Isolation and characterization of tetrachloroethylene- and <Emphasis Type="Italic">cis</Emphasis>-1,2-dichloroethylene-dechlorinating propionibacteria

Two rapidly growing propionibacteria that could reductively dechlorinate tetrachloroethylene (PCE) and cis-1,2-dichloroethylene (cis-DCE) to ethylene were isolated from environmental sediments. Metabolic characterization and partial sequence analysis of their 16S rRNA genes showed that the new isolates, designated as strains Propionibacterium sp. HK-1 and Propionibacterium sp. HK-3, did not match any known PCE- or cis-DCE-degrading bacteria. Both strains dechlorinated relatively high concentrations of PCE (0.3 mM) and cis-DCE (0.52 mM) under anaerobic conditions without accumulating toxic intermediates during incubation. Cell-free extracts of both strains catalyzed PCE and cis-DCE dechlorination; degradation was accelerated by the addition of various electron donors. PCE dehalogenase from strain HK-1 was mediated by a corrinoid protein, since the dehalogenase was inactivated by propyl iodide only after reduction by titanium citrate. The amounts of chloride ions (0.094 and 0.103 mM) released after PCE (0.026 mM) and cis-DCE (0.05 mM) dehalogenation using the cell-free enzyme extracts of both strains, HK-1 and HK-3, were stoichiometrically similar (91 and 100%), indicating that PCE and cis-DCE were fully dechlorinated. Radiotracer studies with [1,2-¹⁴C] PCE and [1,2-¹⁴C] cis-DCE indicated that ethylene was the terminal product; partial conversion to ethylene was observed. Various chlorinated aliphatic compounds (PCE, trichloroethylene, cis-DCE, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,1-dichloroethane, 1,2-dichloroethane, 1,2-dichloropropane, 1,1,2-trichloroethane, and vinyl chloride) were degraded by cell-free extracts of strain HK-1.     

The zinc transporter SLC39A14/ZIP14 controls G-protein coupled receptor-mediated signaling required for systemic growth

Aberrant zinc (Zn) homeostasis is associated with abnormal control of mammalian growth, although the molecular mechanisms of Zn's roles in regulating systemic growth remain to be clarified. Here we report that the cell membrane-localized Zn transporter SLC39A14 controls G-protein coupled receptor (GPCR)-mediated signaling. Mice lacking Slc39a14 (Slc39a14-KO mice) exhibit growth retardation and impaired gluconeogenesis, which are attributable to disrupted GPCR signaling in the growth plate, pituitary gland, and liver. The decreased signaling is a consequence of the reduced basal level of cyclic adenosine monophosphate (cAMP) caused by increased phosphodiesterase (PDE) activity in Slc39a14-KO cells. We conclude that SLC39A14 facilitates GPCR-mediated cAMP-CREB signaling by suppressing the basal PDE activity, and that this is one mechanism for Zn's involvement in systemic growth processes. Our data highlight SLC39A14 as an important novel player in GPCR-mediated signaling. In addition, the Slc39a14-KO mice may be useful for studying the GPCR-associated regulation of mammalian systemic growth.     

Theoretical basis to measure the impact of short-lasting control of an infectious disease on the epidemic peak

Background  

While many pandemic preparedness plans have promoted disease control effort to lower and delay an epidemic peak, analytical methods for determining the required control effort and making statistical inferences have yet to be sought. As a first step to address this issue, we present a theoretical basis on which to assess the impact of an early intervention on the epidemic peak, employing a simple epidemic model.     

Plant regeneration via direct and indirect adventitious shoot formation and chromosome-doubled somaclonal variation in Titanotrichum oldhamii (Hemsl.) Solereder

