A glutathione transferase from Agrobacterium tumefaciens reveals a novel class of bacterial GST superfamily.

In the present work, we report a novel class of glutathione transferases (GSTs) originated from the pathogenic soil bacterium Agrobacterium tumefaciens C58, with structural and catalytic properties not observed previously in prokaryotic and eukaryotic GST isoenzymes. A GST-like sequence from A. tumefaciens C58 (Atu3701) with low similarity to other characterized GST family of enzymes was identified. Phylogenetic analysis showed that it belongs to a distinct GST class not previously described and restricted only in soil bacteria, called the Eta class (H). This enzyme (designated as AtuGSTH1-1) was cloned and expressed in E. coli and its structural and catalytic properties were investigated. Functional analysis showed that AtuGSTH1-1 exhibits significant transferase activity against the common substrates aryl halides, as well as very high peroxidase activity towards organic hydroperoxides. The crystal structure of AtuGSTH1-1 was determined at 1.4 Å resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). Although AtuGSTH1-1 adopts the canonical GST fold, sequence and structural characteristics distinct from previously characterized GSTs were identified. The absence of the classic catalytic essential residues (Tyr, Ser, Cys) distinguishes AtuGSTH1-1 from all other cytosolic GSTs of known structure and function. Site-directed mutagenesis showed that instead of the classic catalytic residues, an Arg residue (Arg34), an electron-sharing network, and a bridge of a network of water molecules may form the basis of the catalytic mechanism. Comparative sequence analysis, structural information, and site-directed mutagenesis in combination with kinetic analysis showed that Phe22, Ser25, and Arg187 are additional important residues for the enzyme's catalytic efficiency and specificity

SILVER-BINDING NUCLEOLAR ORGANIZER REGIONS IN BENIGN AND MALIGNANT PROSTATIC LESIONS

Thirty-six transvesical or transurethral prostatectomy cases were selected from the histopathology files of the Laiko General Hospital. Among the 36 cases, there were 10 with benign prostatic hyperplasia (8 patients having distinct areas of adenosis) and 26 cases of prostatic adenocarcinoma (6 grade I, 13 grades II and III and 7 grade IV carcinomas). From each case, silver-binding nucleolar organizer regions (AgNORs) have been counted in sections of routinely processed paraffin-embedded tissue blocks. The mean AgNOR count per case was calculated. For the cases of prostatic hyperplasia, the mean AgNOR count was found to be 2.95 +/- 0.42, for adenosis 3.45 +/- 0.56, for grade I adenocarcinoma 4.97 +/- 0.74, for grades II and III 7.31 +/- 0.81 and for grade IV adenocarcinomas 11.41 +/- 1.68. This difference in the mean AgNOR count was found to be of statistical significance (p < 0.001) between adenosis and grade I adenocarcinomas and between grade II and III and grade IV adenocarcinomas. It appears that AgNOR counting may prove to be of benefit in differentiating between some benign and malignant prostatic lesions and that it might provide information concerning the biological behavior of prostatic adenocarcinomas

A germline mutation in the POT1 gene is a candidate for familial non-medullary thyroid cancer

Non-medullary thyroid cancer (NMTC) is a common endocrine malignancy with a genetic basis that has yet to be unequivocally established. In a recent whole-genome sequencing study of five families with occurrence of NMTCs, we shortlisted promising variants with the help of bioinformatics tools. Here, we report in silico analyses and in vitro experiments on a novel germline variant (p.V29L) in the highly conserved oligonucleotide/oligosaccharide binding domain of the Protection of Telomeres 1 (POT1) gene in one of the families. The results showed a reduction in telomere-bound POT1 levels in the mutant protein as compared to its wild-type counterpart. HEK293T cells carrying POT1 p.V29L showed increased telomere length in comparison to wild-type cells, suggesting that the mutation causes telomere dysfunction and may play a role in predisposition to NMTC in this family. While one germline mutation in POT1 has already been reported in a melanoma-prone family with prevalence of thyroid cancers, we report the first of such mutations in a family affected solely by NMTCs, thus expanding current knowledge on shelterin complex-associated cancers

