Direct electrochemistry and electrocatalysis of heme-proteins entrapped in agarose hydrogel films

Three heme-proteins, including myoglobin (Mb), hemoglobin (Hb) and horseradish peroxidase (HRP), were immobilized on edge-plane pyrolytic graphite (EPG) electrodes by agarose hydrogel. The proteins entrapped in the agarose film undergo fast direct electron transfer reactions, corresponding to FeIII = e- --> FeII. The formal potential (E degrees'), the apparent coverage (Gamma), the electron transfer coefficient (alpha) and the apparent electron transfer rate constant (ks) were calculated by integrating cyclic voltammograms or performing nonlinear regression analysis of square wave voltammetric (SWV) experimental data. The E degrees's are linearly dependent on solution pH (redox Bohr effect), indicating that the electron transfer was proton-coupled. Ultraviolet visible (UV-Vis) and reflection-absorption infrared (RAIR) spectra suggest that the conformation of proteins in the agarose film are little different from that proteins alone, and the conformation changes reversibly in the range of pH 3.0-10.0. Atomic force microscopy (AFM) images of the agarose film indicate a stable and crystal-like structure formed possibly due to the synergistic interaction of hydrogen bonding between N,N-dimethylformamide (DMF), agarose hydrogel and heme-proteins. This suggests a strong interaction between the heme-proteins and the agarose hydrogel. DMF plays an important role in immobilizing proteins and enhancing electron transfer between proteins and electrodes. The mechanisms for catalytic reduction of hydrogen peroxide and nitric oxide (NO) by proteins entrapped in agarose hydrogel were also explored.     

In vitro propagation of <Emphasis Type="Italic">Pimelea spicata</Emphasis> R.Br (Thymelaeaceae), an endangered species of the Sydney region,Australia

Micropropagation was assessed as an ex situ conservation strategy for the endangered Australian plant Pimelea spicata (Thymelaeaceae). Although regeneration of this species was achieved, several physiological problems were observed and examined. Explants of P. spicata had a higher multiplication rate on MS medium, than on ½ MS, but there was a significantly higher percentage of necrotic shoot tips on the higher salt medium. Increasing calcium concentration and gas exchange exacerbated shoot-tip necrosis. A number of hyperhydrated shoots were produced in all treatments, the cause of which could not be determined, although less hyperhydicity was observed in the ½ MS treatment. Shoots, rooted in vitro on ½ MS in the absence of plant growth regulators, were successfully acclimatised to greenhouse conditions, while direct rooting of microshoots using IBA gel treatment proved unsuccessful. This is the first report of tissue culture propagation of this endangered species.     

Preapoptotic chromatin changes induced by ultraviolet B irradiation in human erythroleukemia K562 cells

Exponentially growing human erythroleukemia K562 cells were permeabilized and the dose dependent decrease of DNA synthesis rate was measured after ultraviolet (UV B, 290 nm) irradiation. Cells were able to overcome 2 and 5 J/m2 UV doses, partial recovery was observed at 15 J/m2, while at high (25 J/m2) UV dose replicative DNA synthesis remained suppressed. K562 cells were subjected to synchronization prior to and after UV irradiation (24 J/m2) and 18 fractions were collected by centrifugal elutriation. Cell cycle analysis by flow cytometry did not show early apoptotic cells after UV irradiation. The gradual increase in DNA content typical for non-irradiated cells was contrasted by an early S phase block between 2.2 and 2.4 C-values after UV irradiation. Cell cycle dependent chromatin changes after ultraviolet irradiation were seen as a fine fibrillary network covering the mainly fibrous chromatin structures and incompletely folded primitive chromosomes. Based on observations after UV irradiation and on earlier results with cadmium treatment and gamma irradiation, we confirm that typical chromatin changes characteristic to genotoxic agents can be recognized and classified.     

