Expression of Human A4V Mutant Cu,Zn Superoxide Dismutase in Schizosaccharomyces pombe: Investigations of its Toxic Properties

Cu,Zn superoxide dismutase (SOD1) is an antioxidant enzyme that catalyzes the removal of superoxide radicals generated in various biological oxidations. Amyotrophic lateral sclerosis (ALS) is one of the most common neurodegenerative disorders, occurring in families (FALS) and sporadically (SALS). FALS and SALS are distinguishable genetically but not clinically. More than 100 point mutations in the human SOD 1 gene have been identified that cause FALS. In order to determine the effects of mutant SOD protein, we first cloned wild-type and A4V mutant human SOD1 into Schizosaccharomyces pombe. This study shows viabilities and some antioxidant properties including SOD, catalase, proteasomal activity, and protein carbonyl levels of transformants in SOD1 deleted strain (MN415); and its parental strain (JY741) at different stress conditions. There was no more oxidative damage in the human mutant SOD carrying the transformant strain compared with other strains. These results may help to explain whether ALS progresses as a consequence of cellular oxidative damage.     

Parkinson's disease candidate gene prioritization based on expression profile of midbrain dopaminergic neurons

Background  

Parkinson's disease is the second most common neurodegenerative disorder. The pathological hallmark of the disease is degeneration of midbrain dopaminergic neurons. Genetic association studies have linked 13 human chromosomal loci to Parkinson's disease. Identification of gene(s), as part of the etiology of Parkinson's disease, within the large number of genes residing in these loci can be achieved through several approaches, including screening methods, and considering appropriate criteria. Since several of the indentified Parkinson's disease genes are expressed in substantia nigra pars compact of the midbrain, expression within the neurons of this area could be a suitable criterion to limit the number of candidates and identify PD genes.     

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades^1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome^3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.Download video file.^{(57M, mov)}     

Proteomic basis of the antibody response to monkeypox virus infection examined in cynomolgus macaques and a comparison to human smallpox vaccination

Monkeypox is a zoonotic viral disease that occurs primarily in Central and West Africa. A recent outbreak in the United States heightened public health concerns for susceptible human populations. Vaccinating with vaccinia virus to prevent smallpox is also effective for monkeypox due to a high degree of sequence conservation. Yet, the identity of antigens within the monkeypox virus proteome contributing to immune responses has not been described in detail. We compared antibody responses to monkeypox virus infection and human smallpox vaccination by using a protein microarray covering 92-95% (166-192 proteins) of representative proteomes from monkeypox viral clades of Central and West Africa, including 92% coverage (250 proteins) of the vaccinia virus proteome as a reference orthopox vaccine. All viral gene clones were verified by sequencing and purified recombinant proteins were used to construct the microarray. Serum IgG of cynomolgus macaques that recovered from monkeypox recognized at least 23 separate proteins within the orthopox proteome, while only 14 of these proteins were recognized by IgG from vaccinated humans. There were 12 of 14 antigens detected by sera of human vaccinees that were also recognized by IgG from convalescent macaques. The greatest level of IgG binding for macaques occurred with the structural proteins F13L and A33R, and the membrane scaffold protein D13L. Significant IgM responses directed towards A44R, F13L and A33R of monkeypox virus were detected before onset of clinical symptoms in macaques. Thus, antibodies from vaccination recognized a small number of proteins shared with pathogenic virus strains, while recovery from infection also involved humoral responses to antigens uniquely recognized within the monkeypox virus proteome.     

Nucleotide sequences of long terminal repeats of the human endogenous retrovirus (LTR HERV-K) on the short arm of chromosome 7: identification,analysis and evaluation of transcriptional activity

Six clones containing long terminal repeat (LTR) sequences of human endogenous retrovirus of the HERV-K family were found in the YAC library (1200 kb) of the short arm of human chromosome 7. The sequence sizes of the three clones corresponded to the full-length LTR (969 bp). The LTR localization was determined using FISH and verified by comparison with the GenBank database. All three DNA fragments containing solitary LTRs were transcribed in normal germline cells (testicular parenchyma tissue). The differences in the expression of these clones in the germline tumor cells (seminoma) were observed.     

