Toxicity of Trichloroethylene to Pseudomonas putida F1 Is Mediated by Toluene Dioxygenase

Trichloroethylene was metabolically activated by toluene dioxygenase to produce toxic effects in Pseudomonas putida F1. Cytotoxicity was indicated by growth inhibition and by the covalent modification of cellular molecules in P. putida F1 exposed to [¹⁴C]trichloroethylene. With a toluene dioxygenase mutant, neither growth inhibition nor alkylation of intracellular molecules was observed.     

Dechlorination of Chloral Hydrate Is Influenced by the Biofilm Adhesin Protein LapA in Pseudomonas putida LF54

LapA is the largest surface adhesion protein of Pseudomonas putida that initiates biofilm formation. Here, by using transposon insertion mutagenesis and a conditional lapA mutant, we demonstrate for the first time that LapA influences chloral hydrate (CH) dechlorination in P. putida LF54.     

DnaK Dependence of the Mycobacterial Stress-Responsive Regulator HspR Is Mediated through Its Hydrophobic C-Terminal Tail

HspR is a repressor known to control expression of heat shock operons in a number of Eubacteria. In mycobacteria and in several other actinobacteria, this protein is synthesized from the dnaKJE-hspR operon. Previous investigations revealed that HspR binds to the operon promoter, thereby controlling its expression in an autoregulatory manner. DnaK, which is a product of the same operon, further aids this autoregulatory process by stimulating the operator binding activity of HspR. The molecular mechanism by which DnaK assists HspR in executing its function is not clearly understood. In this study, it has been shown that DnaK can augment DNA binding activity of HspR by two mechanisms: (i) DnaK can restore the activity of completely denatured HspR by forming a complex with it, and (ii) DnaK can prevent thermal instability of HspR renatured by other means. Unlike the first mechanism, the latter function does not involve complex formation. The C-terminal hydrophobic tail of HspR was found to play a significant role in determining its thermal stability and DnaK dependence properties. A deletion mutant in which this region is removed does not respond to thermal stress and functions independent of DnaK. The hydrophobic C-terminal tails of HspRs of Mycobacterium tuberculosis and related Actinomycetales therefore may have evolved to make these HspRs more sensitive to thermal stress and, at the same time, subject to regulation by DnaK.     

The Inotropic Effect of the Active Metabolite of Levosimendan,OR-1896, Is Mediated through Inhibition of PDE3 in Rat Ventricular Myocardium

Aims

We recently published that the positive inotropic response (PIR) to levosimendan can be fully accounted for by phosphodiesterase (PDE) inhibition in both failing human heart and normal rat heart. To determine if the PIR of the active metabolite OR-1896, an important mediator of the long-term clinical effects of levosimendan, also results from PDE3 inhibition, we compared the effects of OR-1896, a representative Ca²⁺ sensitizer EMD57033 (EMD), levosimendan and other PDE inhibitors.

Methods

Contractile force was measured in rat ventricular strips. PDE assay was conducted on rat ventricular homogenate. cAMP was measured using RII_epac FRET-based sensors.

Results

OR-1896 evoked a maximum PIR of 33±10% above basal at 1 μM. This response was amplified in the presence of the PDE4 inhibitor rolipram (89±14%) and absent in the presence of the PDE3 inhibitors cilostamide (0.5±5.3%) or milrinone (3.2±4.4%). The PIR was accompanied by a lusitropic response, and both were reversed by muscarinic receptor stimulation with carbachol and absent in the presence of β-AR blockade with timolol. OR-1896 inhibited PDE activity and increased cAMP levels at concentrations giving PIRs. OR-1896 did not sensitize the concentration-response relationship to extracellular Ca²⁺. Levosimendan, OR-1896 and EMD all increased the sensitivity to β-AR stimulation. The combination of either EMD and levosimendan or EMD and OR-1896 further sensitized the response, indicating at least two different mechanisms responsible for the sensitization. Only EMD sensitized the α₁-AR response.

Conclusion

The observed PIR to OR-1896 in rat ventricular strips is mediated through PDE3 inhibition, enhancing cAMP-mediated effects. These results further reinforce our previous finding that Ca²⁺ sensitization does not play a significant role in the inotropic (and lusitropic) effect of levosimendan, nor of its main metabolite OR-1896.     