The multiple promoter methylation profile of PR gene and ERalpha gene in tumor cell lines

The changes of methylation status of various gene promoters are a common feature of malignant cells and these changes can occur early in the progression process. Therefore, abnormal methylation can be used as cancer marker. Such studies will first require the development of a panel of methylated markers that are methylated in cancer tissues but unmethylated in normal tissues or methylated status is different between cancer tissues and normal tissues. By using methylation-specific PCR (MSP) assay method, we observed alterations in DNA methylation at the double promoter regions of the progesterone receptor (PR) gene and estrogen receptor (ERalpha) gene in various tumor cell lines. Compared with normal white blood cell, the methylation status of PRA promoter in various cancer cell lines changed from unmethylation pattern to methylation pattern. That of PRB promoter changed from both unmethylated and methylated alleles to only methylated allele. The methylation status of ERalpha-A and ERalpha-B promoter in various cancer cell lines are cell -specific. This study indicates that PR promoter methylation may be a molecular marker in various cancer detections. And the methylation status of ERalpha-A and ERalpha-B is cell-specific.     

Wild-Type Measles Viruses with Non-Standard Genome Lengths

The length of the single stranded, negative sense RNA genome of measles virus (MeV) is highly conserved at 15,894 nucleotides (nt). MeVs can be grouped into 24 genotypes based on the highly variable 450 nucleotides coding for the carboxyl-terminus of the nucleocapsid protein (N-450). Here, we report the genomic sequences of 2 wild-type viral isolates of genotype D4 with genome lengths of 15,900 nt. Both genomes had a 7 nt insertion in the 3′ untranslated region (UTR) of the matrix (M) gene and a 1 nt deletion in the 5′ UTR of the fusion (F) gene. The net gain of 6 nt complies with the rule-of-six required for replication competency of the genomes of morbilliviruses. The insertions and deletion (indels) were confirmed in a patient sample that was the source of one of the viral isolates. The positions of the indels were identical in both viral isolates, even though epidemiological data and the 3 nt differences in N-450 between the two genomes suggested that the viruses represented separate chains of transmission. Identical indels were found in the M-F intergenic regions of 14 additional genotype D4 viral isolates that were imported into the US during 2007–2010. Viral isolates with and without indels produced plaques of similar size and replicated efficiently in A549/hSLAM and Vero/hSLAM cells. This is the first report of wild-type MeVs with genome lengths other than 15,894 nt and demonstrates that the length of the M-F UTR of wild-type MeVs is flexible.     

Triacylglycerol Synthesis Enzymes Mediate Lipid Droplet Growth by Relocalizing from the ER to Lipid Droplets

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Highlights? Triacylglyceride (TG) synthesis is coupled with lipid droplet (LD) growth ? Two LD populations exist: growing LDs, containing TG enzymes, and small LDs ? Specific TG synthesis enzymes move from the ER to LDs through membrane bridges ? LD localization of TG enzymes mediates expansion of a subset of LDs     

Temporal properties of dual-peak responses of mouse retinal ganglion cells and effects of inhibitory pathways

Dual-peak responses of retinal ganglion cells (RGCs) are observed in various species, previous researches suggested that both response peaks were involved in retinal information coding. In the present study, we investigated the temporal properties of the dual-peak responses recorded in mouse RGCs elicited by spatially homogeneous light flashes and the effect of the inhibitory inputs mediated by GABAergic and/or glycinergic pathways. We found that the two peaks in the dual-peak responses exhibited distinct temporal dynamics, similar to that of short-latency and long-latency single-peak responses respectively. Pharmacological studies demonstrated that the application of exogenous GABA or glycine greatly suppressed or even eliminated the second peak of the cells’ firing activities, while little change was induced in the first peak. Co-application of glycine and GABA led to complete elimination of the second peak. Moreover, application of picrotoxin or strychnine induced dual-peak responses in some cells with transient responses by unmasking a second response phase. These results suggest that both GABAergic and glycinergic pathways are involved in the dual-peak responses of the mouse RGCs, and the two response peaks may arise from distinct pathways that would converge on the ganglion cells.