Novel member of the CD209 (DC-SIGN) gene family in primates

Two CD209 family genes identified in humans, CD209 (DC-SIGN) and CD209L (DC-SIGNR/L-SIGN), encode C-type lectins that serve as adhesion receptors for ICAM-2 and ICAM-3 and participate in the transmission of human and simian immunodeficiency viruses (HIV and SIV, respectively) to target cells in vitro. Here we characterize the CD209 gene family in nonhuman primates and show that recent evolutionary alterations have occurred in this family across primate species. All of the primate species tested, specifically, Old World monkeys (OWM) and apes, have orthologues of human CD209. In contrast, CD209L is missing in OWM but present in apes. A third family member, that we have named CD209L2, was cloned from rhesus monkey cDNA and subsequently identified in OWM and apes but not in humans. Rhesus CD209L2 mRNA was prominently expressed in the liver and axillary lymph nodes, although preliminary data suggest that levels of expression may vary among individuals. Despite a high level of sequence similarity to both human and rhesus CD209, rhesus CD209L2 was substantially less effective at binding ICAM-3 and poorly transmitted HIV type 1 and SIV to target cells relative to CD209. Our data suggest that the CD209 gene family has undergone recent evolutionary processes involving duplications and deletions, the latter of which may be tolerated because of potentially redundant functional activities of the molecules encoded by these genes.     

Combined effect of epirubicin and lymphokine-activated killer cells on the resistant human breast cancer cells

Accumulating evidence suggests the concept that epirubicin and lymphokine-activated killer (LAK) cells cytotoxicity may be mediated by free radicals generation and P-glycoprotein-positive (Pg-p⁺) cancer cells are more sensitive for LAK cells than their drug-sensitive parental lines. We tested this hypothesis further by exposing drug-sensitive (WT) and epirubicin-resistant MCF-7 human breast tumor cells to epirubicin and LAK cells. Subsequently, we monitored cell proliferation as a measure of cytotoxicity. The cytotoxicity of epirubicin, LAK, and LAK + epirubicin (1/10 of IC₅₀) was evaluated in 400-fold epirubicin resistant MCF-7 EPI^R (P-glycoprotein overexpressing) and drug-sensitive MCF-7 WT cells. IC₅₀ values were measured using the MTT cytotoxicity test. The MCF-7 EPI^R cells exhibited an increased susceptibility to LAK cells than did the MCF-7 WT cells. P-gp⁺ MCF-7 EPI^R cells were lysed by human LAK cells to a greater extend than were their drug-sensitive counterparts. LAK + epirubicin combined treatment increased susceptibility of MCF-7 WT and MCF-7 EPI^R cells to LAK cells cytotoxicity. For both cell lines, cytotoxicity was dependent upon the concentration of the epirubicin and effector cell/target cell (E/T) ratio. The resistance of MCF-7 EPI^R cells to epirubicin appears to be associated with a developed tolerance to superoxide, most likely because of a tree-fold increase in superoxide dismutase (SOD) activity and 13-fold augmented selenium dependent glutathione peroxidase (GSH-Px) activity. Acting in concert, these two enzymes would decrease the formation of hydroxyl radical from reduced molecular oxygen intermediates. The addition of SOD decreased cytotoxicity of epirubicin and LAK cells. Taken together, these observations support the role of oxygen radicals in the cytotoxicity mechanism of epirubicin and suggest further that the development of resistance to this drug by the MCF-7 EPI^R tumor cells may have a component linked to oxygen free radicals. It is proposed that production of reactive oxygen species by the treatment of epirubicin and LAK cells can cause cytotoxicity of MCF-7 WT and MCF-7 EPI^R cells. SOD, catalase, GSH-Px, GST (glutathione S-transferase), and GSH (reduced glutathione) must be considered as part of the intracellular antioxidant defense mechanism of MCF-7 WT and MCF-7 EPI^R cells against reactive oxygen species.     

