UV-vulnerability of human papilloma virus type-16 E7-expressing astrocytes is associated with mitochondrial membrane depolarization and caspase-3 activation

The human papilloma virus-type 16 (HPV-16) E6 and E7 proteins interact with the p53 and pRb tumor suppressor proteins, respectively. The effect of E6 or E7 expression on UV irradiation (5 and 20 J/m2)-induced genotoxic injury of confluent primary murine astrocytes was determined. Retroviral vectors were used to overexpress E6 and E7. Astrocytes expressing E7 showed increased vulnerability to UV-induced apoptosis while E6 over expressing astrocytes were protected from the same insults. Cell death in the E7 overexpressing cells was apoptotic because it showed DNA ladders, activation of caspase-3, formation of apoptotic bodies and decreased DNA content to less than the G0 level. After UV-irradiation the level of E2F1 in E7-expressing astrocytes was higher than E6-, LXSN- or mock-infected cells, and caspase-3 was activated to a greater extent. E7-expressing astrocytes showed the highest levels of Bax under normal growth conditions. The mitochondrial membrane potential of E7-expressing astrocytes was depolarized by 90% after UV-irradiation while the depolarization in control cells was about 50%. E6 overexpression decreased while E7 overexpression increased UV-induced astrocyte apoptosis.     

Reprogramming of the activity of the activator/dissociation transposon family during plant regeneration in rice

Many aspects of epigenetic phenomena have been elucidated via studies of transposable elements. An active transposable element frequently loses its ability to mobilize and goes into an inactive state during development. In this study, we describe the cyclic activity of a maize transposable element dissociation (Ds) in rice. In rice genome, Ds undergoes the spontaneous loss of mobility. However, an inactive state of Ds can be changed into an active state during tissue culture. The recovery of mobility accompanies not only changes in the methylation patterns of the terminal region of Ds, but also alteration in the steady state level of the activator (Ac) mRNA that is expressed by a constitutive CaMV 35S promoter. Furthermore, the Ds-reactivation process is not random, but stage-specific during plantlet regeneration. Our findings have expanded previous observations on Ac reactivation in the tissue culture of maize.     

Protection by carnosine-related dipeptides against hydrogen peroxide-mediated ceruloplasmin modification

Carnosine, homocarnosine, and anserine are present in high concentrations in the muscle and brain of many animals and humans. Previous studies showed that these compounds have an antioxidant function. We investigated the protective effects of carnosine and related compounds on the modification of human ceruloplasmin that is induced by H2O2. Carnosine, homocarnosine, and anserine significantly inhibited the fragmentation and inactivation of ceruloplasmin that is induced by H2O2. All three compounds also inhibited the release of copper ion from protein, and the formation of hydroxyl radicals in the ceruloplasmin/H2O2 system. These compounds inhibited the fragmentation of human serum albumin that is induced by the copper-catalyzed oxidation system, as well as by the iron-catalyzed oxidation system. These results suggest that carnosine, homocarnosine, and anserine might protect ceruloplasmin against H2O2-mediated oxidative damage through a combination of copper chelation and free radical scavenging.     

A direct repeat of N-type Ca2+ channel alpha1B gene functions as a negative regulatory element in HeLa cells

The expression of the N-type voltage-gated calcium channel alpha1B gene is restricted to neurons by a 5'-upstream region (-3992 to -1788) that contains negative regulatory element(s) that are active in non-neuronal cells. A 39 bp DNA element, which is repeated nine times in a head-to-tail fashion, was found within the same region. To examine whether this direct repeat (DR) may function as a negatively acting cis-regulatory element, several fusion plasmids, DR-110alpha1BLUC (1X), DR-SV40LUC (IX, 2X), in which one or two copies of the DR fragment were subcloned upstream of the homologous and heterologous promoters, were transiently transfected into HeLa and NS20Y cells. The promoter activity of DR-110alpha1BLUC (1X) decreased to approximately 17% of the 110alph(a1B)LUC construct in HeLa cells. The expression of the DR-SV40LUC (1X) and DR-SV40LUC (2X) plasmids was also reduced to 50 to 23% of the levels that were observed in the pGL2-Promoter in the same cells. However, no repression of the DR constructs was observed in NS20Y cells. An electrophoretic mobility shift assay showed that two DR-specific complexes were detected in HeLa cells, but not in NS20Y cells. In addition, Southwestern blotting revealed the presence of approximately 33 and 43 kDa proteins in HeLa cells. Overall, these results suggest that a 39 bp DNA element might act as repressor in non-neuron cells through the specific interactions of the DNA-proteins.     

Fission yeast Rap1 homolog is a telomere-specific silencing factor and interacts with Taz1p

Taz1p is the fission yeast orthologue of human TRF2, a telomeric repeat-binding protein. Delta(taz1) mutants are defective in telomeric silencing, telomere length control, and meiotic recombination events. A recent report demonstrated that the human Rap1p homolog (hRap1) is recruited to telomere by interaction with TRF2, arguing that the telomere control mechanism of higher eukaryotes is distinct from that of the budding yeast. Taz1p showed a significant similarity to human TRF2, but not with the budding yeast Rap1p (scRap1p). This suggests that Taz1p and TRF2 share common features in telomere regulation. To assess the roles of Taz1p in telomere-related functions in detail, we attempted to identify a protein(s) that interacts with Taz1p by using two-hybrid screening. Interestingly, the sequence analysis of a positive clone revealed a perfect match with a Rap1 homolog in S. pombe (spRap1), which showed a significant homology with scRap1p and hRap1p. Here we show that the spRap1 deficiency in haploid cells is viable, which results in increased telomere length regulation, disruption of telomere silencing, and aberrant meiosis (like the delta(taz1) mutant). This suggests that spRap1p might be recruited to the telomere by Taz1p and play crucial roles in telomere function. Interestingly, the delta(rap1) mutants in fission yeast are defective only for telomere silencing. Therefore, the role of spRap1p may be distinct from that of scRap1p, which is involved in the silencing at both the telomere and mating type locus. Our data, therefore, suggest that the regulation mechanisms of telomere in fission yeast resemble that of higher eukaryotic cells rather than the budding yeast.     