Coexistence of early microinvasive endometrioid adenocarcinoma and CIN3 in the uterine cervix in a 32-year-old Japanese woman

<p>Abstract</p> <p>Simultaneous occurrence of early microinvasive endometrioid adenocarcinoma (EMEA) and CIN 3 in the uterine cervix is very rare in Japan. A 32-year-old Japanese woman was pointed out to have atypical cells in the cervical cytology. Colposcopic examination revealed irregular lesions in the cervix, and a biopsy showed simultaneous EMEA and CIN3. The EMEA was grade I and CIN3 corresponded to severe dysplasia/carcinoma in situ. Hysterectomy and lymph nodes dissection were performed. Grossly, mucosal irregularity and erosion were seen in the cervix. No tumor formation was recognized. The cervix was examined by serial sections. Microscopically, there were a tiny adenocarcinoma (0.5 cm in diameter and 0.3 cm in depth) and broad areas of CIN3. The adenocarcinoma was EMEA without mucins. The EMEA was FIGO stage 1A1. Immunohistochemically, the EMEA was positive for pancytokeratins (AE1/2 +++, CAM5.2 ++), cytokeratin (CK) 34βE12 +, CK5/6 +, CK7 +, CK18 +++, CK19 ++, CA19-9 +, CA125 +++, p53 +, ER +++, PgR +++, while it was negative for CK8, CK14, CK20, EMA, vimentin, CEA, desmin, smooth muscle actin, p63, chromogranin, synaptophysin, CD56, CD68, HER2/neu, MUC1, MUC2, MUC5AC, and MUC6. The CIN 3 was positive for pancytokeratins (AE1/2 +++, CAM5.2 +), cytokeratin (CK) 34βE12 +++, CK5/6 +++, CK7 +, EMA, CA19-9 +, CA125 +, p53 +, p63 +++, ER +++, and MUC1 +, while it was negative for CK8, CK14, CK18, CK19, CK20, vimentin, CEA, desmin, smooth muscle actin, chromogranin, synaptophysin, CD56, CD68, PgR, HER2/neu, MUC2, MUC5AC and MUC6. The lymph nodes showed no metastatic lesions (0/34). In conclusion, the author reported a rare case of simultaneous EMEA and CIN 3 with extensive immunohistochemical findings.</p

A glutathione transferase from Agrobacterium tumefaciens reveals a novel class of bacterial GST superfamily.

In the present work, we report a novel class of glutathione transferases (GSTs) originated from the pathogenic soil bacterium Agrobacterium tumefaciens C58, with structural and catalytic properties not observed previously in prokaryotic and eukaryotic GST isoenzymes. A GST-like sequence from A. tumefaciens C58 (Atu3701) with low similarity to other characterized GST family of enzymes was identified. Phylogenetic analysis showed that it belongs to a distinct GST class not previously described and restricted only in soil bacteria, called the Eta class (H). This enzyme (designated as AtuGSTH1-1) was cloned and expressed in E. coli and its structural and catalytic properties were investigated. Functional analysis showed that AtuGSTH1-1 exhibits significant transferase activity against the common substrates aryl halides, as well as very high peroxidase activity towards organic hydroperoxides. The crystal structure of AtuGSTH1-1 was determined at 1.4 Å resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). Although AtuGSTH1-1 adopts the canonical GST fold, sequence and structural characteristics distinct from previously characterized GSTs were identified. The absence of the classic catalytic essential residues (Tyr, Ser, Cys) distinguishes AtuGSTH1-1 from all other cytosolic GSTs of known structure and function. Site-directed mutagenesis showed that instead of the classic catalytic residues, an Arg residue (Arg34), an electron-sharing network, and a bridge of a network of water molecules may form the basis of the catalytic mechanism. Comparative sequence analysis, structural information, and site-directed mutagenesis in combination with kinetic analysis showed that Phe22, Ser25, and Arg187 are additional important residues for the enzyme's catalytic efficiency and specificity