FGF19-induced Hepatocyte Proliferation Is Mediated through FGFR4 Activation

FGF19 and FGF21, unique members of the fibroblast growth factor (FGF) family, are hormones that regulate glucose, lipid, and energy homeostasis. Increased hepatocyte proliferation and liver tumor formation have also been observed in FGF19 transgenic mice. Here, we report that, in contrast to FGF19, FGF21 does not induce hepatocyte proliferation in vivo. To identify the mechanism for FGF19-induced hepatocyte proliferation, we explored similarities and differences in receptor specificity between FGF19 and FGF21. We find that although both are able to activate FGF receptors (FGFRs) 1c, 2c, and 3c, only FGF19 activates FGFR4, the predominant receptor in the liver. Using a C-terminal truncation mutant of FGF19 and a series of FGF19/FGF21 chimeric molecules, we determined that amino acids residues 38–42 of FGF19 are sufficient to confer both FGFR4 activation and increased hepatocyte proliferation in vivo to FGF21. These data suggest that activation of FGFR4 is the mechanism whereby FGF19 can increase hepatocyte proliferation and induce hepatocellular carcinoma formation.     

Antimicrobial Activity of Human Prion Protein Is Mediated by Its N-Terminal Region

Background

Cellular prion-related protein (PrP^c) is a cell-surface protein that is ubiquitously expressed in the human body. The multifunctionality of PrP^c, and presence of an exposed cationic and heparin-binding N-terminus, a feature characterizing many antimicrobial peptides, made us hypotesize that PrP^c could exert antimicrobial activity.

Methodology and Principal Findings

Intact recombinant PrP exerted antibacterial and antifungal effects at normal and low pH. Studies employing recombinant PrP and N- and C-terminally truncated variants, as well as overlapping peptide 20mers, demonstrated that the antimicrobial activity is mediated by the unstructured N-terminal part of the protein. Synthetic peptides of the N-terminus of PrP killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the “classical” human antimicrobial peptide LL-37. In contrast to LL-37, however, no marked helix induction was detected for the PrP-derived peptides in presence of negatively charged (bacteria-mimicking) liposomes. PrP furthermore showed an inducible expression during wounding of human skin ex vivo and in vivo, as well as stimulation of keratinocytes with TGF-α in vitro.

Conclusions

The demonstration of an antimicrobial activity of PrP, localisation of its activity to the N-terminal and heparin-binding region, combined with results showing an increased expression of PrP during wounding, indicate that PrPs could have a previously undisclosed role in host defense.     

Growth Inhibition in Suspension-Cultured Rice Cells under Phosphate Deprivation Is Mediated through Putrescine Accumulation

The effects of phosphate deprivation on the growth and polyamine levels of suspension-cultured rice (Oryza sativa) cells were investigated. When rice suspension cells were deprived of phosphate, cell growth was markedly inhibited. Phosphate deprivation resulted in a higher putrescine level and lower spermidine and spermine levels in rice suspension cells. The growth of rice cells cultured in the absence of phosphate did not recover as a result of spermidine and spermine addition. D-Arginine and [alpha]-methylornithine, inhibitors of putrescine biosynthesis, caused a reduced level of putrescine in rice suspension cells cultured under phosphate deprivation. The growth of rice cells cultured in the absence of phosphate was completely recovered after the addition of D-arginine but not [alpha]-methylornithine. Our results indicate that putrescine accumulation is a factor causing growth inhibition of suspension-cultured rice cells under phosphate deprivation.     

