Formaldehyde Fixation Contributes to Detoxification for Growth of a Nonmethylotroph, Burkholderia cepacia TM1, on Vanillic Acid

During bacterial degradation of methoxylated lignin monomers, such as vanillin and vanillic acid, formaldehyde is released through the reaction catalyzed by vanillic acid demethylase. When Burkholderia cepacia TM1 was grown on vanillin or vanillic acid as the sole carbon source, the enzymes 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) were induced. These enzymes were also expressed during growth on Luria-Bertani medium containing formaldehyde. To understand the roles of these enzymes, the hps and phi genes from a methylotrophic bacterium, Methylomonas aminofaciens 77a, were introduced into B. cepacia TM1. The transformant strain constitutively expressed the genes for HPS and PHI, and these activities were two- or threefold higher than the activities in the wild strain. Incorporation of [¹⁴C]formaldehyde into the cell constituents was increased by overexpression of the genes. Furthermore, the degradation of vanillic acid and the growth yield were significantly improved at a high concentration of vanillic acid (60 mM) in the transformant strain. These results suggest that HPS and PHI play significant roles in the detoxification and assimilation of formaldehyde. This is the first report that enhancement of the HPS/PHI pathway could improve the degradation of vanillic acid in nonmethylotrophic bacteria.     

Cytologic features based on the expression of E-cadherin and catenins in lung adenocarcinoma.

OBJECTIVE: To investigate the relationship between E-cadherin-associated cell-to-cell adhesion and cytologic features in preoperative cytologic lung adenocarcinoma specimens. STUDY DESIGN: Evaluation of the relationship between cell-to-cell adhesion, formation of cellular clusters and frequency of single cells in 31 cases of primary lung adenocarcinoma, collected by brush and needle cytology preoperatively. RESULTS: Most cases with remarkable overlapping of cells in compact cellular clusters and a few solitary cells maintained cell-to-cell adhesion. Cellular clusters that had a slight tendency to overlap, a small cell-to-cell adhesion area and a high frequency of solitary cells tended to lack E-cadherin-associated cell-to-cell adhesion. CONCLUSION: Formation of cellular clusters and the appearance of solitary cancer cells are closely related to E-cadherin-associated cell-to-cell adhesion. Therefore, it is highly likely that cytologic features may indicate malignant behavior, such as local invasion and lymph node metastasis, in primary lung adenocarcinoma.     

Antiproliferative activity of Pterodon pubescens Benth. seed oil and its active principle on human melanoma cells

On a preliminary screening, relevant in vitro antiproliferative activity was observed to the crude ethanolic extract of Pterodon pubescens seed oil against the human melanoma cell line SK MEL 37. The diethyl ether fraction from crude ethanolic extract which exhibited stronger activity was submitted to fractionation by gradient elution with hexane/ethyl acetate. Subfraction A, eluted by hexane/ethyl acetate (80:20), was essentially the most active between all the assayed subfractions with an IC₅₀ of 37 μg/ml calculated by the MTT colorimetric method. At this concentration, subfraction A caused morphological features and internucleosomal DNA fragmentation pattern of apoptosis. Through chromatographic separation, the furane diterpene 1 was isolated from this active subfraction and identified by spectral techniques. Compound 1 showed an IC₅₀ value of 32 μM and fluorescence staining with DAPI revealed some typical nuclear changes which are characteristic of apoptosis. These findings support a role for diterpenoids vouacapan-type skeleton as a model to develop new anticancer agents.     

Conserved protein kinases encoded by herpesviruses and cellular protein kinase cdc2 target the same phosphorylation site in eukaryotic elongation factor 1delta

Earlier studies have shown that translation elongation factor 1delta (EF-1delta) is hyperphosphorylated in various mammalian cells infected with representative alpha-, beta-, and gammaherpesviruses and that the modification is mediated by conserved viral protein kinases encoded by herpesviruses, including UL13 of herpes simplex virus type 1 (HSV-1), UL97 of human cytomegalovirus, and BGLF4 of Epstein-Barr virus (EBV). In the present study, we attempted to identify the site in EF-1delta associated with the hyperphosphorylation by the herpesvirus protein kinases. Our results are as follows: (i) not only in infected cells but also in uninfected cells, replacement of the serine residue at position 133 (Ser-133) of EF-1delta by alanine precluded the posttranslational processing of EF-1delta, which corresponds to the hyperphosphorylation. (ii) A purified chimeric protein consisting of maltose binding protein (MBP) fused to a domain of EF-1delta containing Ser-133 (MBP-EFWt) is specifically phosphorylated in in vitro kinase assays by purified recombinant UL13 fused to glutathione S-transferase (GST) expressed in the baculovirus system. In contrast, the level of phosphorylation by the recombinant UL13 of MBP-EFWt carrying an alanine replacement of Ser-133 (MBP-EFS133A) was greatly impaired. (iii) MBP-EFWt is also specifically phosphorylated in vitro by purified recombinant BGLF4 fused to GST expressed in the baculovirus system, and the level of phosphorylation of MBP-EFS133A by the recombinant BGLF4 was greatly reduced. (iv) The sequence flanking Ser-133 of EF-1delta completely matches the consensus phosphorylation site for a cellular protein kinase, cdc2, and in vitro kinase assays revealed that purified cdc2 phosphorylates Ser-133 of EF-1delta. (v) As observed with EF-1delta, the casein kinase II beta subunit (CKIIbeta) was specifically phosphorylated by UL13 in vitro, while the level of phosphorylation of CKIIbeta by UL13 was greatly diminished when a serine residue at position 209, which has been reported to be phosphorylated by cdc2, was replaced with alanine. These results indicate that the conserved protein kinases encoded by herpesviruses and a cellular protein kinase, cdc2, have the ability to target the same amino acid residues for phosphorylation. Our results raise the possibility that the viral protein kinases mimic cdc2 in infected cells.     

