CD and UV resonance Raman measurements surprisingly find that the charge screening of even 2 M concentrations of NaCl and KCl do not alter the unfolded PPII and 2.51-helix conformations of poly-L-glutamate. These salts appear to be excluded from the region between the side chain charges and the peptide backbone. Furthermore, no direct ion pairing occurs between these salts and the side chain carboxylates.

We used 204 nm excitation UV Resonance Raman (UVRR) spectroscopy to examine the effects of NaClO4 on the conformation of poly–L–lysine (PLL). The presence of NaClO4 induces the formation of α–helix, π–helix/bulge and turn conformations. The dependence of the AmIII3 frequency on the peptide Ψ Ramachandran angle allows us to experimentally determine the conformational population distributions and the energy landscape of PLL along the Ramachandran Ψ angle. We also used UVRR to measure the NaClO4 concentration dependence of PLL amide hydrogen exchange kinetics. Exchange rates were determined by fitting the D2O exchanging PLL UVRR AmII′ band time evolution. Hydrogen exchange is slowed at high NaClO4 concentrations. The PLL AmII′ band exchange kinetics at 0.0, 0.2 and 0.35 M NaClO4 can be fit by single exponentials, but the AmII′ band kinetics of PLL at 0.8 M NaClO4 requires a double exponential fit. The exchange rates for the extended conformations were monitored by measuring the Cα–H band kinetics. These kinetics are identical to those of the AmII′ band until 0.8 M NaClO4 whereupon the extended conformation exchange becomes clearly faster than that of the α–helix–like conformations. Our results indicate that ClO4− binds to the PLL backbone to protect it from OH− exchange catalysis. In addition, ClO4− binding also slows the conformational exchange between the extended and α–helix–like conformations, probably by increasing the activation barriers for conformational interchanges.

Recently, a multicenter randomized controlled trial (RCT) by Cooper and colleagues indicated that decompressive craniectomy (DC) may be associated with a worse functional outcome in patients with diffuse traumatic brain injury (TBI), although DC can immediately and constantly reduce intracranial pressure (ICP). As this trial is well planned and of high quality, the unexpected result is meaningful. However, the evidence of the study is insufficient and the effect of DC in severe TBI is still uncertain. Additional multicenter RCTs are necessary to provide class I evidence on the role of DC in the treatment of refractory raised ICP after severe TBI.

We used 204 nm excitation UV Resonance Raman (UVRR) spectroscopy to examine the role of side chain electrostatic interactions in determining the conformation of poly-L-lysine (PLL). We examined the pH and ionic strength dependence of the UVRR. The pH dependence of PLL UVRR spectra between pH 7.1 and 11.7 cannot be described by a two-state model, but requires at least one additional state. The AmIII3 region fitting with pH 7.1 and 11.7 basis spectra reveals a small pH induced decrease in the relative fraction of the 2.51-helix conformation compared to the PPII conformation. We performed a 2D general correlation analysis on the PLL pH dependence UVRR spectra. The asynchronous spectrum shows enhanced spectral resolution. The 2D asynchronous spectrum reveals multiple components in the Cα-H b band and the AmII band whose origins are unclear. The cross peaks in the 2D asynchronous spectrum between the AmIII band and the other bands reveals that increasing pH induces three new structures: π-helix, α-helix and some turn structure. We find that 2.5 M NaCl does not change the equilibrium between the PPII and 2.51-helix conformations by screening sidechain electrostatic repulsion. The result indicates that NaCl does not penetrate the region between the sidechain and the peptide backbone. We also compared PLL conformations induced by high pH to that induced by 0.8 M ClO4−. Both conditions induce α-helix-like conformations. 0.8 M ClO4− induces 6% more α-helix-like conformations than at pH 12.4. Higher pH gives rise to longer α-helices and less turn structures.

