Protein transduction domain of HIV-1 Tat protein promotes efficient delivery of DNA into mammalian cells

The plasma membrane of mammalian cells is one of the tight barriers against gene transfer by synthetic delivery systems. Various agents have been used to facilitate gene transfer by destabilizing the endosomal membrane under acidic conditions, but their utility is limited, especially for gene transfer in vivo. In this article, we report that the protein transduction domain of human immunodeficiency virus type 1 Tat protein (Tat peptide) greatly facilitates gene transfer via membrane destabilization. We constructed recombinant lambda phage particles displaying Tat peptide on their surfaces and carrying mammalian marker genes as part of their genomes (Tat-phage). We demonstrate that, when animal cells are briefly exposed to Tat-phage, significant expression of phage marker genes is induced with no harmful effects to the cells. In contrast, recombinant phage displaying other functional peptides, such as the integrin-binding domain or a nuclear localization signal, could not induce detectable marker gene expression. The expression of marker genes induced by Tat-phage is not affected by endosomotropic agents but is partially impaired by inhibitors of caveolae formation. These data suggest that Tat peptide will become a useful component of synthetic delivery vehicles that promote gene transfer independently of the classical endocytic pathway.     

Loss of CpG methylation is strongly correlated with loss of histone H3 lysine 9 methylation at DMR-LIT1 in patients with Beckwith-Wiedemann syndrome

To clarify the chromatin-based imprinting mechanism of the p57(KIP2)/LIT1 subdomain at chromosome 11p15.5 and the mouse ortholog at chromosome 7F5, we investigated the histone-modification status at a differentially CpG methylated region of Lit1/LIT1 (DMR-Lit1/LIT1), which is an imprinting control region for the subdomain and is demethylated in half of patients with Beckwith-Wiedemann syndrome (BWS). Chromatin-immunoprecipitation assays revealed that, in both species, DMR-Lit1/LIT1 with the CpG-methylated, maternally derived inactive allele showed histone H3 Lys9 methylation, whereas the CpG-unmethylated, paternally active allele was acetylated on histone H3/H4 and methylated on H3 Lys4. We have also investigated the relationship between CpG methylation and histone H3 Lys9 methylation at DMR-LIT1 in patients with BWS. In a normal individual and in patients with BWS with normal DMR-LIT1 methylation, histone H3 Lys9 methylation was detected on the maternal allele; however, it disappeared completely in the patients with the DMR-LIT1 imprinting defect. These findings suggest that the histone-modification status at DMR-Lit1/LIT1 plays an important role in imprinting control within the subdomain and that loss of histone H3 Lys9 methylation, together with CpG demethylation on the maternal allele, may lead to the BWS phenotype.     

Relationship between Locomotor Activity and Monoamines Following Single and Double Transient Forebrain Ischemia in Gerbils

The relationship between locomotor activity and monoamine levels in gerbils after single and/or double forebrain ischemic insult was studied. Locomotor hyperactivity was observed after the first ischemic episode, but the gerbils failed to show hyperactivity after the second ischemic episode induced one week later. The monoamine levels were determined in order to clarify the biochemical basis of post-ischemic locomotor hyperactivity. Norepinephrine increased in response to first ischemic episode but remained at normal levels after the second episode of ischemia. Metabolites of dopamine and serotonin increased after both the first and second ischemic insults, which indicates that these monoamines do not play significant roles in post-ischemic locomotor activity. Therefore, increases in norepinephrine after first ischemic insult may play a role in increasing locomotor activity during the period following such an episode.     

Effects of divalent metal ions on chlorophyll a fluorescence in isolated spinach chloroplasts

The effects of divalent metal ions on the yields of chlorophyll a fluorescence were investigated in isolated spinach chloroplasts at room and liquid nitrogen temperatures. Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺ and Mn²⁺ increased the yields of fluorescence emission at 684 and 695 nm from pigment system II and decreased that at 735 nm from pigment system I. Al³⁺ showed similar but less significant effects on the fluorescence yields. Zn²⁺ and Cd²⁺ showed no significant effect on the fluorescence yields at concentrations lower than 5 mM.

In accordance with the results of our previous study concerning the effects of Mg²⁺ on the excitation transfer in the chloroplasts, it was concluded that ions of alkaline earths and manganese suppress the excitation transfer from bulk chlorophylla of pigment system II to that of pigment system I.     

Control of excitation transfer in photosynthesis. IV. Kinetics of chlorophyll a fluorescence in Porphyra yezoensis

The kinetics of chlorophyll a fluorescence were measured at 685 nm in intact cells of Porphyra yezoensis during alternate illumination of the organism with two colors of light, one absorbed by phycoerythrin and the other by chlorophyll a. Two components of fluorescence change overlapping each other in time were separated; the fast component may be controlled by the rate of Photoreaction II which competes with the fluorescence emission process, and the slow component by the light-induced change in excitation transfer between two pigment systems as suggested in our previous study⁶. The kinetics of the slow change in fluorescence yield were extensively investigated.

