Nozomi Ohuchi

Insights into β-Lactamases from Burkholderia Species, Two Phylogenetically Related yet Distinct Resistance Determinants

Background: Resistance to β-lactams in Burkholderia is mediated by different β-lactamases (e.g. PenA and PenI). Results: PenA from B. multivorans is a carbapenemase, and PenI from B. pseudomallei is an extended-spectrum enzyme. Conclusion: Subtle changes within the active site of β-lactamases result in major phenotypic changes. Significance: Future antibiotic design must consider the distinctive phenotypes of PenA and PenI β-lactamases. Burkholderia cepacia complex and Burkholderia pseudomallei are opportunistic human pathogens. Resistance to β-lactams among Burkholderia spp. is attributable to expression of β-lactamases (e.g. PenA in B. cepacia complex and PenI in B. pseudomallei). Phylogenetic comparisons reveal that PenA and PenI are highly related. However, the analyses presented here reveal that PenA is an inhibitor-resistant carbapenemase, most similar to KPC-2 (the most clinically significant serine carbapenemase), whereas PenI is an extended spectrum β-lactamase. PenA hydrolyzes β-lactams with kcat values ranging from 0.38 ± 0.04 to 460 ± 46 s−1 and possesses high kcat/kinact values of 2000, 1500, and 75 for β-lactamase inhibitors. PenI demonstrates the highest kcat value for cefotaxime of 9.0 ± 0.9 s−1. Crystal structure determination of PenA and PenI reveals important differences that aid in understanding their contrasting phenotypes. Changes in the positioning of conserved catalytic residues (e.g. Lys-73, Ser-130, and Tyr-105) as well as altered anchoring and decreased occupancy of the deacylation water explain the lower kcat values of PenI. The crystal structure of PenA with imipenem docked into the active site suggests why this carbapenem is hydrolyzed and the important role of Arg-220, which was functionally confirmed by mutagenesis and biochemical characterization. Conversely, the conformation of Tyr-105 hindered docking of imipenem into the active site of PenI. The structural and biochemical analyses of PenA and PenI provide key insights into the hydrolytic mechanisms of β-lactamases, which can lead to the rational design of novel agents against these pathogens.

Proliferative effects of angiotensin II and endothelin-1 on guinea pig gingival fibroblast cells in culture.

We investigated whether phenytoin (PHT) and nifedipine (NIF) induce angiotensin II (Ang II) and endothelin-1 (ET-1) generation by cultured gingival fibroblasts derived from guinea pigs and whether Ang II and ET-1 induce proliferation of these cells. Immunohistochemical experiments showed that PHT (250 nM) and NIF (250 nM) increased the immunostaining intensities of immunoreactive Ang II and ET-1 (IRET-1) in these cells. Captopril (3 microM), an angiotensin-converting enzyme inhibitor, reduced these enhanced intensities to control levels. Ang II (100 nM) enhanced the immunostaining intensity of IRET-1. PHT (250 nM) and NIF (250 nM)-induced cell proliferation. Both PHT- and NIF-induced proliferation was inhibited by captopril (3 microM). Ang II (100 nM) and ET-1 (100 nM) also induced cell proliferation. Ang II-induced proliferation was inhibited by CV11974 (1 microM), an AT(1) receptor antagonist and saralasin (1 microM), an AT(1)/AT(2) receptor antagonist, but not by PD123,319 (1 microM), an AT(2) receptor antagonist. ET-1-induced proliferation was inhibited by BQ123 (10 microM), an ET(A) receptor antagonist, but not by BQ788 (1 microM), an ET(B) receptor antagonist. These findings suggest that PHT- and NIF-induced gingival fibroblast proliferation is mediated indirectly through the induction of Ang II and ET-1 and probably mediated through AT(1) and ET(A) receptors present in or on gingival fibroblasts.

Effects of endothelin-1 and nitric oxide on proliferation of cultured guinea pig bronchial smooth muscle cells

The proliferative effects of endothelin-1 (ET-1), both alone and in combination with epidermal growth factor (EGF), and the effect of nitric oxide (NO) on the cell proliferation were investigated in cultured guinea pig bronchial smooth muscle cells. ET-1 (10-100 nM) alone augmented cell proliferation, and was additive to the effect of EGF (0.48 nM) in a concentration-dependent manner. An ET(A) antagonist, BQ-123 (10 microM), reduced the cell-proliferative effect of ET-1, whereas an ET(B) antagonist, BQ-788 (10 microM), did not influence the effect. A NO donor, SIN-1 (10 nM-1 microM), reduced the cell-proliferative effect of ET-1 in a concentration-dependent manner. The effect of SIN-1 (1 microM) was partly, but significantly, reversed by a soluble guanylyl cyclase inhibitor, ODQ (1 microM). These results suggest that ET-1 acts not only as a co-mitogen with EGF but also as a mitogen alone, and that its action is mediated through activation of ET(A) receptors. Therefore, ET-1 may contribute to airway remodeling, a pathophysiological hallmark of asthma. In addition, NO, which is produced mainly in the airway epithelium and is partly mediated through cGMP-dependent pathway, may reduce the phenomenon.