The X protein (HBx) of hepatitis B virus (HBV) is involved in the development of hepatocellular carcinoma (HCC), and methionine adenosyltransferase 2A (MAT2A) promotes the growth of liver cancer cells through altering S-adenosylmethionine homeostasis. Thus, we speculated that a link between HBx and MAT2A may contribute to HCC development. In this study, the effects of HBx on MAT2A expression and cell apoptosis were investigated, and the molecular mechanism by which HBx and MAT2A regulate tumorigenesis was evaluated. Results from immunohistochemistry analyses of 37 pairs of HBV-associated liver cancer tissues/corresponding peritumor tissues showed that HBx and MAT2A are highly expressed in most liver tumor tissues. Our in vitro results revealed that HBx activates MAT2A expression in a dose-dependent manner in hepatoma cells, and such regulation requires the cis-regulatory elements NF-κB and CREB on the MAT2A gene promoter. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) further demonstrated that HBx facilitates the binding of NF-κB and CREB to MAT2A gene promoter. In addition, overexpression of HBx or MAT2A inhibits cell apoptosis, whereas knockdown of MAT2A expression stimulates apoptosis in hepatoma cells. Furthermore, we demonstrated that HBx reduces MAT1A expression and AdoMet production but enhances MAT2β expression. Thus, we proposed that HBx activates MAT2A expression through NF-κB and CREB signaling pathways to reduce AdoMet production, inhibit hepatoma cell apoptosis, and perhaps enhance HCC development. These findings should provide new insights into our understanding how the molecular mechanisms underline the effects of HBV infection on the production of MAT2A and the development of HCC.

Purpose

To obtain and analyze early retinal changes at the molecular level 24 h after a radiation injury to the ipsilateral intraorbital nerve using gamma knife surgery (GKS), and to examine the morphological changes in bilateral optic nerves.

Methods

Unilateral intraorbital optic nerves of three rhesus macaques were treated by GKS with irradiated doses of 15 Gy, while contralateral optic nerves and retinas served as the control. Gene expression profiles of the control and affected retinas were analyzed with Affymetrix Rhesus Macaque Genome arrays. To verify the results, a quantitative real-time polymerase chain reaction (qRT–PCR) was performed to test the expression patterns of five function-known genes. Morphological changes in the bilateral optic nerves were examined using a transmission electron microscope (TEM) and light microscopy. The glial cell reaction in bilateral optic nerves was studied using immunohistochemistry.

Results

Of the probe sets, 1,597 (representing 1,081 genes) met the criteria for differential expression, of which 82 genes were significantly up-or down-regulated in treated retinas. There was prominent upregulation of genes associated with glial cell activation in the treated retina. Genes related to an early inflammatory reaction and to cell death were also significantly regulated in response to a radiation injury to the intraorbital optic nerve. In contrast, the messenger ribonucleic acid (mRNA) expression levels of retinal ganglion cell (RGC)-specific genes were low. Morphologically, cytoplasmic processes of astrocytes in treated nerves were shorter than those of the control and were not straight, while also being accompanied by decreased GFAP immunostaining. More oligodendrocytes and inflammatory cells were apparent in treated nerves than in the control. In addition, swollen mitochondria and slight chromation condensation could be seen in the glial cells of treated nerves.

Conclusions

We conclude that the current irradiated dose of 15 Gy was sufficient to lead to a radiation injury of the optic nerve and retina. Several transcripts deregulated in retinas after a radiation injury play a key role in radiation-induced neurogenic visual loss, especially for genes associated with RGC, glial cell, and cell death. Glial cells in optic nerves might be the primary target of a radiation injury in the optic nerve.

Background

Modifications of DNA and histones in various combinations are correlated with many cellular processes. In this study, we investigated the possible relationship between histone H4 tetraacetylation, DNA methylation and histone H3 dimethylation at lysine 9 during mitosis in maize root meristems.

Results

Treatment with trichostatin A, which inhibits histone deacetylases, resulted in increased histone H4 acetylation accompanied by the decondensation of interphase chromatin and a decrease in both global H3K9 dimethylation and DNA methylation during mitosis in maize root tip cells. These observations suggest that histone acetylation may affect DNA and histone methylation during mitosis. Treatment with 5-azacytidine, a cytosine analog that reduces DNA methylation, caused chromatin decondensation and mediated an increase in H4 acetylation, in addition to reduced DNA methylation and H3K9 dimethylation during interphase and mitosis. These results suggest that decreased DNA methylation causes a reduction in H3K9 dimethylation and an increase in H4 acetylation.

Conclusions

The interchangeable effects of 5-azacytidine and trichostatin A on H4 acetylation, DNA methylation and H3K9 dimethylation indicate a mutually reinforcing action between histone acetylation, DNA methylation and histone methylation with respect to chromatin modification. Treatment with trichostatin A and 5-azacytidine treatment caused a decrease in the mitotic index, suggesting that H4 deacetylation and DNA and H3K9 methylation may contain the necessary information for triggering mitosis in maize root tips.

