LET dependence of lethality in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> irradiated by heavy ions

To clarify the effect of heavy ions in plants, dry seeds of Arabidopsis were irradiated with carbon, neon, and argon ions with various linear energy transfer (LET) values. The relative biological effectiveness (RBE) for lethality peaked at LET values over 350 keV/microns for neon and argon ions. This LET giving the peak RBE was higher than the LET of 100-200 keV/microns which was reported to have a maximum RBE for other types of cells, such as mammalian cells. Furthermore, sterility showed a higher RBE at an LET of 354 keV/microns with neon ions than that at an LET of 113 keV/microns with carbon ions. Lethality and sterility are both considered to be caused by damage to DNA. The results indicate that the LET having a maximum of RBE for lethality is higher in Arabidopsis seeds than in other unicellular systems. The most likely explanation for this shift of LET is that the DNA in dry seeds has a different chemical environment and/or hydration state than the DNA in cells in culture.     

Decline in insulin action with age in endurance-trained humans.

We tested the hypothesis that regular endurance exercise prevents the age-related decline in insulin action typically observed in healthy, sedentary adults. An index of whole body insulin sensitivity (ISI), obtained from minimal model analysis of insulin and glucose concentrations during a frequently sampled intravenous glucose tolerance test, was determined in 126 healthy adults: 25 young [27 +/- 1 (SE) yr; 13 men/12 women] and 43 older (59 +/- 1 yr; 20/13) sedentary and 25 young (29 +/- 1 yr; 12/13) and 33 older (60 +/- 1 yr; 20/13) endurance trained. ISI values were lower in the older vs. young adults in both sedentary (-53%; 3.9 +/- 0.3 vs. 7.0 +/- 0.7 x10(-4) x min(-1) x microU(-1) x ml(-1); P < 0.01) and endurance-trained (-36%; 7.9 +/- 0.6 vs. 12.4 +/- 1.0 x 10(-4) min(-1) x microU(-1) x ml(-1); P < 0.01) groups, but the value was 72-102% higher in the trained subjects at either age (P < 0.01). In subgroup analysis of sedentary and endurance-trained adults with similar body fat levels (n = 62), the age-related reduction in ISI persisted only in the endurance-trained subjects (12.9 +/- 1.9 vs. 8.7 +/- 1.2 x 10(-4) x min(-1) x microU(-1) x ml(-1); P < 0.01). The results of the present study suggest that habitual endurance exercise does not prevent the age-associated decline insulin action. Moreover, the age-related reduction in ISI in endurance-trained adults appears to be independent of adiposity.     

Effect of catecholamine on aldosterone release in isolated rat glomerulosa cell suspensions

Effect of adrenergic activity on the adrenal steroidogenesis and the modulation by catecholamines of aldosterone release were studied in isolated rat adrenal cell suspensions. Isoproterenol, norepinephrine and epinephrine, but not dopamine, caused statistically significant increase in aldosterone release. Both prazosin (alpha 1 antagonist) and yohimbine (alpha 2 antagonist) suppressed the norepinephrine-induced aldosterone release in a dose dependent manner, respectively. Both atenolol (beta 1 antagonist) and ICI 118-551 (beta 2 antagonist) also blocked (-)-isoproterenol-induced aldosterone release in a dose dependent manner, respectively. Neither (-)-isoproterenol nor (+/-)-norepinephrine at concentrations of 10(-6) M potentiated aldosterone release stimulated by angiotensin II or ACTH. These results suggest that catecholamines stimulate aldosteroidogenesis, but it appears unlikely that aldosterone release induced by ACTH or angiotensin-II is modulated by adrenergic stimulation.     

Importance of purine-pyridine hydroxyl and purine amino groups for hammerhead ribozyme cleavage

The importance of the 2′-hydroxyl and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. The three guanosines in the central core of a hammerhead ribozyme were replaced by deoxyinosine, inosine, and deoxyguanosine, and ribozymes containing these analogues were chemically synthesized. Most of the modified ribozymes are drastically descreased in their cleavage efficiency. However. deletion of the 2-amino group at G₈ (replacement with inosine, deoxyguanosine, deoxyinosine) caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. Whereas, deletion of the 2′-amino group at G₁₂ and G₅ (replacement with inosine, deoxyinosine, and deoxyguanosine) resulted in ribozymes with drastic decrease in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U⁷ and U⁴, in the ribozyne sequence were replaced by deoxyuridine (dU). The dU4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that ws about half that observed for the native complex. By comparison, the dU⁷ complex exhibited a relative cleavage activity within 3.3-fold of that observed with native ribozyme/substrate complex. This result suggests that the 2′-hydroxyl group at U ⁷ is not essential for activity.

The importance of the 2′-hydroxyl, and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead roibozyme has been investigated. Most of the modified rybozymes are drastically decreased in their cleavage efficiency. However, deletion of the 2-amino group at G₈ or deletion of the 2′-hydroxyl group at G₁₂ caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U₇ and U₄, in the ribozyme sequence were replaced by deoxyuridine (dU). The U4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that was about half that observed for the native complex.     

