Effect of double-stranded DNA on maturation of dendritic cells in vitro

A preparation of human genomic fragmented double-stranded DNA (dsDNA) was used as maturation stimulus in cultures of human dendritic cells (DCs) generated in compliance with the interferon protocol. Culturing of the DCs in medium with 5 μg/ml of the DNA preparation was associated with a decrease in the relative proportion of CD14 + cells and an increase in that of CD83 + cells. These changes are markers of DC maturation. The efficiency with which the DNA preparation was able to elicit DC maturation was commensurate with that of lypopolysaccharide from bacterial cell, the standard inducer of DC maturation. Generated ex vivo, matured in the presence of the human DNA preparation, pulsed with tumor antigens mouse DCs were used as a vaccine in biological tests for its antitumor activity. The experimental results demonstrate that reinfusion of mature pulsed with tumor antigens DCs cause a statistically significant suppression of tumor graft growth.     

Cutting edge: gamma delta T cells provide help to B cells with altered clonotypes and are capable of inducing Ig gene hypermutation

It has not been resolved whether gammadelta T cells can collaborate with germinal center B cells and support Ig hypermutation during an Ab response to a truly defined T-dependent Ag. In this study, we show that in the absence of alphabeta T cells, immunization with the well-defined T-dependent Ag, (4-hydroxy-3-nitrophenyl) acetyl (NP) conjugate, was able to induce Ig hypermutation. However, the clonotypes of B cells responding to NP were dramatically altered in TCR beta(-/-) mice. Unlike B cells in wild-type mice that use canonical VDJ rearrangements, most NP-responding B cells in mutant mice use analog genes of the J558 gene family. In addition, the majority of anti-NP Abs produced in mutant mice use kappaL chain instead of lambda1L chain, which dominates in mice of Igh(b) background. Thus, the B cell population that collaborates with gammadelta T cells is distinct from B cells interacting with conventional alphabeta Th cells.     

Toll-like receptor 3 activation promotes desensitization of histamine response in human gingival fibroblasts: Poly (I:C) induces histamine receptor desensitization in human gingival fibroblasts

Viruses are associated with the development of periodontal disease, particularly during periods of suppressed cellular immunity. For this reason, we evaluated the hypothesis that viral components regulate the actions of histamine, an important mediator of immune responses. We assessed the effect of Poly (I:C) on histamine-mediated intracellular calcium mobilization in human gingival fibroblasts. Our results show that histamine induces an increase in intracellular calcium concentrations in a dose-dependent manner. This response was blocked when cells were incubated in the presence of Poly (I:C). In addition, phorbol esters, a diacylglycerol analog, mimics the inhibitory actions of Poly (I:C) in response to histamine. The effect of Poly (I:C) was reversed by Stuarosporine (1 μM), GÖ6983 (7 μM), Bisindolylmaleimide (1 μM) [a protein inhibitor (PKC)], and SB 203580 (3 μM) (a p38-MAPK inhibitor). These findings suggest that Poly (I:C) regulates histamine-induced calcium mobilization through activation of PKC and p38.     

Carnosine protects neurons against oxidative stress and modulates the time profile of MAPK cascade signaling

Carnosine is a known protector of neuronal cells against oxidative injury which prevents both apoptotic and necrotic cellular death. It was shown earlier that carnosine serves as an intracellular buffer of free radicals. Using the model of ligand-dependent oxidative stress in neurons, we have shown that homocysteine (HC) initiates long-term activation of extracellular signal regulated kinase, isoforms 1 and 2 (ERK 1/2) and Jun N-terminal kinase (JNK) which corresponds to exitotoxic effect resulting in cellular death. L-carnosine (β-alanyl-L-histidine) protects neurons from both excitotoxic effect of homocysteine and cellular death. Its analogs, β-alanyl-D-histidine (D-carnosine) and L-histidyl-β-alanine, restricted accumulation of free radicals and delayed activation of ERK1/2 and JNK in neuronal cells, but did not promote neuronal viability.     

Tautomerism,acid-base equilibria,and H-bonding of the six histidines in subtilisin BPN' by NMR

