC), respectively. The aims of this study are to evaluate the expressions of AMACR and claudin-7 in RCCs and to investigate whether they are helpful for making the histological classification of RCCs. METHODS: Immunohistochemistry for CD10, RCC marker, cytokeratin (CK)7, CD117, AMACR and claudin-7 was performed for 104 RCCs, and these consisted of 54 clear cell RCCs (CCRCC), 26 PRCCs and 24 ChRCCs. RESULTS: For diagnosing PRCC, the sensitivity and specificity of AMACR were 92.3% and 71.8%, respectively, and using AMACR(+)/CK7(+), the specificity was increased by 23.1% to 94.9%. For diagnosing ChRCC, the sensitivity and specificity of claudin-7 were 91.7% and 78.8%, respectively, and using claudin-7(+)/AMACR(-), the specificity was significantly improved (to 96.3%). For diagnosing CCRCC, CK7(-)/claudin-7(-)/CD117(-) was the most useful immunohistochemical panel (sensitivity, 96.3%; specificity, 98%). CONCLUSIONS: AMACR and claudin-7 are helpful markers for the histologic classification of RCCs, and their diagnostic utility is strengthened when they are used as an immunohistochemical panel, AMACR(+)/CK7(+) for PRCC, claudin-7(+)/AMACR(-) for ChRCC and CK7(-)/claudin-7(-)/CD117(-) for CCRCC.
Carcinoma, Renal Cell ; Immunohistochemistry ; Keratins ; Racemases and Epimerases ; Sensitivity and Specificity

OBJECTIVETo study the Gleason histologic grading of prostate carcinoma in relation to the serum prostate-specific antigen (PSA) level and the PSA in situ of the tumor, and the immunohistochemical staining of basal cell-specific cytokertain(34 beta E12) and alpha-Methylacyl-CoA racemase(P504S) of the tumor.

METHODSThe serum PSA levels were measured in 40 cases of prostate carcinoma. The Gleason histologic grading was based on histopathologic examination of the tumors, and the immunohistochemical staining including PSA in situ (35 cases), 34 beta E12(12 cases) and P504S(10 cases) was examined.

RESULTSThe higher the Gleason histologic grading of prostate carcinoma, the higher the serum PSA level(P < 0.01), and the weaker the positive reaction of the immunohistochemical staining of PSA of the tumor(P < 0.05). And the tumor cells displayed positive reaction for P504S and negative for 34 beta E12.

CONCLUSIONThe Gleason histologic grading of prostate carcinoma is positively related to the serum PSA level and negatively to PSA in situ of the tumor immunohistochemically. It is important to use immunohistochemical staining for 34 beta E12 and P504S in the pathologic diagnosis of prostate carcinoma.

Aged ; Aged, 80 and over ; Humans ; Immunohistochemistry ; Keratins ; analysis ; Male ; Middle Aged ; Neoplasm Staging ; Prostate-Specific Antigen ; blood ; Prostatic Neoplasms ; chemistry ; pathology ; Racemases and Epimerases ; analysis

Diagnosis, Differential ; Humans ; Immunohistochemistry ; Keratins ; analysis ; Male ; Prostatic Hyperplasia ; diagnosis ; Prostatic Intraepithelial Neoplasia ; diagnosis ; Prostatic Neoplasms ; diagnosis ; Racemases and Epimerases ; analysis ; Staining and Labeling ; methods

Antigens, Surface ; blood ; Diagnosis, Differential ; Glutamate Carboxypeptidase II ; blood ; Humans ; Male ; Neoplasm Staging ; Prostate-Specific Antigen ; blood ; Prostatic Hyperplasia ; pathology ; Prostatic Neoplasms ; etiology ; metabolism ; pathology ; Racemases and Epimerases ; analysis

OBJECTIVETo investigate the clinical manifestations, pathological characteristics, and treatments of urothelial-type mucinous adenocarcinoma of the prostate (UMAP).

METHODSWe reported a case of UMAP, reviewed relevant literature, and analyzed the clinicopaothological features, diagnosis, treatment, and prognosis of the disease.

RESULTSThe patient was a 60-year-old male and underwent transurethral resection of the prostate for dysuria. Postoperative pathology indicated mucinous adenocarcinoma and sigmoidoscopy revealed no primary colon cancer. Immunohistochemical staining showed the negative expressions of PSA and P504s and positive expressions of CK7, CK34 β E12, CK20, and CDX2. Thus UMAP was confirmed and treated by intensity-modulated radiotherapy. Then the patient was followed up for 30 months, which showed desirable therapeutic result, with neither local progression nor distant metastasis.

CONCLUSIONUMAP has a bad prognosis and its diagnosis depends on pathological and immunohistocchemical examinations. It responds well to radical prostatectomy but is not sensitive to endocrine therapy. Radiotherapy can be considered for those who are not fit to receive radical prostatectomy.