The gesneriaceous perennial plant Titanotrichum oldhamii has beautiful foliage and attractive bright yellow flowers. However, breeding of T. oldhamii by conventional sexual hybridization may be difficult because sexual reproduction of this species is very rare. In the present study, plant regeneration systems via both direct and indirect formation of adventitious shoots from leaf explants were established as the first step toward breeding T. oldhamii by using biotechnological techniques. Adventitious shoots were formed efficiently on medium containing 0.1 mg l⁻¹ benzyladenine. Histological observation showed that shoot formation on this medium occurred directly from leaf epidermal cells without callus formation. On the other hand, leaf explants formed calluses on medium containing 0.1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid. The calluses could be maintained by monthly subculturing to fresh medium of the same composition. When the calluses were transferred to plant growth regulator-free medium, they formed adventitious shoots. Directly and indirectly formed shoots rooted well on medium containing 0.1 mg l⁻¹ indole-3-butyric acid. Plantlets thus obtained were successfully acclimatized and grew vigorously in the greenhouse. Flow cytometry analysis indicated that no variation in the ploidy level was observed in plants regenerated via direct shoot formation, whereas chromosome doubling occurred in several plants regenerated via indirect shoot formation. Regenerated plants with the same ploidy level as the mother plants showed almost the same phenotype as the mother plants, whereas chromosome-doubled plants showed apparent morphological alterations: they had small and crispate flowers, and round and deep green leaves.     

Positive narrowing pharyngoplasty with forearm flap for functional restoration after extensive soft palate resection

The restoration of velopharyngeal function after extensive soft palate resection to treat malignant oropharyngeal tumors is a major challenge to reconstructive surgeons. The authors had previously reconstructed soft palatal defects routinely with the folded forearm flap. A patient who had more than half of the soft palate excised experienced postoperative velopharyngeal dysfunction. To restore efficient velopharyngeal function, pharyngoplasty was additively applied where the folded ridge of the forearm flap was sutured to the posterior pharyngeal wall in an inverse manner of the pharyngeal flap technique. The essence of the procedure was positive narrowing of the nasopharyngeal space. Five patients who underwent this pharyngoplasty and another five who did not were evaluated for postoperative functions of speech intelligibility and of nasal regurgitation during oral feeding. The velopharyngeal movements of all patients were examined under a nasopharyngeal endoscope. The evaluations demonstrated that this surgical procedure afforded satisfactory results. This positive narrowing pharyngoplasty technique is simple, easy, and minimally invasive to the remaining healthy tissue, and it is the method of choice for the reconstruction of the soft palate after malignant tumor resection.     

Involvement of P1 adhesin in gliding motility of Mycoplasma pneumoniae as revealed by the inhibitory effects of antibody under optimized gliding conditions

To examine the participation of P1 adhesin in gliding of Mycoplasma pneumoniae, we examined the effects of an anti-P1 monoclonal antibody on individual gliding mycoplasmas. The antibody reduced the gliding speed and removed the gliding cells from the glass over time in a concentration-dependent manner but had only a slight effect on nongliding cells, suggesting that the conformational changes of P1 adhesin and its displacement are involved in the gliding mechanism.     

Identification of a 521-kilodalton protein (Gli521) involved in force generation or force transmission for Mycoplasma mobile gliding

Several mycoplasma species are known to glide on solid surfaces such as glass in the direction of the membrane protrusion, but the mechanism underlying this movement is unknown. To identify a novel protein involved in gliding, we raised monoclonal antibodies against a detergent-insoluble protein fraction of Mycoplasma mobile, the fastest glider, and screened the antibodies for inhibitory effects on gliding. Five monoclonal antibodies stopped the movement of gliding mycoplasmas, keeping them on the glass surface, and all of them recognized a large protein in immunoblotting. This protein, named Gli521, is composed of 4,738 amino acids, has a predicted molecular mass of 520,559 Da, and is coded downstream of a gene for another gliding protein, Gli349, which is known to be responsible for glass binding during gliding. Edman degradation analysis indicated that the N-terminal region is processed at the peptide bond between the amino acid residues at positions 43 and 44. Analysis of gliding mutants isolated previously revealed that the Gli521 protein is missing in a nonbinding mutant, m9, where the gli521 gene is truncated by a nonsense mutation at the codon for the amino acid at position 1170. Immunofluorescence and immunoelectron microscopy indicated that Gli521 localizes all around the base of the membrane protrusion, at the "neck," as previously observed for Gli349. Analysis of the inhibitory effects of the anti-Gli521 antibody on gliding motility revealed that this protein is responsible for force generation or force transmission, a role distinct from that of Gli349, and also suggested conformational changes of Gli349 and Gli521 during gliding.     