Terms, “State I” and “State II” are used to describe the state of excitation transfer. In the State I a lesser amount of excitation energy is delivered in Pigment System I and greater to Pigment System II than in the State II. The conversion of the states is achieved by the selective illumination of pigment systems.

The conversion from the State I toward the State II occurred under Light II (light absorbed by Pigment System II) with a half time of about 10 sec, and it saturated at a light intensity of less than 1000 ergs×cm⁻²×sec⁻¹. The reverse conversion occurred under Light I (light absorbed by Pigment System I) with a half time of about 5 sec, and it saturated at about 10 000 ergs×cm⁻²×sec⁻¹.

Light I and Light II competed with each other in the interconversion of the states.     

Synthesis and structural analysis of five novel oligosaccharides prepared by glucosyltransfer from beta-D-glucose 1-phosphate to isokestose and nystose using Thermoanaerobacter brockii kojibiose phosphorylase

Five novel oligosaccharides (tetra-, penta- and hexa-saccharides) were synthesized by glucosyltransfer from beta-D-glucose 1-phosphate to isokestose (O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside) or nystose (O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside) using Thermoanaerobacter brockii kojibiose phosphorylase. The oligosaccharides were identified as 2(2-alpha-D-glucopyranosyl)(m)isokestose; [O-alpha-D-glucopyranosyl-(1-->2)](m)-O-[beta-D-fructofuranosyl-(2-->1)](2)-alpha-D-glucopyranoside: m=1, 2, and 3, and 2(2-alpha-D-glucopyranosyl)(n)nystose; [O-alpha-D-glucopyranosyl-(1-->2)](n)-O-[beta-D-fructofuranosyl-(2-->1)](3)-alpha-D-glucopyranoside: n=1 and 2 using gas liquid chromatography analysis of the methyl derivatives, and MALDI-TOF-MS and NMR measurements of the newly formed oligosaccharides. 1H, 13C NMR signals of each saccharide were assigned using 2D-NMR techniques, including COSY, HSQC, HSQC-TOCSY, HMBC, CH(2)-selected E-HSQC, and CH(2)-selected E-HSQC-TOCSY.     

Dissection of photodamage at low temperature and repair in darkness suggests the existence of an intermediate form of photodamaged photosystem II

Irradiation of the photosynthetic machinery with strong light induces damage to the photosystem II complex (PSII), and this phenomenon is referred to as photodamage. In an attempt to characterize the mechanism of photodamage to PSII, we examined the events associated with photodamage by monitoring the phenomenon in Synechocystis sp. PCC 6803 at a low temperature. After the activity of PSII had been reduced to 10% of the original activity by exposure of Synechocystis cells to strong light at 10 degrees C, recovery was allowed to proceed at 34 degrees C in darkness. Under these conditions, approximately 50% of the activity of PSII was restored within 60 min. The recovery in darkness did not require protein synthesis, as demonstrated by Western blotting analysis and a radiolabeling experiment with [(35)S]methionine. We also observed a similar recovery of PSII in darkness in isolated thylakoid membranes. Our findings, together with those of other studies, suggest the presence of an intermediate form of photodamaged PSII that is generated prior to the formation of photodamaged PSII.     

Hypoxia induces apoptosis in SV40-immortalized rat proximal tubular cells through the mitochondrial pathways,devoid of HIF1-mediated upregulation of Bax

Chronic hypoxia is a major contributor to tubulointerstitial injury in various renal diseases and apoptosis is apparently involved. Although many studies report hypoxia-induced apoptosis in cultured tubular cells, information has been limited in proximal tubular cells, those from the most susceptible portion of renal tubules against hypoxia. This study was to confirm a role for apoptosis in hypoxic proximal tubular cells and to investigate its association with HIF-1. Temperature-sensitive SV40-immortalized rat proximal tubular cells (IRPTCs) showed apoptosis in 21.9+/-2.9% by hypoxia (0.2% O(2), 48h), with alterations in mitochondrial signaling such as Bcl2 and caspase-9. Bax mRNA was unaffected during the process. However, treating IRPTCs at the nonpermissive temperature showed an upregulation of Bax by hypoxia, which was abrogated by overexpressing dominant-negative HIF-1alpha. These findings extend previous reports on hypoxia-mediated tubular cell apoptosis and demonstrate the possible involvement of HIF-1 as an upstream molecule of Bax.