Survival of Pathogenic Mycobacteria in Macrophages Is Mediated through Autophosphorylation of Protein Kinase G

Pathogenic mycobacteria survive within macrophages through the inhibition of phagosome-lysosome fusion. A crucial factor for avoiding lysosomal degradation is the mycobacterial serine/threonine protein kinase G (PknG). PknG is released into the macrophage cytosol upon mycobacterial infection, suggesting that PknG might exert its activity by interfering with host signaling cascades, but the mode of action of PknG remains unknown. Here, we show that PknG undergoes autophosphorylation on threonine residues located at the N terminus. In contrast to all other mycobacterial kinases investigated thus far, autophosphorylation of PknG was not involved in the regulation of its kinase activity. However, autophosphorylation was crucial for the capacity of PknG to promote mycobacterial survival within macrophages. These results will contribute to a better understanding of the virulence mechanisms of pathogenic mycobacteria and may help to design improved inhibitors of PknG to be developed as antimycobacterial compounds.The genome of Mycobacterium tuberculosis comprises 11 serine/threonine protein kinases which are considered key players in signaling cascades by transferring phosphate groups from γ-ATP to the side chain hydroxyl group of serine and threonine residues in their substrates (1, 25). One of these kinases, protein kinase G (PknG), is secreted into the cytosol of infected macrophages and prevents the intracellular destruction of mycobacteria by blocking phagosome-lysosome fusion (30). Its essential role in mycobacterial virulence makes PknG an attractive drug target; the precise mode of action of PknG remains unclear, however (24, 30).Kinases in general can be classified into so-called RD and non-RD kinases based on the presence of a conserved arginine (R) and aspartate (D) sequence in the catalytic loop of the kinase domain. The RD arginine interacts with the phosphorylated activation loop and controls its fold, finally resulting in the activation of kinase activity (13, 18). Interestingly, PknG is unique among mycobacterial kinases in that the critical arginine residue in the kinase domain is lacking. The fact that PknG is classified as a non-RD kinase therefore suggests the absence of autophosphorylation in the activation loop (24).Although PknG is known to undergo autophosphorylation (14, 30), neither the localization of the autophosphorylation sites of PknG nor the importance of autophosphorylation for PknG activity is known. Here, we identify the autophosphorylation sites of PknG and provide evidence that autophosphorylation is essential for the capacity of PknG to modulate the survival of intracellularly residing mycobacteria.     

Transovarial Transmission of a Plant Virus Is Mediated by Vitellogenin of Its Insect Vector

Most plant viruses are transmitted by hemipteroid insects. Some viruses can be transmitted from female parent to offspring usually through eggs, but the mechanism of this transovarial transmission remains unclear. Rice stripe virus (RSV), a Tenuivirus, transmitted mainly by the small brown planthopper (Laodelphax striatellus), is also spread to the offspring through the eggs. Here, we used the RSV–planthopper system as a model to investigate the mechanism of transovarial transmission and demonstrated the central role of vitellogenin (Vg) of L. striatellus in the process of virus transmission into the eggs. Our data showed Vg can bind to pc3 in vivo and in vitro and colocalize in the germarium. RSV filamentous ribonucleoprotein particles (RNPs) only accumulated in the terminal filaments and pedicel areas prior to Vg expression and was not present in the germarium until Vg was expressed, where RSV RNPs and Vg had colocalized. Observations by immunoelectron microscopy (IEM) also indicated that these two proteins colocalized in nurse cells. Knockdown of Vg expression due to RNA interference resulted in inhibition of the invasion of ovarioles by RSV. Together, the data obtained indicated that RSV RNPs may enter the nurse cell of the germarium via endocytosis through binding with Vg. Finally, the virus enters the oocytes through nutritive cords, using the same route as for Vg transport. Our results show that the Vg of L. striatellus played a critical role in transovarial transmission of RSV and shows how viruses can use existing transovarial transportation systems in insect vectors for their own purposes.     

Specific Encapsidation of Nodavirus RNAs Is Mediated through the C Terminus of Capsid Precursor Protein Alpha

