Group-Specific Monitoring of Phenol Hydroxylase Genes for a Functional Assessment of Phenol-Stimulated Trichloroethylene Bioremediation

The sequences of the largest subunit of bacterial multicomponent phenol hydroxylases (LmPHs) were compared. It was found that LmPHs formed three phylogenetic groups, I, II, and III, corresponding to three previously reported kinetic groups, low-K_s (the half-saturation constant in Haldane's equation for trichloroethylene [TCE]), moderate-K_s, and high-K_s groups. Consensus sequences and specific amino acid residues for each group of LmPH were found, which facilitated the design of universal and group-specific PCR primers. PCR-mediated approaches using these primers were applied to analyze phenol/TCE-degrading populations in TCE-contaminated aquifer soil. It was found that the aquifer soil harbored diverse genotypes of LmPH, and the group-specific primers successfully amplified LmPH fragments affiliated with each of the three groups. Analyses of phenol-degrading bacteria isolated from the aquifer soil confirmed the correlation between genotype and phenotype. Competitive PCR assays were used to quantify LmPHs belonging to each group during the enrichment of phenol/TCE-degrading bacteria from the aquifer soil. We found that an enrichment culture established by batch phenol feeding expressed low TCE-degrading activity at a TCE concentration relevant to the contaminated aquifer (e.g., 0.5 mg liter⁻¹); group II and III LmPHs were predominant in this batch enrichment. In contrast, group I LmPHs overgrew an enrichment culture when phenol was fed continuously. This enrichment expressed unexpectedly high TCE-degrading activity that was comparable to the activity expressed by a pure culture of Methylosinus trichosporium OB3b. These results demonstrate the utility of the group-specific monitoring of LmPH genes in phenol-stimulated TCE bioremediation. It is also suggested that phenol biostimulation could become a powerful TCE bioremediation strategy when bacteria possessing group I LmPHs are selectively stimulated.     

Eldecalcitol (ED-71), an analog of 1α,25(OH)<Subscript>2</Subscript>D<Subscript>3</Subscript>, inhibits the growth of squamous cell carcinoma (SCC) cells in vitro and in vivo by down-regulating expression of heparin-binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1) and FGF-2

Heparin-binding protein 17 (HBp17)/fibroblast growth factor-binding protein-1 (FGFBP-1) was first purified from medium conditioned by A431 cells for its capacity to bind to fibroblast growth factors 1 and 2 (FGF-1 and -2). Among FGF family members, FGF-2 is a potent mitogen for various cell types, including vascular endothelial cells, fibroblasts, and cancer cells such as oral squamous cell carcinoma (OSCC) cells. Besides being well known in bone metabolism, the active form of vitamin D₃, i.e., 1α,25(OH)₂D₃ (1,25D₃), was reported to have protective effects for heart disease and cancer. Previously, we reported that 1,25D₃ inhibited HBp17/FGFBP-1 expression in OSCC cell lines through NF-κB inhibition (IκBα activation) and resulted in the inactivation of FGF-2. In this study, we examined the potential anti-tumor effect of ED-71, an analog of 1α,25(OH)₂D₃, for squamous cell carcinoma cells in vitro and in vivo. The cell lines used were OSCC cell lines (NA—HO-1-n-1 and UE—HO-1-u-1), established from oral cancer patients in our laboratory, and an epidermoid carcinoma/SCC cell line (A431). The growth assay in serum-free culture revealed that ED-71 inhibited the growth of the cancer cell lines in a dose-dependent manner. In addition, ED-71 suppressed HBp17/FGFBP-1 expression by inhibiting the NF-κB pathway as did 1,25D₃. Furthermore, a luciferase reporter assay revealed that the promoter activity of HBp17/FGFBP-1 (region between ?217 and +61) was down-regulated by ED-71. Oral administration of ED-71 significantly inhibited the growth of A431-derived tumors in athymic nude mice. Immunohistochemical analysis revealed that the expression of HBp17/FGFBP-1, FGF-2, CD31, and Ki-67 in the tumors of ED71-treated group was down-regulated in comparison to control. These results suggest that ED-71 possesses potential anti-tumor activity for SCCs both in vitro and in vivo. This compound may act directly on the tumor cells or on endothelial cells by modulating the tumor microenvironment.     