We have determined by (15)N, (1)H, and (13)C NMR, the chemical behavior of the six histidines in subtilisin BPN' and their PMSF and peptide boronic acid complexes in aqueous solution as a function of pH in the range of from 5 to 11, and have assigned every (15)N, (1)H, C(epsilon 1), and C(delta2) resonance of all His side chains in resting enzyme. Four of the six histidine residues (17, 39, 67, and 226) are neutrally charged and do not titrate. One histidine (238), located on the protein surface, titrates with pK(a) = 7.30 +/- 0.03 at 25 degrees C, having rapid proton exchange, but restricted mobility. The active site histidine (64) in mutant N155A titrates with a pK(a) value of 7.9 +/- 0.3 and sluggish proton exchange behavior, as shown by two-site exchange computer lineshape simulation. His 64 in resting enzyme contains an extremely high C(epsilon 1)-H proton chemical shift of 9.30 parts per million (ppm) owing to a conserved C(epsilon 1)-H(.)O=C H-bond from the active site imidazole to a backbone carbonyl group, which is found in all known serine proteases representing all four superfamilies. Only His 226, and His 64 at high pH, exist as the rare N(delta1)-H tautomer, exhibiting (13)C(delta1) chemical shifts approximately 9 ppm higher than those for N(epsilon 2)-H tautomers. His 64 in the PMSF complex, unlike that in the resting enzyme, is highly mobile in its low pH form, as shown by (15)N-(1)H NOE effects, and titrates with rapid proton exchange kinetics linked to a pK(a) value of 7.47 +/- 0.02.     

Expression of an extracellular ribonuclease gene increases resistance to Cucumber mosaic virus in tobacco

Background

The apoplast plays an important role in plant defense against pathogens. Some extracellular PR-4 proteins possess ribonuclease activity and may directly inhibit the growth of pathogenic fungi. It is likely that extracellular RNases can also protect plants against some viruses with RNA genomes. However, many plant RNases are multifunctional and the direct link between their ribonucleolytic activity and antiviral defense still needs to be clarified. In this study, we evaluated the resistance of Nicotiana tabacum plants expressing a non-plant single-strand-specific extracellular RNase against Cucumber mosaic virus.

Results

Severe mosaic symptoms and shrinkage were observed in the control non-transgenic plants 10 days after inoculation with Cucumber mosaic virus (CMV), whereas such disease symptoms were suppressed in the transgenic plants expressing the RNase gene. In a Western blot analysis, viral proliferation was observed in the uninoculated upper leaves of control plants, whereas virus levels were very low in those of transgenic plants. These results suggest that resistance against CMV was increased by the expression of the heterologous RNase gene.

Conclusion

We have previously shown that tobacco plants expressing heterologous RNases are characterized by high resistance to Tobacco mosaic virus. In this study, we demonstrated that elevated levels of extracellular RNase activity resulted in increased resistance to a virus with a different genome organization and life cycle. Thus, we conclude that the pathogen-induced expression of plant apoplastic RNases may increase non-specific resistance against viruses with RNA genomes.

     

Diindolilmethane (DIM) selectively inhibits cancer stem cells

Epidemiologic studies repeatedly have shown chemopreventive effects of cruciferous vegetables. Indole-3-carbinol (I3C) and its metabolite diindolylmethane (DIM) were identified in these plants as active ingredients and theirs anti-tumor activities were confirmed in multiple in vitro and in vivo experiments. Here, we demonstrate that DIM is a selective and potent inhibitor of cancer stem cells (CSCs). In several cancer cell lines, DIM inhibited tumor sphere formation at the concentrations 30-300 times lower than concentrations required for growth inhibition of parental cells cultured as adherent culture. We also found that treatment with DIM overcomes chemoresistance of CSCs to cytotoxics, such as paclitaxel, doxorubicin, and SN-38. Pre-treatment of tumor spheres with DIM before implantation to mice significantly retarded the growth of primary tumors compared to tumors formed by untreated tumor spheres. The concentrations of DIM required to suppress CSCs formation are in the close range to those achievable in human plasma after oral dosing of the compound. Therefore, DIM can potentially be used in cancer patients, either alone, or in combinations with existing drugs.     

N‐acetylcysteine increases susceptibility of HeLa cells to bacterial invasion

Serratia grimesii are non‐pathogenic bacteria capable, however, to invade eukaryotic cells provided that they synthesize intracellular metalloprotease grimelysin (Bozhokina et al. [2011] Cell. Biol. Int. 35: 111–118). To elucidate how invasion of grimelysin containing bacteria depends on physiological state of host cells, we studied the effect of N‐acetylcysteine (NAC) on susceptibility of HeLa cells to invasion by the wild‐type S. grimesii and recombinant E. coli expressing grimelysin gene. Incubation of HeLa cells with 10 mM NAC resulted in changes of cell morphology and disassembly of actin cytoskeleton that were reversed when NAC was removed from the culture medium. Both in the presence of NAC and upon its removal, the entry of grimelysin producing bacteria increased by a factor of 1.5–2 and 3–3.5 for wild‐type S. grimesii and recombinant E. coli, respectively. This effect does not correlate with cytoskeleton rearrangements but may be due to the NAC‐induced up‐regulation of cell surface receptors playing a role in cell adhesion and cell–cell junctions. A twofold difference in the efficiency of S. grimesii and recombinant E. coli to enter the NAC‐treated cells suggests that the entry of the wild‐type and recombinant bacteria occurs via different receptors which activity is differently affected by NAC. J. Cell. Biochem. 114: 1568–1574, 2013. © 2013 Wiley Periodicals, Inc.