DNA damage-induced ubiquitylation of RFC2 subunit of replication factor C complex

Many proteins involved in DNA replication and repair undergo post-translational modifications such as phosphorylation and ubiquitylation. Proliferating cell nuclear antigen (PCNA; a homotrimeric protein that encircles double-stranded DNA to function as a sliding clamp for DNA polymerases) is monoubiquitylated by the RAD6-RAD18 complex and further polyubiquitylated by the RAD5-MMS2-UBC13 complex in response to various DNA-damaging agents. PCNA mono- and polyubiquitylation activate an error-prone translesion synthesis pathway and an error-free pathway of damage avoidance, respectively. Here we show that replication factor C (RFC; a heteropentameric protein complex that loads PCNA onto DNA) was also ubiquitylated in a RAD18-dependent manner in cells treated with alkylating agents or H(2)O(2). A mutant form of RFC2 with a D228A substitution (corresponding to a yeast Rfc4 mutation that reduces an interaction with replication protein A (RPA), a single-stranded DNA-binding protein) was heavily ubiquitylated in cells even in the absence of DNA damage. Furthermore RFC2 was ubiquitylated by the RAD6-RAD18 complex in vitro, and its modification was inhibited in the presence of RPA. The inhibitory effect of RPA on RFC2 ubiquitylation was relatively specific because RAD6-RAD18-mediated ubiquitylation of PCNA was RPA-insensitive. Our findings suggest that RPA plays a regulatory role in DNA damage responses via repression of RFC2 ubiquitylation in human cells.     

Drought induction of Arabidopsis 9-cis-epoxycarotenoid dioxygenase occurs in vascular parenchyma cells

The regulation of abscisic acid (ABA) biosynthesis is essential for plant responses to drought stress. In this study, we examined the tissue-specific localization of ABA biosynthetic enzymes in turgid and dehydrated Arabidopsis (Arabidopsis thaliana) plants using specific antibodies against 9-cis-epoxycarotenoid dioxygenase 3 (AtNCED3), AtABA2, and Arabidopsis aldehyde oxidase 3 (AAO3). Immunohistochemical analysis revealed that in turgid plants, AtABA2 and AAO3 proteins were localized in vascular parenchyma cells most abundantly at the boundary between xylem and phloem bundles, but the AtNCED3 protein was undetectable in these tissues. In water-stressed plants, AtNCED3 was detected exclusively in the vascular parenchyma cells together with AtABA2 and AAO3. In situ hybridization using the antisense probe for AtNCED3 showed that the drought-induced expression of AtNCED3 was also restricted to the vascular tissues. Expression analysis of laser-microdissected cells revealed that, among nine drought-inducible genes examined, the early induction of most genes was spatially restricted to vascular cells at 1 h and then some spread to mesophyll cells at 3 h. The spatial constraint of AtNCED3 expression in vascular tissues provides a novel insight into plant systemic response to drought stresses.     

Contrasting effects of presynaptic alpha2-adrenergic autoinhibition and pharmacologic augmentation of presynaptic inhibition on sympathetic heart rate control

Presynaptic alpha2-adrenergic receptors are known to exert feedback inhibition on norepinephrine release from the sympathetic nerve terminals. To elucidate the dynamic characteristics of the inhibition, we stimulated the right cardiac sympathetic nerve according to a binary white noise signal while measuring heart rate (HR) in anesthetized rabbits (n = 6). We estimated the transfer function from cardiac sympathetic nerve stimulation to HR and the corresponding step response of HR, with and without the blockade of presynaptic inhibition by yohimbine (1 mg/kg followed by 0.1 mg.kg(-1).h(-1) iv). We also examined the effect of the alpha2-adrenergic receptor agonist clonidine (0.3 and 1.5 mg.kg(-1).h(-1) iv) in different rabbits (n = 5). Yohimbine increased the maximum step response (from 7.2 +/- 0.8 to 12.2 +/- 1.7 beats/min, means +/- SE, P < 0.05) without significantly affecting the initial slope (0.93 +/- 0.23 vs. 0.94 +/- 0.22 beats.min(-1).s(-1)). Higher dose but not lower dose clonidine significantly decreased the maximum step response (from 6.3 +/- 0.8 to 6.8 +/- 1.0 and 2.8 +/- 0.5 beats/min, P < 0.05) and also reduced the initial slope (from 0.56 +/- 0.07 to 0.51 +/- 0.04 and 0.22 +/- 0.06 beats.min(-1).s(-1), P < 0.05). Our findings indicate that presynaptic alpha2-adrenergic autoinhibition limits the maximum response without significantly compromising the rapidity of effector response. In contrast, pharmacologic augmentation of the presynaptic inhibition not only attenuates the maximum response but also results in a sluggish effector response.     

Intra-arterial transplantation of bone marrow mononuclear cells immediately after reperfusion decreases brain injury after focal ischemia in rats

AIMS: Transplantation of bone marrow cells has been reported to exert neuroprotection against cerebral ischemia. However, the effect of bone marrow mononuclear cells (BMMCs) administered immediately after reperfusion has rarely been investigated. The present study was designed to examine whether brain injury in response to transient focal ischemia can be ameliorated by BMMC administration immediately after reperfusion in rats, and to determine whether there are differences in the route of administration. MAIN METHODS: Autologous BMMCs were obtained from each rat. Rats were then subjected to transient focal ischemia followed by BMMC administration via the ipsilateral carotid artery (IA group) or the femoral vein (IV group) immediately after reperfusion. Control rats underwent the same procedure but received vehicle injection. Infarct volume was compared among the groups 24 h and 7 days after reperfusion. BMMCs were fluorescently labeled with PKH26 prior to administration to track transplanted cells. KEY FINDINGS: Total infarct volume decreased in the IA group, but not in the IV group, when compared to the vehicle group. In the ipsilateral hemisphere, PKH26 positive cell count was greater in the IA group than in the IV group. Motor function, assessed with a rotarod test, improved in the IA group compared to the vehicle group. SIGNIFICANCE: These results show significant neuroprotection after transient focal ischemia by 1x10(7) autologous BMMCs administered intra-arterially, but not intravenously, immediately after reperfusion in rats. The larger number of transplanted BMMCs in the brain during the early stage of reperfusion may be responsible for the protective effect.     

