Hypervalent iodine-catalyzed oxylactonization of ketocarboxylic acids to ketolactones

The hypervalent iodine-catalyzed oxylactonization of ketocarboxylic acids to ketolactones was achieved in the presence of iodobenzene (10 mol %), p-toluenesulfonic acid monohydrate (20 mol %) and meta-chloroperbenzoic acid as a stoichiometric co-oxidant.     

Effect of insulin-mimetic vanadyl sulfate on cytochrome P450 2E1-dependent p-nitrophenol hydroxylation in the liver microsomes of streptozotocin-induced type 1 diabetic rats

CYP2E1 is known to be induced in streptozotocin (STZ)-treated diabetic rats (STZ rats), and its induction is improved by insulin. We have examined the age-dependent changes of CYP2E1 in the liver microsomes of type 1 diabetic STZ rats, the effects of VOSO4 on the contents of total P450 and CYP2E1, and the activities of CYP2E1 in terms of p-nitrophenol hydroxylation. The contents of P450 and CYP2E1 and CYP2E1 activity were enhanced with the development of diabetes. When the hyperglycemia of STZ rats was improved by daily intraperitoneal injections of VOSO4 for 10 days at the doses of 7 mg/kg body weight for 5 days, 5 mg/kg for the following 3 days, and then 2.5 mg/kg for 2 days, the P450 and CYP2E1 levels and CYP2E1 activity were lowered than those in the untreated STZ rats. To understand the mechanism underlying CYP2E1-dependent hydroxylation activity, the production of reactive oxygen species was examined in the NADPH-liver microsomal systems by ESR spin-trapping. Singlet oxygen (1O2) was detected in all microsomal systems, while superoxide anion radical(*O2-) and hydroxyl radical (*OH) were not. On the basis of these results, we conclude that (1) CYP2E1 level and activity are enhanced in the diabetic state, however, they are improved by VOSO4 treatment, and (2) 1O2 is generated during CYP2E1-dependent substrate oxygenation.     

S-adenosylmethionine (SAM)-accumulating sake yeast suppresses acute alcohol-induced liver injury in mice

The suppressive effects on acute alcoholic liver injury of S-adenosylmethionine (SAM) and the sake yeast, Saccharomyces cerevisiae Kyokai No. 9, have been shown previously. To enhance the suppression of acute alcoholic liver injury by sake yeast, we prepared SAM-accumulating sake yeast (SAM yeast). Male C57BL/6 mice that had been fed on a diet containing 0.25% SAM yeast or sake yeast for two weeks received three doses of ethanol (5 g/kg BW). In the mice fed on the SAM yeast, the ethanol-induced increases in both triglyceride (TG) and alanine aminotransferase (ALT) were significantly repressed. In addition, the SAM yeast-fed mice did not show an ethanol-induced decrease in hepatic SAM level, suggesting that a disorder of methionine metabolism in the liver caused by ethanol was relieved by the SAM yeast. These results suggest that the SAM yeast had a stronger effect suppressing acute alcoholic liver injury in mice than the sake yeast.     

Thermoadaptation-Directed Enzyme Evolution in an Error-Prone Thermophile Derived from Geobacillus kaustophilus HTA426

Thermostability is an important property of enzymes utilized for practical applications because it allows long-term storage and use as catalysts. In this study, we constructed an error-prone strain of the thermophile Geobacillus kaustophilus HTA426 and investigated thermoadaptation-directed enzyme evolution using the strain. A mutation frequency assay using the antibiotics rifampin and streptomycin revealed that G. kaustophilus had substantially higher mutability than Escherichia coli and Bacillus subtilis. The predominant mutations in G. kaustophilus were A · T→G · C and C · G→T · A transitions, implying that the high mutability of G. kaustophilus was attributable in part to high-temperature-associated DNA damage during growth. Among the genes that may be involved in DNA repair in G. kaustophilus, deletions of the mutSL, mutY, ung, and mfd genes markedly enhanced mutability. These genes were subsequently deleted to construct an error-prone thermophile that showed much higher (700- to 9,000-fold) mutability than the parent strain. The error-prone strain was auxotrophic for uracil owing to the fact that the strain was deficient in the intrinsic pyrF gene. Although the strain harboring Bacillus subtilis pyrF was also essentially auxotrophic, cells became prototrophic after 2 days of culture under uracil starvation, generating B. subtilis PyrF variants with an enhanced half-denaturation temperature of >10°C. These data suggest that this error-prone strain is a promising host for thermoadaptation-directed evolution to generate thermostable variants from thermolabile enzymes.     