Galectin-1, a member of a family of evolutionarily conserved glycan-binding proteins, binds specifically to poly-N-acetyllactosamine-enriched glycoconjugates. Through interactions with these glycoconjugates, this protein modulates inflammatory responses and contributes to tumor progression and immune cell homeostasis. The carbohydrate recognition domain includes the single protein tryptophan (Trp68). UV Resonance Raman spectroscopy and molecular dynamic simulation were used to examine the change in the environment of the Trp on ligand binding. The UV Raman spectra and the calculated water radial distribution functions show that, while no large structural changes in the protein follows lactose binding, substantial solvent reorganization occurs. These new insights into the microscopic role of water molecules on Gal-1 binding to its specific carbohydrate ligands provides a better understanding of the physicochemical properties of Gal-1-saccharide interactions, which will be useful for the design of synthetic inhibitors for therapeutic purposes.

Background

In humans, chromosome fragile sites are regions that are especially prone to forming non-staining gaps, constrictions or breaks in one or both of the chromatids on metaphase chromosomes either spontaneously or following partial inhibition of DNA synthesis and have been well identified. So far, no plant chromosome fragile sites similar to those in human chromosomes have been reported.

Methods and Results

During the course of cytological mapping of rDNA on ryegrass chromosomes, we found that the number of chromosomes plus chromosome fragments was often more than the expected 14 in most cells for Lolium perenne L. cv. Player by close cytological examination using a routine chromosome preparation procedure. Further fluorescent in situ hybridization (FISH) using 45S rDNA as a probe indicated that the root-tip cells having more than a 14-chromosome plus chromosome fragment count were a result of chromosome breakage or gap formation in vitro (referred to as chromosome lesions) at 45S rDNA sites, and 86% of the cells exhibited chromosome breaks or gaps and all occurred at the sites of 45S rDNA in Lolium perenne L. cv. Player, as well as in L. multiflorum Lam. cv. Top One. Chromatin depletion or decondensation occurred at various locations within the 45S rDNA regions, suggesting heterogeneity of lesions of 45S rDNA sites with respect to their position within the rDNA region.

Conclusions

The chromosome lesions observed in this study are very similar cytologically to that of fragile sites observed in human chromosomes, and thus we conclude that the high frequency of chromosome lesions in vitro in Lolium species is the result of the expression of 45S rDNA fragile sites. Possible causes for the spontaneous expression of fragile sites and their potential biological significance are discussed.

Objectives

Particulate air pollution is a serious problem all over the world, and the development of a method to evaluate the health effects of ambient particles is necessary. In this study, cells cultured in vitro were exposed to particles sampled at the side of a main road, and their protein expression levels were examined.

Methods

Ambient particles were collected at the side of a main road using a high-volume air sampler. Some of the collected particles (crude particles) were treated with an organic solvent to remove chemical components, and the resulting residues were used as residual particles. Cells from the mouse alveolar epithelial cell line LA-4 were inoculated into tissue-culture dishes at 1.4×104/cm2, exposed to each type of particle or artificial carbon particles (Printex 90) that were dispersed using an ultrasonic homogenizer by mixing in the medium twice at 24 and 48 hours, and incubated for up to 72 hours after the start of inoculation. After exposure, the number of cells and intracellular dehydrogenase activity were measured. Proteins extracted from the cells were subjected to two-dimensional gel electrophoresis with isoelectric focusing at pHs 4–7 using a 10% acrylamide gel, and their expression levels were analyzed after fluorescent staining.

Results

The intracellular dehydrogenase activity of the cells significantly decreased as a result of exposure to the residual (0.70-fold) and crude (0.84-fold) particles compared with that of the control, but it showed no change as a result of exposure to Printex 90. The protein expression levels in the cells exposed to the particles increased or decreased similarly, but different expression levels were also observed. There were differences in the effects observed between the cells exposed to the artificial carbon particles and those exposed to particles collected from ambient air.

Conclusion

This study indicates that protein expression levels in cells change in response to exposure to particles collected from ambient air. To evaluate the effects of particles on health, it is considered necessary to use particles collected from ambient air.

Interactions between the MinD and MinE proteins are required for proper placement of the Escherichia coli division septum. The site within MinE that is required for interaction with MinD was mapped by studying the effects of site-directed minE mutations on MinD-MinE interactions in yeast two-hybrid and three-hybrid experiments. This confirmed that the MinE N-terminal domain is responsible for the interaction of MinE with MinD. Mutations that interfered with the interaction defined an extended surface on one face of the α-helical region of the MinE N-terminal domain, consistent with the idea that the MinE-MinD interaction involves formation of a coiled-coil structure by interaction with a complementary helical surface within MinD.