Adenocarcinoma, Mucinous ; metabolism ; pathology ; therapy ; Humans ; Keratins ; metabolism ; Male ; Middle Aged ; Neoplasm Proteins ; metabolism ; Prognosis ; Prostatectomy ; Prostatic Neoplasms ; metabolism ; pathology ; therapy ; Racemases and Epimerases ; metabolism

Early-onset progressive encephalopathy is a lethal encephalopathy caused by NAXE gene mutations. This paper reports the clinical and genetic features of a patient with early-onset progressive encephalopathy. A 4-year-old boy admitted to the hospital had repeated walking instability and limb weakness for 2 years. The patient and his elder brother (already dead) had clinical onset at 2 years of age. Both of them showed symptoms such as strabismus, ataxia, reduced muscle tone, delayed development, and repeated respiratory failure after infection. The NAXE gene of the patient showed new compound heterozygous mutations, i.e., c.255 (exon 2) A>T from his mother and c.361 (exon 3) G>A from his father. The NAXE gene encodes an epimerase that is essential for the repair of cellular metabolites of NADHX and NADPHX. This disease is associated with a deficiency of the mitochondrial NAD(P)HX repair system. Patients usually have rapid disease progression. They are also quite likely to have respiratory failure immediately after infection.
Adult ; Age of Onset ; Base Sequence ; Brain Diseases ; enzymology ; genetics ; Child, Preschool ; Disease Progression ; Female ; Heterozygote ; Humans ; Male ; Mutation ; Racemases and Epimerases ; genetics

D-psicose 3-epimerase (DPEase) is demonstrated to be useful in the bioproduction of D-psicose, a rare hexose sugar, from D-fructose, found plenty in nature. Clostridium cellulolyticum H10 has recently been identified as a DPEase that can epimerize D-fructose to yield D-psicose with a much higher conversion rate when compared with the conventionally used DTEase. In this study, the crystal structure of the C. cellulolyticum DPEase was determined. The enzyme assembles into a tetramer and each subunit shows a (β/α)(8) TIM barrel fold with a Mn(2+) metal ion in the active site. Additional crystal structures of the enzyme in complex with substrates/products (D-psicose, D-fructose, D-tagatose and D-sorbose) were also determined. From the complex structures of C. cellulolyticum DPEase with D-psicose and D-fructose, the enzyme has much more interactions with D-psicose than D-fructose by forming more hydrogen bonds between the substrate and the active site residues. Accordingly, based on these ketohexose-bound complex structures, a C3-O3 proton-exchange mechanism for the conversion between D-psicose and D-fructose is proposed here. These results provide a clear idea for the deprotonation/protonation roles of E150 and E244 in catalysis.
Binding Sites ; Biocatalysis ; Catalytic Domain ; Clostridium cellulolyticum ; enzymology ; Hexoses ; chemistry ; Manganese ; chemistry ; Protein Structure, Quaternary ; Racemases and Epimerases ; chemistry ; metabolism ; Substrate Specificity

Larval excretory-secretory products of Anisakis simplex are known to cause allergic reactions in humans. A cDNA library of A. simplex 3rd-stage larvae (L3) was immunoscreened with polyclonal rabbit serum raised against A. simplex L3 excretory-secretory products to identify an antigen that elicits the immune response. One cDNA clone, designated as alpha-methylacyl CoA racemase (Amacr) contained a 1,412 bp cDNA transcript with a single open reading frame that encoded 418 amino acids. A. simplex Amacr showed a high degree of homology compared to Amacr orthologs from other species. Amacr mRNA was highly and constitutively expressed regardless of temperature (10-40degrees C) and time (24-48 hr). Immunohistochemical analysis revealed that Amacr was expressed mainly in the ventriculus of A. simplex larvae. The Amacr protein produced in large quantities from the ventriculus is probably responsible for many functions in the development and growth of A. simplex larvae.
Amino Acid Sequence ; Animals ; Anisakis/*enzymology/genetics ; Cloning, Molecular ; Cluster Analysis ; Gene Expression Profiling ; Gene Library ; Humans ; Immunohistochemistry ; Larva/enzymology/genetics ; Mice ; Mice, Inbred ICR ; Molecular Sequence Data ; Phylogeny ; Rabbits ; Racemases and Epimerases/genetics/*metabolism ; Sequence Homology, Amino Acid

OBJECTIVETo investigate the value of detection of AMACR (P504S), P63 and 34betaE12 cocktail in the early diagnosis of prostate cancer (PCa).

METHODSThe expressions of AMACR, P63 and 34betaE12 were examined in the biopsy specimens of 42 cases of prostate cancer, 12 cases of high-grade prostatic intraepithelial neoplasia (HGPIN) and 30 cases of benign prostatic hyperplasia (BPH) using the Maxvision single-step immunohistochemical method with triple-antibody cocktail (AMACR/P63/34betaE12) staining and double-color chromogens in single paraffin sections .

RESULTSThe expressions of AMACR, P63 and 34betaE12 were significantly different between PCa and BPH (P < 0.01). The staining of PCa was positive for AMACR and negative for P63 and 34betaE12, and the positivity rate of AMACR was 100%. BPH was strongly expressed for P63 and 34betaE12, but negatively for AMACR. The expression of AMACR was significantly different between HGPIN and BPH (P < 0.01), but not between HGPIN and PCa (P > 0.05), and the positivity rate of AMACR in HGPIN was 91.67%. However, the expressions of P63 and 34betaE12 were significantly different between HGPIN and PCa (P < 0.01), but not between HGPIN and BPH (P > 0.05), and the positivity rate of AMACR in HGPIN was 100%. The level of AMACR expression was not correlated with PCa Gleason score (P > 0.05).

CONCLUSIONAMACR is a sensitive and specific marker for PCa. P63 and 34betaE12 cocktail staining can increase the sensitivity and specificity of the basal cell detection. The immunohistochemical analysis with triple-antibody cocktail (AMACR/P63/34betaE12) staining and double-color chromogens can improve diagnostic accuracy and has an important applied value for the early diagnosis of prostate cancer.

Aged ; Carcinoma, Basal Cell ; diagnosis ; metabolism ; Early Diagnosis ; Humans ; Immunohistochemistry ; Keratins ; biosynthesis ; Male ; Membrane Proteins ; biosynthesis ; Middle Aged ; Prostatic Hyperplasia ; diagnosis ; metabolism ; Prostatic Neoplasms ; diagnosis ; metabolism ; Racemases and Epimerases ; biosynthesis