Thrombin‐stimulated proliferation is mediated by endothelin‐1 in cultured rat gingival fibroblasts

Endothelin‐1 (ET‐1) appears to be involved in drug‐induced proliferation of gingival fibroblasts. Thrombin induces proliferation of human gingival fibroblasts via protease‐activated receptor 1 (PAR1). In this study, using cultured rat gingival fibroblasts, we investigated whether thrombin‐induced proliferation of gingival fibroblasts is mediated by ET‐1. Thrombin‐induced proliferation (0.05–2.5 U/mL). Proliferation was also induced by a PAR1‐specific agonist (TFLLR‐NH2, 0.1–30 μm), but not by a PAR2‐specific agonist (SLIGRL‐NH2). Thrombin (2.5 U/mL) induced an increase in immunoreactive ET‐1 expression, which was inhibited by cycloheximide (10 μg/mL), and an increase in preproET‐1 mRNA expression, as assessed by reverse transcription polymerase chain reaction. TFLLR‐NH2 increased ET‐1 release into the culture medium in both a concentration (0.01–10 μm)‐ and time (6–24 h)‐dependent manner, as assessed by solid phase sandwich enzyme‐linked immunosorbent assay. The thrombin (2.5 U/mL)‐induced proliferation was inhibited by a PAR1‐selective inhibitor, SCH79797 (0.1 μm) and an ETA antagonist, BQ‐123 (1 μm), but not by an ETB antagonist, BQ‐788 (1 μm). These findings suggest that thrombin, acting via PAR1, induced proliferation of cultured rat gingival fibroblasts that was mediated by ET‐1 acting via ETA.

Proliferative response to phenytoin and nifedipine in gingival fibroblasts cultured from humans with gingival fibromatosis.

The response of gingival fibroblasts cultured from humans with gingival fibromatosis to phenytoin (PHT) and nifedipine (NIF) was investigated. PHT and NIF induced proliferation, and increased the expression of immunoreactive endothelin‐1 (ET‐1). ET‐1 (0.1 nm–1 μm) itself also induced proliferation in a concentration‐dependent manner. The proliferation was inhibited by BQ‐123 (ETA receptor antagonist; 1 μm) and TAK044 (ETA/ETB receptor antagonist; 1 μm), but not by BQ‐788 (ETB receptor antagonist; 1 μm). The proliferation induced by PHT (0.25 μm) and NIF (0.25 μm) was inhibited by BQ‐123 (1 μm). In addition, the results of Western blot analysis indicated the presence of ETA and ETB receptors in/on the fibroblasts. These findings suggest that PHT‐ and NIF‐induced gingival proliferation may be mediated by endogenously generated ET‐1, possibly via ETA receptors.

Inhibition of angiotensin II- and endothelin-1-stimulated proliferation by selective mek inhibitor in cultured rabbit gingival fibroblasts

We investigated the implication of extracellular signal‐regulated protein kinases 1 and 2 (ERK1/2) in the proliferation stimulated by angiotensin II (Ang II) and endothelin‐1 (ET‐1) in cultured rabbit gingival fibroblasts (CRGF). Ang II stimulated activation of ERK1/2 and the activation was inhibited by CV‐11974, an AT1 antagonist, and saralasin, an AT1/AT2 antagonist, but not by PD123,319, an AT2 antagonist in the CRGF. Ang II‐stimulated proliferation was inhibited by PD98059 or U0126, selective MEK inhibitors. Furthermore, ET‐1 stimulated proliferation via G‐protein‐coupled ETA receptors, which were identified by Western blot analysis of membrane protein from the CRGF. ET‐1 also stimulated activation of ERK1/2 and the activation was inhibited by BQ‐123, an ETA inhibitor, and TAK044, an ETA/ETB inhibitor, but not by BQ‐788, an ETB inhibitor. ET‐1‐stimulated proliferation was inhibited by PD98059 or U0126. These findings suggest that ERK1/2 play a role in the signaling process leading to proliferation stimulated by Ang II and ET‐1 via G‐protein‐coupled receptors, AT1 and ETA in CRGF.