Inactivation and sedimentation of mercury from organomercurials by Klebsiella pneumoniae

Abstract A susceptibility of 63 clinical isolates of Klebsiella pneumoniae to inorganic and organic mercuric compounds was determined. 18 of them were found to be resistant to fluorescein mercuric acetate (FMA) and merbromin (MB). Moreover, all the resistant strains inactivate the antibacterial effect of FMA. The changes in the amount of organic mercury at the time of inactivation of the drug and the structures of the end products were examined in detail with the plasmid-bearing strain JK9 and its transconjugants of Escherichia coli .
The results showed that FMA was inactivated by an intracellular enzyme produced inducively and was degraded to fluorescein (sodium salt, uranine), which led to the sedimentation of metallic mercury. The discovery of the genes conferring inducible organic mercury-inactivating enzymes determined by plasmids was the next step and their application in the recovery of metallic mercury from organomercurials is now imminent.     

Linkage of Mercury, Cadmium, and Arsenate and Drug Resistance in Clinical Isolates of Pseudomonas aeruginosa

Of the 787 isolates, 99.8% were metal resistant, with most (99.5%) showing multiple resistance. Fifty-three percent of the isolates were both metal and drug resistant, whereas only 19% were metal resistant and drug sensitive.     

Vascular endothelial growth factor-D is a key molecule that enhances lymphatic metastasis of soft tissue sarcomas

Studies on lymph node metastasis of soft tissue sarcomas are insufficient because of its rarity. In this study, we examined the expressions of vascular endothelial growth factor (VEGF)-C and VEGF-D in soft tissue sarcomas metastasized to lymph nodes. In addition, the effects of the two molecules on the barrier function of a lymphatic endothelial cell monolayer against sarcoma cells were analyzed. We examined 7 patients who had soft tissue sarcomas with lymph node metastases and who had undergone neither chemotherapy nor radiotherapy before lymphadenectomy. Immunohistochemistry revealed that 2 of 7 sarcomas that metastasized to lymph nodes expressed VEGF-C both in primary and metastatic lesions. On the other hand, VEGF-D expression was detected in 4 of 7 primary and 7 of 7 metastatic lesions, respectively. Interestingly, 3 cases that showed no VEGF-D expression at primary sites expressed VEGF-D in metastatic lesions. Recombinant VEGF-C at 10(-8) and VEGF-D at 10(-7)and 10(-8)g/ml significantly increased the random motility of lymphatic endothelial cells compared with controls. VEGF-D significantly increased the migration of sarcoma cells through lymphatic endothelial monolayers. The fact that VEGF-D induced the migration of fibrosarcomas through the lymphatic endothelial monolayer is the probable reason for the strong relationship between VEGF-D expression and lymph node metastasis in soft tissue sarcomas. The important propensities of this molecule for the increase of lymph node metastases are not only lymphangiogenesis but also down-regulation of the barrier function of lymphatic endothelial monolayers, which facilitates sarcoma cells entering the lymphatic circulation.     

Obligate gut symbiotic association in the sloe bug <Emphasis Type="Italic">Dolycoris baccarum</Emphasis> (Hemiptera: Pentatomidae)

A number of phytophagous stinkbugs are associated with specific bacterial symbionts in their alimentary tracts. The sloe bug Dolycoris baccarum (Linnaeus), a notorious pest of diverse crops, possesses a number of sac-like tissues, called crypts, in a posterior section of the midgut, wherein a specific bacterial symbiont colonizes. Here we characterized the symbiotic bacterium of D. baccarum by histological analysis, molecular phylogeny, and diagnostic PCR with a specific primer set. The cloning and sequencing analyses of bacterial 16S rRNA genes and fluorescent in situ hybridization demonstrated that the sloe bug is associated with a single species of Gammaproteobacteria in the midgut crypts. Molecular phylogenetic analysis strongly suggested that the symbiont should be placed in the genus Pantoea of the Enterobacteriaceae. Diagnostic PCR and egg surface sterilization with formalin indicated the stinkbug vertically transmits the Pantoea symbiont via egg-smearing. The sterilization-produced aposymbiotic nymphs showed high mortality and no insects reached adulthood. In addition, the Pantoea symbiont was uncultivable outside the insect host, indicating an obligate and intimate host-symbiont association.     

The autocrine motility factor (AMF) and AMF-receptor combination needs sugar chain recognition ability and interaction using the C-terminal region of AMF

The autocrine motility factor (AMF) promotes cellular locomotion or invasion, and regulates tumor angiogenesis or ascites accumulation. These signals are triggered by binding between AMF and its receptor (AMFR), a glycoprotein on the cell surface. AMF has been identified as phosphohexose isomerase (PHI). Previous reports have suggested that the substrate-recognition of exo-PHI is significant for receptor binding. Crystallographic studies have shown that AMF consists of three domains, and that the substrate or inhibitor of PHI is stored between the large and small domains, corresponding to approximately residues 117-288. Here, site-directed mutagenesis was used to investigate 18 recombinant human AMF point mutants involving critical amino acid residues for substrate or enzyme inhibitor recognition or binding. Mutation of residues that interact with the phosphate group of the PHI substrate significantly reduced the cell motility-stimulating activity. Their binding capacities for AMFR were also lower than wild-type human AMF. Mutants that retained the enzymic activity showed the motility-stimulating effect and receptor binding and had sensitivity to a PHI inhibitor. Mutant AMFR lacking the N-sugar chain was expressed on the cell membrane but did not respond to AMF-stimulation, and N-glycosidase-treated AMFR did not compete with receptor binding of AMF. Furthermore, the AMF domains that contain the substrate storage domain and C-terminal region stimulate cell locomotion. These results suggest that the N-glyco side-chain of AMFR is a trigger and that interaction between the 117-C-terminal part of AMF and the extracellular core protein of AMFR is needed during AMF-AMFR interactions.     