Constituents of the Argentine Liverwort Plagiochila diversifolia and Their Insecticidal Activities

Three new compounds, a bicyclogermacrene ( 1 ) and two 2,3‐secoaromadendrane esters ( 2 and 3 ), together with (13S)‐13‐hydroxylabda‐8,14‐diene ( 4 ), fusicogigantone B ( 5 ), 3α,14‐diacetoxy‐2‐hydoxybicyclogermacrene ( 6 ), fusicogigantone A ( 7 ), neofuranoplagiochilal ( 8 ), plagiochiline B ( 9 ), furanoplagiochilal ( 10 ), trans‐nerolidol, spathulenol, α‐tocopherol, and (+)‐globulol were isolated from an Argentine collection of Plagiochila diversifolia. Their structures were elucidated by extensive mono and bidimensional NMR studies. Compounds 4 , 5 , and 6 , incorporated to the larval diet at 100 μg per g of diet, reduced the larval growth of Spodoptera frugiperda (Lepidoptera: Noctuidae) by 70 ± 25, 57 ± 23, and 33 ± 16%, respectively. Compounds 4 and 5 produced 70% and 60% larval mortality at early instars. The latter also showed antifeedant properties in the Choice Test, with a feeding ratio of 0.54 ± 0.16.     

Subcellular localization and targeting of glucocorticoid receptor protein fusions expressed in transgenic Arabidopsis thaliana

An animal system of inducible activation of protein fusions with the binding domain of glucocorticoid receptor (BDGR) was tested in Arabidopsis thaliana by monitoring dexamethasone (DEX)-induced nuclear targeting of reporter constructs. Two constructs containing green fluorescent protein (GFP), human homeobox protein Hanf-1 and Xenopus laevis BDGR were used, GFP/Hanf-1/BDGR and GFP/BDGR. The control construct contained GFP alone. In the absence of DEX both fusion proteins were uniformly distributed in the cytoplasm of root cells, but showed strong association with plastids in plant aerial parts. DEX treatment of roots prompted a strong and reversible nuclear accumulation of GFP/Hanf-1/BDGR, but not GFP/BDGR. Thus, in roots, the specific nuclear translocation of GFP/Hanf-1/BDGR was driven by Hanf-1 and tightly regulated by BDGR. However, in plant aerial parts treated with DEX, nuclear translocation of GFP/Hanf-1/BDGR was observed only in a few cases, and most part of the fusion protein was incorrectly and irreversibly targeted to plastids. Protease X digestion of isolated chloroplasts showed that BDGR fusion proteins were translocated into the chloroplast envelope and bound to envelope membranes, probably due to association with the chloroplast import apparatus. Thus, for efficient use of the glucocorticoid-inducible system in plants, it will be necessary to modify BDGR structure to prevent incorrect targeting of fusion proteins.     

Flavonoid glucuronides from Helicteres isora

Five flavonoid glucuronides were obtained from the fruit of Helicteres isora, three of which were previously unknown compounds: isoscutellarein 4'-methyl ether 8-O-beta-D-glucuronide 6"-n-butyl ester. isoscutellarein 4'-methyl ether 8-O-beta-D-glucuronide 2", 4"-disulfate and isoscutellarein 8-O-beta-D-glucuronide 2",4"-disulfate. The structures were determined on the basis of spectroscopy and hydrolysis experiments.     

Arg660Ser mutation in Thermus aquaticus DNA polymerase I suppresses T-->C transitions: implication of wobble base pair formation at the nucleotide incorporation step

We examined the replication fidelity of an Arg660Ser (R660S) mutant of Thermus aquaticus DNA polymerase I (Taq pol I). In a forward mutation assay, R660S showed a marked reduction in T→C transitions, one of the most frequent errors made by the wild-type enzyme. Steady-state kinetics showed that R660S discriminates against dGTP incorporation at a template T 13-fold better than the wild-type. R660S was also 3.2-fold less efficient than the wild-type at extending a T:dG mismatch. These results indicate that R660S has enhanced fidelity during incorporation and extension, which reduces its T→C transition frequency. Interestingly, R660S also discriminated correct from incorrect nucleotides at the incorporation step of C:dATP, A:dATP, G:dATP and C:8-OH-dGTP mispairs 28-, 6.0-, 4.1- and 6.8-fold better, respectively, than the wild-type, although it may not always be as accurate as the wild-type at the extension step. A structural model suggests that Arg660 may participate in two interactions that influence fidelity; the guanidinium group of Arg660 might interact with the incoming guanine base at the major groove and it might compete for forming another interaction with the primer terminus. Substituting Arg with Ser may eliminate or alter these interactions and destabilize the closed complex with incorrect substrates. Our data also suggest that T:dGTP and C:dATP base pairs form ‘wobble’ structures at the incorporation step of Taq pol I.