N-glycan structures of a recombinant mouse soluble Fcγ receptor II

N-glycans of a recombinant mouse soluble Fc receptor II (sFcRII) expressed in baby hamster kidney cells were released from glycopeptides by digestion with glycoamidase A (from sweet almond), and the reducing ends of the oligosaccharides were reductively aminated with 2-aminopyridine. The derivatized N-glycans were separated and structurally identified by a three-dimensional high-performance liquid chromatography (HPLC) mapping technique on three kinds of HPLC columns [Takahashi, et al. (1995) Anal. Biochem. 226: 139–46]. Eighteen different major N-glycan structures were identified, of which six were neutral (45%), five mono-sialyl (49%), one di-sialyl (4.6%), five tri-sialyl (1.1%), and one tetra-sialyl (0.3%). All N-glycan structures determined were complex type with fucosylation at the N-acetylglucosamine residue of the reducing end, and N-acetylneuraminic acid, when present, was -(2,3)-linked. The existence of a unique structure containing both N-acetylgalactosamine and -(2,3)-N-acetylneuraminic acid residues at the reducing ends, as below, was confirmed by MALDI-TOF mass spectrometry.     

Occurrence of Yersinia enterocolitica in wild-living birds and Japanese serows.

Yersinia spp. were isolated from 34 of 500 birds representing nine species. The highest isolation rate, 5 of 21 (23.8%), was found in blue magpies (Cyanopia cyanus), followed by pheasants (Phasianus colchicus tohkaidi), 5 of 33 (15.2%); gray starlings (Sturnus cineraceus), 6 of 57 (10.5%); tree sparrows (Passer montanus), 1 of 14 (7.1%); bulbuls (Hypsipetes amaurotis), 4 of 57 (7.0%); crows (Corvus levailantii or Corvus corone), 7 of 117 (6.0%); eastern turtledoves (Streptopelia orientalis), 4 of 118 (3.4%); Chinese bamboo pheasants (Bumbusicola thoracica thoracica), 1 of 36 (2.8%); and domestic pigeons (Columba livia domestica), 1 of 47 (2.1%). The isolates were identified as Yersinia enterocolitica O:3, O:4, O:4,32, O:5A, O:6,30, O:7,8, and O:14, Yersinia frederiksenii, Yersinia intermedia, and Yersinia kristensenii. Yersinia spp. were isolated from 35 of 157 wild-living Japanese serows (Capricornis cripus). The isolates were identified as Y. enterocolitica O:4, O:4,32, O:5A, O:7, O:7,8, O:9, O:14, O:18, and O:34, Y. frederiksenii, Y. intermedia, and Y. kristensenii.     

Antimicrobial susceptibility and genetic relatedness of bovine Stenotrophomonas maltophilia isolates from a mastitis outbreak

Aims: To evaluate the antimicrobial susceptibility and genetic relatedness of 11 Stenotrophomonas maltophilia isolates from an outbreak of bovine clinical mastitis in one herd and two isolates from two separate mastitis cases in two other herds. Methods and Results: Thirteen S. maltophilia isolates were obtained from milk samples from 11 cows from three dairy herds in Japan during 2008. We tested their susceptibility to 14 antimicrobials by broth microdilution and identified their genotypes by enterobacterial repetitive intergenic consensus 2 (ERIC2)‐PCR. Every cow had acute mild mastitis (slightly watery foremilk with flakes) without systemic symptoms and all resolved within 3–5 weeks of diagnosis. Eleven of the 13 isolates derived from nine cows in one herd over a 7‐month period exhibited a closely related ERIC2 type (A). The remaining two isolates derived from two cows from two other herds exhibited two distinct ERIC2 types (B and C). Most of the 13 isolates exhibited susceptibility to trimethoprim‐sulfamethoxazole, chloramphenicol, minocycline and levofloxacin; however, they were resistant to four β‐lactams, kanamycin, gentamicin and oxytetracycline. They were intermediate to enrofloxacin. Conclusions: Eleven closely related S. maltophilia isolates were involved in a herd outbreak of mastitis to some extent. Bovine S. maltophilia isolates exhibited resistance to many classes of antimicrobials. Significance and impact of study: This is a rare report of a herd outbreak of bovine mastitis involving closely related S. maltophilia isolates.     

mTORC1 serves ER stress-triggered apoptosis via selective activation of the IRE1-JNK pathway

Mammalian target of rapamycin (mTOR) has a key role in the regulation of an array of cellular function. We found that rapamycin, an inhibitor of mTOR complex 1 (mTORC1), attenuated endoplasmic reticulum (ER) stress-induced apoptosis. Among three major branches of the unfolded protein response, rapamycin selectively suppressed the IRE1-JNK signaling without affecting PERK and ATF6 pathways. ER stress rapidly induced activation of mTORC1, which was responsible for induction of the IRE1-JNK pathway and apoptosis. Activation of mTORC1 reduced Akt phosphorylation, which was an event upstream of IRE-JNK signaling and consequent apoptosis. In vivo, administration with rapamycin significantly suppressed renal tubular injury and apoptosis in tunicamycin-treated mice. It was associated with enhanced phosphorylation of Akt and suppression of JNK activity in the kidney. These results disclosed that, under ER stress conditions, mTORC1 causes apoptosis through suppression of Akt and consequent induction of the IRE1-JNK pathway.