Fatty acid hydroperoxide lyase in tomato fruits: cloning and properties of a recombinant enzyme expressed in Escherichia coli

Fatty acid hydroperoxide lyase (HPL) is a member of a novel subfamily of cytochrome P450 and catalyzes a cleavage reaction of fatty acid hydroperoxides to form short-chain aldehydes and oxo-acids. A cDNA encoding tomato fruit HPL (LeHPL) was obtained. An active LeHPL was expressed in E. coli and purified. It showed highest activity against the 13-hydroperoxide of linolenic acid, followed by that of linoleic acid. 9-Hydroperoxides were poor substrates. The absorption spectrum of the purified LeHPL in the native form was similar to that of most P450s although a CO-adduct having a lambda max at 450 nm could not be obtained. LeHPL activity is reversibly inhibited by nordihydroguaiaretic acid, while salicylic acid irreversibly inhibited it. LeHPL is kinetically inactivated by fatty acid hydroperoxides, especially 9-hydroperoxides. The inactivation is prevented by inhibitors of LeHPL. Thus, HPL catalytic activity is thought to be essential to its inactivation. During the inactivation, an abolition of the Soret band was evident, indicating that inactivation is caused mainly by degradation of the prosthetic heme in LeHPL.     

Crystal structures of Delta1-piperideine-2-carboxylate/Delta1-pyrroline-2-carboxylate reductase belonging to a new family of NAD(P)H-dependent oxidoreductases: conformational change, substrate recognition, and stereochemistry of the reaction

Delta(1)-Piperideine-2-carboxylate/Delta(1)-pyrroline-2-carboxylate reductase from Pseudomonas syringae pv. tomato belongs to a novel sub-class in a large family of NAD(P)H-dependent oxidoreductases distinct from the conventional MDH/LDH superfamily characterized by the Rossmann fold. We have determined the structures of the following three forms of the enzyme: the unliganded form, the complex with NADPH, and the complex with NADPH and pyrrole-2-carboxylate at 1.55-, 1.8-, and 1.7-A resolutions, respectively. The enzyme exists as a dimer, and the subunit consists of three domains; domain I, domain II (NADPH binding domain), and domain III. The core of the NADPH binding domain consists of a seven-stranded predominantly antiparallel beta-sheet fold (which we named SESAS) that is characteristic of the new oxidoreductase family. The enzyme preference for NADPH over NADH is explained by the cofactor binding site architecture. A comparison of the overall structures revealed that the mobile domains I and III change their conformations to produce the catalytic form. This conformational change plays important roles in substrate recognition and the catalytic process. The active site structure of the catalytic form made it possible to identify the catalytic Asp:Ser:His triad and investigate the catalytic mechanism from a stereochemical point of view.     

Purification, identification, and cDNA cloning of Jun a 2, the second major allergen of mountain cedar pollen

The second major allergen of Juniperus ashei (mountain cedar) pollen, Jun a 2, has been purified and its cDNA cloned. The purified protein has a molecular mass of 43 kDa and its N-terminal 9-residue amino acid sequence is highly homologous to those of Cry j 2 and Cha o 2, the second major allergen of Cryptomeria japonica and Chamaecyparis obtusa pollen, respectively. cDNA clones encoding Jun a 2 were isolated after PCR based amplification, and their nucleotide sequences were determined. The cDNA contains an open reading frame of 507 amino acid residues, and encodes a putative 54-residue signal sequence and a 453-residue intermediate, which releases a C-terminal fragment upon maturation. Three possible N-linked glycosylation sites and 20 cystein-residues are found in the deduced amino acid sequence. The amino acid sequence of Jun a 2 shows 70.7 and 82.0% identity with those of Cry j 2 and Cha o 2, respectively. Immunological observations that IgE antibodies in sera of Japanese pollinosis patients bind not only to Cry j 2 and Cha o 2 but also to Jun a 2 strongly suggest that Jun a 2 is an allergen of mountain cedar pollen, and that allergenic epitopes of these three allergens are similar.     

Restriction endonuclease PshAI from Plesiomonas shigelloides with the novel recognition site 5'-GACNN/NNGTC

A new restriction endonuclease (ENase), PshAI, has been isolated from Plesiomonas shigelloides 319-73, an organism that causes food poisoning in humans. The enzyme was stable and produced a yield of 410 units/g of cells. In the presence of 10 mM MgCl2, PshAI recognizes and cleaves the nucleotide sequence 5'-GACNN/NNGTC, producing blunt ends. PshAI will be useful for structural analysis and molecular cloning of DNA, because no ENases recognizing sequence GACNNNNGTC have been previously described.     