Conserved protein kinases encoded by herpesviruses and cellular protein kinase cdc2 target the same phosphorylation site in eukaryotic elongation factor 1delta

Earlier studies have shown that translation elongation factor 1delta (EF-1delta) is hyperphosphorylated in various mammalian cells infected with representative alpha-, beta-, and gammaherpesviruses and that the modification is mediated by conserved viral protein kinases encoded by herpesviruses, including UL13 of herpes simplex virus type 1 (HSV-1), UL97 of human cytomegalovirus, and BGLF4 of Epstein-Barr virus (EBV). In the present study, we attempted to identify the site in EF-1delta associated with the hyperphosphorylation by the herpesvirus protein kinases. Our results are as follows: (i) not only in infected cells but also in uninfected cells, replacement of the serine residue at position 133 (Ser-133) of EF-1delta by alanine precluded the posttranslational processing of EF-1delta, which corresponds to the hyperphosphorylation. (ii) A purified chimeric protein consisting of maltose binding protein (MBP) fused to a domain of EF-1delta containing Ser-133 (MBP-EFWt) is specifically phosphorylated in in vitro kinase assays by purified recombinant UL13 fused to glutathione S-transferase (GST) expressed in the baculovirus system. In contrast, the level of phosphorylation by the recombinant UL13 of MBP-EFWt carrying an alanine replacement of Ser-133 (MBP-EFS133A) was greatly impaired. (iii) MBP-EFWt is also specifically phosphorylated in vitro by purified recombinant BGLF4 fused to GST expressed in the baculovirus system, and the level of phosphorylation of MBP-EFS133A by the recombinant BGLF4 was greatly reduced. (iv) The sequence flanking Ser-133 of EF-1delta completely matches the consensus phosphorylation site for a cellular protein kinase, cdc2, and in vitro kinase assays revealed that purified cdc2 phosphorylates Ser-133 of EF-1delta. (v) As observed with EF-1delta, the casein kinase II beta subunit (CKIIbeta) was specifically phosphorylated by UL13 in vitro, while the level of phosphorylation of CKIIbeta by UL13 was greatly diminished when a serine residue at position 209, which has been reported to be phosphorylated by cdc2, was replaced with alanine. These results indicate that the conserved protein kinases encoded by herpesviruses and a cellular protein kinase, cdc2, have the ability to target the same amino acid residues for phosphorylation. Our results raise the possibility that the viral protein kinases mimic cdc2 in infected cells.     

pH-induced conformational change in an alpha-helical coiled-coil is controlled by His residues in the hydrophobic core

An alpha-helical coiled-coil structure is one of the basic structural units in proteins. Hydrophilic residues at the hydrophobic positions in the coiled-coil structure play important roles in structures and functions of natural proteins. We reported here a peptide that formed a triple stranded alpha-helical coiled-coil showing the pH-dependent structural change. The peptide was designed to have two His residues at the hydrophobic positions of the center of the coiled-coil structure. The peptide folded into a triple stranded coiled-coil at neutral pH, while it unfolded at acidic pH. This construct is useful to create a protein that the structure or function is controlled by pH.     

Fluorescent-Based Methods for Gene Knockdown and Functional Cardiac Imaging in Zebrafish

A notable advantage of zebrafish as a model organism is the ease of gene knockdown using morpholino antisense oligonucleotide (MO). However, zebrafish morphants injected with MO for a target protein often show heterogeneous phenotypes, despite controlling the injection volume of the MO solution in all embryos. We developed a method for estimating the quantity of MO injected into each living morphant, based on the co-injection of a control MO labeled with the fluorophore lissamine. By applying this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2a-depleted zebrafish with a decreased heart rate and impairment of cardiac contraction. To investigate cardiac impairment of the tnnt2a morphant, we performed fluorescent cardiac imaging using Bodipy-ceramide. Cardiac image analysis showed moderate reduction of tnnt2a impaired diastolic distensibility and decreased contraction and relaxation velocities. To the best of our knowledge, this is the first report to analyze the role of tnnt2a in cardiac function in tnnt2a-depleted living animals. Our combinatorial approach can be applied for analyzing the molecular function of any protein associated with human cardiac diseases.     

Transformation of Polyoxins D,E, and F with Sodium Bisulfite

Polyoxins D, E, and F which possess 5-carboxyuracil as the nucleobase were reacted selectively with sodium bisulfite at pH 4.0 resulting in facile decarboxylation to afford corresponding 5,6-dihydrouracil-6-sulfonates and uracil type polyoxins (polyoxins L, M, and K) in good yield. The former compounds were also converted to the latter almost quantitatively with mild alkali treatment. Biological activities of the transformed compounds were described.     

Isolation and Characterization of Two Plasmids in Bacillus subtilis var. amylosacchariticus

Five bufadienolides (1-5) isolated from the leaves of Kalanchoe pinnata and K. daigremontiana×tubiflora (Crassulaceae) were examined for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate. All bufadienolides showed inhibitory activity, and bryophyllin A (1) exhibited the most marked inhibition (IC₅₀=0.4 μM) among the tested compounds. Bryophyllin C (2), a reduction analogue of 1, and bersaldegenin-3-acetate (3) lacking the orthoacetate moiety were less active. These results strongly suggest that bufadienolides are potential cancer chemopreventive agents.     

Preparation of 8-Chloropurine Nucleosides Through the Reaction Between their C-8 Lithiated Species and p-Toluenesulfonyl Chloride

Abstract

Chlorination of purine nucleosides protected with tert-butyldimethylsilyl (TBDMS) group was examined by the reaction of the C-8 lithiated species, generated by LDA, with p-toluenesulfonyl chloride as an electrophile. This provides a new method for the preparation of 8-chloropurine nucleosides.