Frizzled3a and Celsr2 function in the neuroepithelium to regulate migration of facial motor neurons in the developing zebrafish hindbrain

Migration of neurons from their birthplace to their final target area is a crucial step in brain development. Here, we show that expression of the off-limits/frizzled3a (olt/fz3a) and off-road/celsr2 (ord/celsr2) genes in neuroepithelial cells maintains the facial (nVII) motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. In the absence of olt/fz3a expression in the neuroepithelium, nVII motor neurons extended aberrant radial processes towards the ventricular surface and mismigrated radially to the dorsomedial part of the hindbrain. Our findings reveal a novel role for these genes, distinctive from their already known functions, in the regulation of the planar cell polarity (i.e. preventing integration of differentiated neurons into the neuroepithelial layer). This contrasts markedly with their reported role in reintegration of neuroepithelial daughter cells into the neuroepithelial layer after cell division.     

Increased Resistance to Nitric Oxide Cytotoxicity Associated with Differentiation of Neuroblastoma-Glioma Hybrid (NG108-15) Cells

Nitric oxide (NO), synthesized by the enzyme nitric oxide synthase (NOS), acts as an intercellular messenger associated with various physiological and pathological events. In this study, we investigated whether there exits a difference in the vulnerability to NO-induced cytotoxicity between undifferentiated and differentiated NG108-15 cells, and if so, the mechanisms responsible for the difference. Following a 7- to 8-day exposure to dibutyryl cAMP (dbcAMP), NG108-15 cells exhibited a neuron-like morphology associated with the expression of the neuronal protein, synaptophysin, and with increased NADPH-d activity. Neuron-like differentiated NG108-15 cells acquired resistance to exogenously applied NO. This increased resistance to NO toxicity in differentiated cells was almost completely cancelled out by inhibiting the activity of superoxide dismutase (SOD), but not by inhibiting the activity of NOS. The present study suggested that the activity of SOD increased in parallel with the activity of NOS associated with differentiation and was crucial for the acquired resistance to NO toxicity in differentiated cells.     

Functional role of a putative carbonic anhydrase II-binding domain in the electrogenic Na+ -HCO₃- cotransporter NBCe1 expressed in Xenopus oocytes

The electrogenic Na+ -HCO?? cotransporter NBCe1 plays essential roles in the regulation of systemic and/or local pH. Homozygous inactivating mutations in NBCe1 cause proximal renal tubular acidosis associated with ocular abnormalities. We recently showed that defective membrane expression of NBCe1, caused by several mutations such as Delta65bp (S982NfsX4), is also associated with familial migraine. The Delta65bp mutant is quite unique in that it lacks a putative carbonic anhydrase (CA) II-binding domain but still shows an apparently normal transport activity in Xenopus oocytes. In this addendum, we show that the co-expression of CAII together with the wild-type NBCe1 or the Delta65bp mutant does not enhance the NBCe1 activities in oocytes. Moreover, a carbonic anhydrase inhibitor acetazolamide fails to inhibit the wild-type or the Delta65bp activities co-expressed with CAII. These results indicate that a bicarbonate transport metabolon proposed for the interaction between CAII and NBCe1 does not work at least in Xenopus oocytes.     

Phosphorylation of Dpsyl2 (CRMP2) and Dpsyl3 (CRMP4) is required for positioning of caudal primary motor neurons in the zebrafish spinal cord

Dpysls (CRMPs) that were initially identified as mediator proteins of Semaphorin3a (Sema3a) signaling are involved in neuronal polarity and axon elongation in cultured neurons. Previous studies have shown that knockdown of neuropilin1a, one of the sema3a receptors, exhibited ectopic primary motor neurons (PMNs) outside of the spinal cord in zebrafish. However, downstream molecules of sema3a signaling involved in the positioning of motor neurons are largely unknown. Here, we addressed the role of Dpysl2 (CRMP2) and Dpysl3 (CRMP4) in the positioning of PMNs in the zebrafish spinal cord. We found that the knockdown of dpysls by antisense morpholino oligonucleotides (AMO) causes abnormal positioning of caudal primary (CaP) motor neurons outside the spinal cord. The knockdown of cdk5 and dyrk2 by AMO also caused similar phenotype in the positioning of CaP motor neurons, and this phenotype was rescued by co‐injection of phosphorylation‐mimic type dpysl2 mRNA. These results suggest that the phosphorylation of Dpysl2 and Dpysl3 by Cdk5 and Dyrk2 is required for correct positioning of CaP motor neurons in the zebrafish spinal cord. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 911–920, 2013