Role of Diffusion Weighted Imaging and Contrast-Enhanced MRI in the Evaluation of Intrapelvic Recurrence of Gynecological Malignant Tumor

Background and Purpose

To investigate the diagnostic performance of diffusion-weighted imaging (DWI) and contrast-enhanced imaging in combination with T2-weighted imaging (T2WI) for magnetic resonance imaging (MRI) evaluation of intrapelvic recurrence of gynecological malignancies.

Materials and Methods

Sixty-two patients with suspected intrapelvic recurrence of gynecological malignancies underwent pelvic MRI including T2WI DWI, and contrast-enhanced imaging. Diagnostic performance for detection of local recurrence, pelvic lymph node and bone metastases, and peritoneal lesions was evaluated by consensus reading of two experienced radiologists using a 5-point scoring system, and compared among T2WI with unenhanced T1-weighted imaging (T1WI) (protocol A), a combination of protocol A and DWI (protocol B), and a combination of protocol B and contrast-enhanced imaging (protocol C). Final diagnoses were obtained by histopathological examinations, radiological imaging and clinical follow-up for at least 6 months. Receiver operating characteristic (ROC) analysis and McNemar test were employed for statistical analysis.

Results

Locally recurrent disease, lymph node recurrence, peritoneal dissemination and bone metastases were present in 48.4%, 29.0%, 16.1%, and 6.5% of the patients, respectively. The patient-based sensitivity, specificity, accuracy, and area under the ROC curve (AUC) for detection of intrapelvic recurrence were 55.0, 81.8, 64.5% and 0.753 for protocol A, 80.0, 77.3, 79.0% and 0.838 for protocol B, and 80.0, 90.9, 83.9% and 0.862 for protocol C, respectively. The sensitivity, accuracy, and AUC were significantly better for protocols B and C than for protocol A (p<0.001). There was no significant difference between protocols B and C.

Conclusion

MRI using a combination of DWI and T2WI gives comparatively acceptable results for assessment of intrapelvic recurrence of gynecological malignancies.     

Highly Diastereoselective Synthesis of (2′S)-[2′-2H]-2′-Deoxyribonucleosides from the Corresponding Ribonucleosides

Abstract

The four (2′S)-[2′-²H]-2′-deoxynucleosides (>90 atom % ²H), were synthesized from the corresponding ribonucleosides involving six steps of reactions, i.e., oxidation of their 2′-hydroxyl group, stereoselective reductive deuteration of the resulting 2′-ketonucleoside intermediates with NaB²H₄ in EtOH-H₂O or EtOH, triflation, bromination with LiBr, highly stereoselective Bu₃SnH-Et₃B reduction of the resulting bromide, and, finally, unmasking.     

Beta-adrenergic pathway activation enhances aggressiveness and inhibits stemness in head and neck cancer

Chronic stress leads to the activation of the beta-adrenergic pathway. Its activation has been implicated in the progression of different types of cancer but its role on head and neck squamous cell carcinomas (HNSCCs) remains undefined. The aim of this study was to investigate the influence of the beta-adrenergic pathway activation in the progression of HNSCCs and offer a panel of potential treatments for patients with the active beta-adrenergic pathway. Five hundred and twenty TCGA patients with primary HNSCCs were divided in two groups: ADRB2^low / SLC6A2^low and ADRB2^high / SLC6A2^high. Differentially expressed genes (DEGs) were identified through differential expression analysis. The association of clinicopathological and genomic features between the groups was analyzed using a bioinformatic approach. Potential drugs for treatment of HNSCC were identified based on the DEGs. There was association between ADRB2 and SLC6A2 expressions with age, race, tumor site, histologic grade, perineural invasion, and HPV p16 status. It was identified 898 DEGs between the groups. High ADRB2/SLC6A2 expression stimulated HNSCC proliferation, adhesion, invasion, and angiogenesis. On the other hand, genes related to cell stemness were downregulated in patients with activation of the beta- adrenergic pathway. Finally, 56 FDA-approved antineoplastic and immunotherapeutic drugs were identified as potential targets for the personalized treatment.