Toll-like receptor 3 and STAT-1 contribute to double-stranded RNA+ interferon-gamma-induced apoptosis in primary pancreatic beta-cells

Viral infections and local production of cytokines probably contribute to the pathogenesis of Type 1 diabetes. The viral replicative intermediate double-stranded RNA (dsRNA, tested in the form of polyinosinic-polycytidylic acid, PIC), in combination with the cytokine interferon-gamma (IFN-gamma), triggers beta-cell apoptosis. We have previously observed by microarray analysis that PIC induces expression of several mRNAs encoding for genes downstream of Toll-like receptor 3 (TLR3) signaling pathway. In this report, we show that exposure of beta-cells to dsRNA in combination with IFN-alpha, -beta, or -gamma significantly increases apoptosis. Moreover, dsRNA induces TLR3 mRNA expression and activates NF-kappaB and the IFN-beta promoter in a TRIF-dependent manner. dsRNA also induces an early (1 h) and sustained increase in IFN-beta mRNA expression, and blocking IFN-beta with a specific antibody partially prevents PIC plus IFN-gamma-induced beta-cell death. On the other hand, dsRNA plus IFN-gamma does not induce apoptosis in INS-1E cells, and expression of TLR3 and type I IFNs mRNAs is not detected in these cells. Of note, disruption of the STAT-1 signaling pathway protects beta-cells against dsRNA plus IFN-gamma-induced beta-cell apoptosis. This study suggests that dsRNA plus IFN-gamma triggers beta-cell apoptosis by two complementary pathways, namely TLR3-TRIF-NF-kappaB and STAT-1.     

Intracellular cAMP controls a physical association of V-1 with CapZ in cultured mammalian endocrine cells

V-1, an ankyrin repeat protein with the activity to control tyrosine hydroxylase (TH) gene expression and transmitter release in PC12D cells, associates with CapZ, an actin capping protein, and thereby regulates actin polymerization in vitro. In this study, immunoprecipitation and Western blot analysis showed that V-1 was physically associated with CapZ-beta in PC12D transfectants overexpressing V-1. These proteins were co-localized in the soma of Purkinje cells of rat cerebellum as assayed by immunohistochemistry. Furthermore, in the V-1 transfectants, the amount of CapZ which physically associated with V-1 was steeply reduced at 2h after treatment with forskolin, but was thereafter increased to reach its initial level at 12h after forskolin-treatment. These results suggest that the association of V-1 with CapZ is controlled by a cAMP-dependent signalling pathway probably to play a functional role in the regulatory mechanism of actin dynamics in the endocrine system and the central nervous system.     

High glucose enhances interleukin-6-induced vascular endothelial growth factor 165 expression via activation of gp130-mediated p44/42 MAPK-CCAAT/enhancer binding protein signaling in gingival fibroblasts

Diabetic patients are susceptible to severe inflammatory periodontitis manifesting as swollen gingiva with bleeding, but the underlying mechanism is not well understood. Our purpose was to determine the effect of a high glucose (HG) condition on the interleukin-6/soluble interleukin-6 receptor (IL-6/sIL-6R)-induced activation of signaling and vascular endothelial growth factor (VEGF) expression in human gingival fibroblasts (HGFs). In this study, HGFs were cultured for at least two passages under a normal glucose (NG; 5.5 mM) condition or high glucose (25 mM) condition. Importantly, the HG condition significantly induced expression of gp130 mRNA in HGFs compared with levels in control cells. Consistent with the expression of its mRNA, the HG condition also increased the expression of gp130 protein, and phosphorylation of the tyrosine residue by gp130 was enhanced significantly by IL-6/sIL-6R stimulation. Furthermore, the HG condition enhanced the IL-6/sIL-6R-induced phosphorylation of p44/42 MAPK and led to subsequent activation of CCAAT/enhancer binding protein in nuclei. In contrast, there was no significant difference in phosphorylation of JNK between the HG and NG condition. Interestingly, HGFs increased IL-6/sIL-6R-induced VEGF165 mRNA expression and VEGF165 secretion under the HG condition compared with levels under the NG condition. In contrast, the induction of VEGF165 secretion was partially inhibited by PD98059 (selective p44/42 MAPK inhibitor) under the HG condition. In addition, the VEGF165 secretion was completely inhibited by the combination of PD98059 and SP600125 (JNK inhibitor). Our findings suggest that the HG condition indirectly increases VEGF expression via activation of gp130-mediated p44/42 MAPK-CCAAT/enhancer binding protein signaling in HGFs. Thus, elevated VEGF secretion in HGFs under the HG condition may play a role in the development of the severe periodontitis observed in diabetic patients.