Flock house virus (FHV) is a small icosahedral insect virus with a bipartite, messenger-sense RNA genome. Its T=3 icosahedral capsid is initially assembled from 180 subunits of a single type of coat protein, capsid precursor protein alpha (407 amino acids). Following assembly, the precursor particles undergo a maturation step in which the alpha subunits autocatalytically cleave between Asn363 and Ala364. This cleavage generates mature coat proteins beta (363 residues) and gamma (44 residues) and is required for acquisition of virion infectivity. The X-ray structure of mature FHV shows that gamma peptides located at the fivefold axes of the virion form a pentameric helical bundle, and it has been suggested that this bundle plays a role in release of viral RNA during FHV uncoating. To provide experimental support for this hypothesis, we generated mutant coat proteins that carried deletions in the gamma region of precursor protein alpha. Surprisingly, we found that these mutations interfered with specific recognition and packaging of viral RNA during assembly. The resulting particles contained large amounts of cellular RNAs and varying amounts of the viral RNAs. Single-site amino acid substitution mutants showed that three phenylalanines located at positions 402, 405, and 407 of coat precursor protein alpha were critically important for specific recognition of the FHV genome. Thus, in addition to its hypothesized role in uncoating and RNA delivery, the C-terminal region of coat protein alpha plays a significant role in recognition of FHV RNA during assembly. A possible link between these two functions is discussed.     

Differential Effects of Chloral Hydrate and Pentobarbital Sodium on a Cocaine Level and Its Catecholamine Response in the Medial Prefrontal Cortex: A Comparison with Conscious Rats

Abstract: Adult male Sprague-Dawley rats anesthetized with chloral hydrate and pentobarbital sodium were used as two different treatment groups. Conscious rats were used as a control group. By using baseline (precocaine) concentration as 100%, after cocaine administration (3.0 mg/kg i.v.), the maximal dopamine (DA) increase occurring at the first microdialysis collection period (20 min) in the medial prefrontal cortex was 299 ± 46% for the chloral hydrate group, 168 ± 12% for the pentobarbital sodium group, and 325 ± 23% for the conscious group. At the same time, norepinephrine (NA) increases reached a maximum and were 162 ± 20%, 100 ± 5%, and 141 ± 17%, respectively. The maximal changes of DA and NA in the chloral hydrate group and in the control group were both significantly higher than that in the pentobarbital sodium group. Meanwhile, the cocaine concentration was higher over a 100-min period of time in the chloral hydrate group when compared with the pentobarbital group and the control group. The peak cocaine concentration in dialysate occurred in the same time slot of maximal DA and NA responses, which were 0.65 ± 0.08, 0.30 ± 0.02, and 0.41 ± 0.05 µ M , respectively. Anesthetics suppress the pharmacologic response of neurons, which may explain the difference in catecholamine response between the pentobarbital sodium and the conscious groups. Conversely, because there was no significant difference in DA and NA response between the chloral hydrate group and the conscious group, it may possibly be due to the balancing effect between the higher existing cocaine concentration and the anesthetic suppression on pharmacological response of neurons in the chloral hydrate group. The effect of guide cannula implantation on the cocaine-induced catecholamine response was also evaluated.     

NGX6a Is Degraded through a Proteasome-dependent Pathway without Ubiquitination Mediated by Ezrin,a Cytoskeleton-Membrane Linker

Our previous study demonstrated that the NGX6b gene acts as a suppressor in the invasion and migration of nasopharyngeal carcinoma (NPC). Recently, we identified the novel isoform NGX6a, which is longer than NGX6b. In this study, we first found that NGX6a was degraded in NPC cells and that this degradation was mediated by ezrin, a linker between membrane proteins and the cytoskeleton. Specific siRNAs against ezrin increase the protein level of NGX6a in these cells. During degradation, NGX6a is not ubiquitinated but is degraded through a proteasome-dependent pathway. The distribution pattern of ezrin was negatively associated with NGX6a in an immunochemistry analysis of a nasopharyngeal carcinoma tissue microarray and fetus multiple organ tissues and Western blot analysis in nasopharyngeal and NPC cell lines, suggesting that ezrin and NGX6a are associated and are involved in the progression and invasion of NPC. By mapping the interacting binding sites, the seven-transmembrane domain of NGX6a was found to be the critical region for the degradation of NGX6a, and the amino terminus of ezrin is required for the induction of NGX6a degradation. The knockdown of ezrin or transfection of the NGX6a mutant CO, which has an EGF-like domain and a transmembrane 1 domain, resulted in no degradation, significantly reducing the ability of invasion and migration of NPC cells. This study provides a novel molecular mechanism for the low expression of NGX6a in NPC cells and an important molecular event in the process of invasion and metastasis of nasopharyngeal carcinoma cells.