Antisense expression of carnation cDNA encoding ACC synthase or ACC oxidase enhances polyamine content and abiotic stress tolerance in transgenic tobacco plants

The amount of polyamines (such as putrescine, spermidine, and spermine) increased under environmental stress conditions. We used transgenic technology in an attempt to evaluate their potential for mitigating the adverse effects of several abiotic stresses in plants. Because there is a metabolic competition for S-adenosylmethionine as a precursor between polyamine (PA) and ethylene biosyntheses, it was expected that the antisense-expression of ethylene biosynthetic genes could result in an increase in PA biosynthesis. Antisense constructs of cDNAs for senescence-related 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (CAS) and ACC oxidase (CAO) were isolated from carnation flowers that were introduced into tobacco by Agrobacterium-mediated transformation. Several transgenic lines showed higher PA contents than wild-type plants. The number and weight of seeds also increased. Stress-induced senescence was attenuated in these transgenic plants in terms of total chlorophyll loss and phenotypic changes after oxidative stress with hydrogen peroxide (H2O2), high salinity, acid stress (pH 3.0), and ABA treatment. These results suggest that the transgenic plants with antisense CAS and CAO cDNAs are more tolerant to abiotic stresses than wild-type plants. This shows a positive correlation between PA content and stress tolerance in plants.     

Self-assembled ternary complex of cationic dendrimer,cucurbituril, and DNA: noncovalent strategy in developing a gene delivery carrier

A ternary complex of PPI-DAB dendrimer [(1,4-diaminobutane); Gen = N; dendri-poly(propyleneimine); -[NHC(=O)CH(2)NH(2)(+)(CH(2))(4)NH(3)(+)](z)()], DNA, and cucurbituril (CB) was evaluated as an example of a totally self-assembled gene delivery carrier. The complex was formed in a noncovalent way in which DNA interacts with PPI-DAB electrostatistically and CB with PPI-DAB through multiple noncovalent interactions. Dynamic light scattering data indicated that the diameter and size distributions of the complexes were dependent upon the sequence of mixing of each component with unimodal distribution ranging from 150.8 to 210.2 nm under favorable conditions. Fluorescence studies showed the quantitative binding of CB to PPI-DAB after ternary complex formation. The complex was able to transfect mammalian cells with high efficiency and the cytotoxicity of the PPI-DAB/CB complex was relatively low.     

Biodegradable,endosome disruptive,and cationic network-type polymer as a highly efficient and nontoxic gene delivery carrier

The success of gene therapy is largely dependent on the delivery vector system. Efficient transfection and nontoxicity are two of the most important requirements of an ideal gene delivery vector. To generate both an efficient and nontoxic vector, we rationally constructed polymeric vectors to have simultaneous multiple functions, i.e., controlled degradation, an endosome disruptive function, and positive charges. Remarkably, the transfection efficiency of network poly(amino ester) (n-PAE) synthesized in this manner was comparable to that of polyethylenimine (PEI), one of the most efficient polymeric gene delivery vectors reported to date. However, there was a marked difference in cytotoxicity between the polymers. The majority of PEI-transfected cells were granulated and dead, whereas most of the cells transfected with n-PAE were viable and healthy. Successive events of efficient endosome escape of n-PAE/DNA polyplex and n-PAE biodegradation should result in high transfection efficiency and favorable cell viability response. The n-PAE-mediated transfection was also very efficient in the presence of serum. These data show that the approach we applied is a very appropriate way of making an ideal gene delivery carrier.     

A phytochrome-associated protein phosphatase 2A modulates light signals in flowering time control in Arabidopsis

Reversible protein phosphorylation, which is catalyzed by functionally coupled protein kinases and protein phosphatases, is a major signaling mechanism in eukaryotic cellular functions. The red and far-red light-absorbing phytochrome photoreceptors are light-regulated Ser/Thr-specific protein kinases that regulate diverse photomorphogenic processes in plants. Here, we demonstrate that the phytochromes functionally interact with the catalytic subunit of a Ser/Thr-specific protein phosphatase 2A designated FyPP. The interactions were influenced by phosphorylation status and spectral conformation of the phytochromes. Recombinant FyPP efficiently dephosphorylated oat phytochrome A in the presence of Fe(2+) or Zn(2+) in a spectral form-dependent manner. FyPP was expressed predominantly in floral organs. Transgenic Arabidopsis plants with overexpressed or suppressed FyPP levels exhibited delayed or accelerated flowering, respectively, indicating that FyPP modulates phytochrome-mediated light signals in the timing of flowering. Accordingly, expression patterns of the clock genes in the long-day flowering pathway were altered greatly. These results indicate that a self-regulatory phytochrome kinase-phosphatase coupling is a key signaling component in the photoperiodic control of flowering.