Cutting edge: expansion of the KIR locus by unequal crossing over

The killer Ig-like receptor (KIR) genes have high sequence similarity and are organized in a head-to-tail fashion. These properties may enhance misalignment of homologous chromosomes during synapsis preceding meiotic recombination, resulting in unequal crossing over. We have identified an extended KIR haplotype that contains a novel hybrid gene and two copies of each of two previously described KIR genes. A parsimonious mechanism for the derivation of this haplotype invokes unequal crossing over between two known ancestral KIR haplotypes. These data raise the possibility that unequal crossing over may be responsible in part for the expansion/contraction of KIR haplotypes as well as other homologous gene families that map in tandem.     

Physiological and Biochemical Mechanisms of Salinity Tolerance in <i>Carex morrowii</i> Boott

Carex species are widely used in many parts of the world and contain a large number of ecologically diverse species. Among the Carex species, some of them are known to be glycophytes, while others are halophytes. Carex morrowii Boott (Cyperaceae) is resistant to trample through their root structure and has an essential ornamental value in the landscape with their leaves. However, no information was found about the level of salinity tolerance/ sensitivity of the Carex morrowii among these species. In the present study, changes in trace element contents (Na, K, Ca, Cu, Mn, Mg, Ni, Fe, P, Zn, and N) and their transport from roots to leaves, osmotic regulation, alterations in chlorophyll and carotenoid contents, nitrogen assimilation (nitrate reductase activity; NRA) and total soluble protein content in both roots and leaves of Carex morrowii under different salinity concentrations (50 mM, 100 mM, 200 mM and 300 mM NaCl) were examined in detail. Our study provides the first detailed data concerning the responses of leaves and roots and the determination of the level of salinity tolerance/sensitivity of the Carex morrowii. The K+ /Na+ ratio was preserved up to 200 mM NaCl, and accordingly, the element uptake and transport ratios showed that they could control moderate NaCl levels. Ca homeostasis that is maintained even in 200 mM NaCl concentration can be effective in maintaining the structural integrity and selective permeability of the cell membranes, while 300 mM NaCl concentration caused decreased photosynthetic pigments, and deterioration in element content and compartmentation. Moreover, these data suggest that plant parts of Carex morrowii respond differently against varied levels of salinity stress. Although the decrease in NR activity at 200 mM and 300 mM NaCl concentrations in the leaves, NR activity was maintained in the roots. Consequently, Carex morrowii is moderately tolerant to salinity and the carotenoid content and osmotic regulation of Carex morrowii appears to be instrumental in its survival at different salinity levels. Especially the roots of Carex morrowii have a remarkable role in salinity tolerance.     

Cellulose nanocrystals from celery stalk as quercetin scaffolds: A novel perspective of human holo-transferrin adsorption and digestion behaviours

In this study, cellulose nanocrystals (CNCs) were synthesized from celery stalks to be used as the platform for quercetin delivery. Additionally, CNCs and CNCs–quercetin were characterized using the results of scanning electron microscope (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and zeta potential, while their interactions with human holo-transferrin (HTF) were also investigated. We examined their interaction under physiological conditions through the exertion of fluorescence, resonance light scattering, synchronized fluorescence spectroscopy, circular dichroism, three-dimensional fluorescence spectroscopy, and fluorescence resonance energy transfer techniques. The data from SEM and TEM exhibited the spherical shape of CNCs and CNCs–quercetin and also, a decrease was detected in the size of quercetin-loaded CNCs from 676 to 473 nm that indicated the intensified water solubility of quercetin. The success of cellulose acid hydrolysis was confirmed based on the XRD results. Apparently, the crystalline index of CNCs–quercetin was reduced by the interaction of CNCs with quercetin, which also resulted in the appearance of functional groups, as shown by FTIR. The interaction of CNCs–quercetin with HTF was also demonstrated by the induced quenching in the intensity of HTF fluorescence emission and Stern–Volmer data represent the occurrence of static quenching. Overall, the effectiveness of CNCs as quercetin vehicles suggests its potential suitability for dietary supplements and pharmaceutical products.