Bacteriochlorophyll-c Homolog Composition in Green Sulfur Photosynthetic Bacterium Chlorobium vibrioformeDependent on the Concentration of Sodium Sulfide in Liquid Cultures

Green sulfur photosynthetic bacteria Chlorobium (Chl.) vibrioforme (DSM 263 strain and NCIB 8327 substrain possessing BChl-c) and Chl. tepidum (ATCC 49652) were photoautotrophically grown in liquid cultures containing different concentrations of sodium sulfide (Na₂S). BChl-c homologs possessing a methyl group at the 12-position tended to increase in cells of the two strains of Chl. vibrioforme cultured under high Na₂S concentrations. In contrast, the Na₂S concentration in liquid cultures did not affect the relative composition of BChl-c homologs in Chl. tepidum. 8-Propyl-12-methyl([P,M])-BChl-c homolog, which has been little observed in usual cultivations, could be isolated by reverse-phase high-performance liquid chromatography from the cells of Chl. vibrioforme grown under high Na₂S contents. The [P,M]-BChl-c homolog has the R-configuration at the 3¹-position, which was determined by ¹H-NMR analyses.     

Androgen receptor gene knockout male mice exhibit impaired cardiac growth and exacerbation of angiotensin II-induced cardiac fibrosis

Androgen has anabolic effects on cardiac myocytes and has been shown to enhance left ventricular enlargement and function. However, the physiological and patho-physiological roles of androgen in cardiac growth and cardiac stress-induced remodeling remains unclear. We aimed to clarify whether the androgen-nuclear androgen receptor (AR) system contributes to the cardiac growth and angiotensin II (Ang II)-stimulated cardiac remodeling by using systemic AR-null male mice. AR knock-out (ARKO) male mice, at 25 weeks of age, and age-matched wild-type (WT) male mice were treated with or without Ang II stimulation (2.0 mg/kg/day) for 2 weeks. ARKO mice with or without Ang II stimulation showed a significant reduction in the heart-to-body weight ratio compared with those of WT mice. In addition, echocardiographic analysis demonstrated impairments of both the concentric hypertrophic response and left ventricular function in Ang II-stimulated ARKO mice. Western blot analysis of the myocardium revealed that activation of extracellular signal-regulated kinases (ERK) 1/2 and ERK5 by Ang II stimulation were lower in ARKO mice than those of WT mice. Ang II stimulation caused more prominent cardiac fibrosis in ARKO mice than in WT mice with enhanced expression of types I and III collagen and transforming growth factor-beta1 genes and with increased Smad2 activation. These results suggest that, in male mice, the androgen-AR system participates in normal cardiac growth and modulates cardiac adaptive hypertrophy and fibrosis during the process of cardiac remodeling under hypertrophic stress.     

Two distinct pathways responsible for the loading of CENP-A to centromeres in the fission yeast cell cycle

CENP-A is a centromere-specific histone H3 variant that is- essential for faithful chromosome segregation in all eukaryotes thus far investigated. We genetically identified two factors, Ams2 and Mis6, each of which is required for the correct centromere localization of SpCENP-A (Cnp1), the fission yeast homologue of CENP-A. Ams2 is a cell-cycle-regulated GATA factor that localizes on the nuclear chromatin, including on centromeres, during the S phase. Ams2 may be responsible for the replication-coupled loading of SpCENP-A by facilitating nucleosomal formation during the S phase. Consistently, overproduction of histone H4, but not that of H3, suppressed the defect of SpCENP-A localization in Ams2-deficient cells. We demonstrated the existence of at least two distinct phases for SpCENP-A loading during the cell cycle: the S phase and the late-G2 phase. Ectopically induced SpCENP-A was efficiently loaded onto the centromeres in G2-arrested cells, indicating that SpCENP-A probably undergoes replication-uncoupled loading after the completion of S phase. This G2 loading pathway of SpCENP-A may require Mis6, a constitutive centromere-binding protein that is also implicated in the Mad2-dependent spindle attachment checkpoint response. Here, we discuss the functional relationship between the flexible loading mechanism of CENP-A and the plasticity of centromere chromatin formation in fission yeast.     

Cooperative effect of two surface amino acid mutations (Q252L and E170K) in glucose dehydrogenase from Bacillus megaterium IWG3 on stabilization of its oligomeric state

A thermostable glucose dehydrogenase (GlcDH) mutant of Bacillus megaterium IWG3 harboring the Q252L substitution (Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Biol. Chem. 264:6381-6385, 1989) is stable at pH values above 9, but only in the presence of 2 M NaCl. Another GlcDH mutant exhibiting increased stability at an alkaline pH in the absence of NaCl has been isolated previously (S.-H. Baik, T. Ide, H. Yoshida, O. Kagami, and S. Harayama, Appl. Microbiol. Biotechnol. 61:329-335, 2003). This mutant had two amino acid substitutions, Q252L and E170K. In the present study, we characterized three GlcDH mutants harboring the substitutions Q252L, E170K, and Q252L/E170K under low-salt conditions. The GlcDH mutant harboring two substitutions, Q252L/E170K, was stable, but mutants harboring a single substitution, either Q252L or E170K, were unstable at an alkaline pH. Gel filtration chromatography analyses demonstrated that the oligomeric state of the Q252/E170K enzyme was stable, while the tetramers of the enzymes harboring a single substitution (Q252L or E170K) dissociated into dimers at an alkaline pH. These results indicated that the Q252L and E170K substitutions synergistically strengthened the interaction at the dimer-dimer interface. The crystal structure of the E170K/Q252L mutant, determined at 2.0-angstroms resolution, showed that residues 170 and 252 are located in a hydrophobic cavity at the subunit-subunit interface. We concluded that these residues in the wild-type enzyme have thermodynamically unfavorable effects, while the Q252L and E170K substitutions increase the subunit-subunit interactions by stabilizing the hydrophobic cavity.     

Expressions of hepatobiliary organic anion transporters and bilirubin-conjugating enzyme in differentiating embryonic stem cells

Mouse embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of developing blastocysts and have multi-lineage differentiation ability. We previously reported that ES cells can be made to differentiate into hepatocytes possessing high metabolic activities by transfection of hepatocyte nuclear factor-3beta (HNF-3beta). In the present study, we investigated the expression of hepatobiliary organic anion transporters and bilirubin uridine diphosphate glucuronosyltransferase (ugt1a1) in undifferentiated and differentiating HNF-3beta-transfected ES (HNF-3beta-ES) cells. The expression of organic anion transporting polypeptide 1 (oatp1), multidrug resistance-associated protein 1 (mrp1), mrp2, mrp3, and ugt1a1 was not seen in the undifferentiated HNF-3beta-ES cells by RT-PCR, whereas all were expressed in differentiating HNF-3beta-ES cells. Protein expression for oatp1, mrp1, mrp2, mrp3, and ugt1a1 was also observed in the differentiating HNF-3beta-ES cells by Western blotting. An immunofluorescence examination revealed that oatp1 was co-located with desmoplakin, a marker for the basolateral (sinusoidal) membrane, and mrp2 was co-localized with CD26, a marker for the apical (canalicular) membrane, though they were both expressed throughout most of the cell membranes.     

Molecular characterization and substrate preference of a polycyclic aromatic hydrocarbon dioxygenase from Cycloclasticus sp. strain A5

Cycloclasticus sp. strain A5 is able to grow with petroleum polycyclic aromatic hydrocarbons (PAHs), including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. A set of genes responsible for the degradation of petroleum PAHs was isolated by using the ability of the organism to oxidize indole to indigo. This 10.5-kb DNA fragment was sequenced and found to contain 10 open reading frames (ORFs). Seven ORFs showed homology to previously characterized genes for PAH degradation and were designated phn genes, although the sequence and order of these phn genes were significantly different from the sequence and order of the known PAH-degrading genes. The phnA1, phnA2, phnA3, and phnA4 genes, which encode the alpha and beta subunits of an iron-sulfur protein, a ferredoxin, and a ferredoxin reductase, respectively, were identified as the genes coding for PAH dioxygenase. The phnA4A3 gene cluster was located 3.7 kb downstream of the phnA2 gene. PhnA1 and PhnA2 exhibited moderate (less than 62%) sequence identity to the alpha and beta subunits of other aromatic ring-hydroxylating dioxygenases, but motifs such as the Fe(II)-binding site and the [2Fe-2S] cluster ligands were conserved. Escherichia coli cells possessing the phnA1A2A3A4 genes were able to convert phenanthrene, naphthalene, and methylnaphthalene in addition to the tricyclic heterocycles dibenzofuran and dibenzothiophene to their hydroxylated forms. Significantly, the E. coli cells also transformed biphenyl and diphenylmethane, which are ordinarily the substrates of biphenyl dioxygenases.     

Inverted papilloma of the nasal cavity presenting with massive amounts of squamous metaplastic cells in sputum. A case report

BACKGROUND: Squamous metaplasic cells are rarely seen in sputum of female nonsmokers. CASE: A 47-year-old female nonsmoker presented with massive amounts of squamous metaplasic cells in sputum and an elevated level of squamous cell carcinoma (SCC) antigen in serum present for months, while no causative lesion was detected either by lung computed tomography or bronchoscopy. The patient was eventually diagnosed as having inverted papilloma in the right nasal cavity. Resection of the tumor brought about disappearance of squamous metaplastic cells in sputum and return of serum SCC antigen to the normal range. CONCLUSION: This case clearly demonstrates that squamous metaplastic cells in sputum can originate in lesions in the nasal cavity, although they are rare. It should be kept in mind that the nasal cavity is a potential site producing squamous metaplastic cells in sputum.