Mineralocorticoid receptor activation: a major contributor to salt-induced renal injury and hypertension in young rats

Excessive salt intake is known to preferentially increase blood pressure (BP) and promote kidney damage in young, salt-sensitive hypertensive human and animal models. We have suggested that mineralocorticoid receptor (MR) activation plays a major role in kidney injury in young rats. BP and urinary protein were compared in young (3-wk-old) and adult (10-wk-old) uninephrectomized (UNx) Sprague-Dawley rats fed a high (8.0%)-salt diet for 4 wk. The effects of the MR blocker eplerenone on BP and renal injury were examined in the high-salt diet-fed young UNx rats. Renal expression of renin-angiotensin-aldosterone (RAA) system components and of inflammatory and oxidative stress markers was also measured. The effects of the angiotensin receptor blocker olmesartan with or without low-dose aldosterone infusion, the aldosterone synthase inhibitor FAD286, and the antioxidant tempol were also studied. Excessive salt intake induced greater hypertension and proteinuria in young rats than in adult rats. The kidneys of young salt-loaded rats showed marked histological injury, overexpression of RAA system components, and an increase in inflammatory and oxidative stress markers. These changes were markedly ameliorated by eplerenone treatment. Olmesartan also ameliorated salt-induced renal injury but failed to do so when combined with low-dose aldosterone infusion. FAD286 and tempol also markedly reduced urinary protein. UNx rats exposed to excessive salt at a young age showed severe hypertension and renal injury, likely primarily due to MR activation and secondarily due to angiotensin receptor activation, which may be mediated by inflammation and oxidative stress.     

Adherent monomer-misfolded SOD1

Background

Multiple cellular functions are compromised in amyotrophic lateral sclerosis (ALS). In familial ALS (FALS) with Cu/Zn superoxide dismutase (SOD1) mutations, the mechanisms by which the mutation in SOD1 leads to such a wide range of abnormalities remains elusive.

Methodology/Principal Findings

To investigate underlying cellular conditions caused by the SOD1 mutation, we explored mutant SOD1-interacting proteins in the spinal cord of symptomatic transgenic mice expressing a mutant SOD1, SOD1^Leu126delTT with a FLAG sequence (DF mice). This gene product is structurally unable to form a functional homodimer. Tissues were obtained from both DF mice and disease-free mice expressing wild-type with FLAG SOD1 (WF mice). Both FLAG-tagged SOD1 and cross-linking proteins were enriched and subjected to a shotgun proteomic analysis. We identified 34 proteins (or protein subunits) in DF preparations, while in WF preparations, interactions were detected with only 4 proteins.

Conclusions/Significance

These results indicate that disease-causing mutant SOD1 likely leads to inadequate protein-protein interactions. This could be an early and crucial process in the pathogenesis of FALS.     

RAD18 Activates the G2/M Checkpoint through DNA Damage Signaling to Maintain Genome Integrity after Ionizing Radiation Exposure

The ubiquitin ligase RAD18 is involved in post replication repair pathways via its recruitment to stalled replication forks, and its role in the ubiquitylation of proliferating cell nuclear antigen (PCNA). Recently, it has been reported that RAD18 is also recruited to DNA double strand break (DSB) sites, where it plays novel functions in the DNA damage response induced by ionizing radiation (IR). This new role is independent of PCNA ubiquitylation, but little is known about how RAD18 functions after IR exposure. Here, we describe a role for RAD18 in the IR-induced DNA damage signaling pathway at G2/M phase in the cell cycle. Depleting cells of RAD18 reduced the recruitment of the DNA damage signaling factors ATM, γH2AX, and 53BP1 to foci in cells at the G2/M phase after IR exposure, and attenuated activation of the G2/M checkpoint. Furthermore, depletion of RAD18 increased micronuclei formation and cell death following IR exposure, both in vitro and in vivo. Our data suggest that RAD18 can function as a mediator for DNA damage response signals to activate the G2/M checkpoint in order to maintain genome integrity and cell survival after IR exposure.     

Inducible ablation of melanopsin-expressing retinal ganglion cells reveals their central role in non-image forming visual responses

Rod/cone photoreceptors of the outer retina and the melanopsin-expressing retinal ganglion cells (mRGCs) of the inner retina mediate non-image forming visual responses including entrainment of the circadian clock to the ambient light, the pupillary light reflex (PLR), and light modulation of activity. Targeted deletion of the melanopsin gene attenuates these adaptive responses with no apparent change in the development and morphology of the mRGCs. Comprehensive identification of mRGCs and knowledge of their specific roles in image-forming and non-image forming photoresponses are currently lacking. We used a Cre-dependent GFP expression strategy in mice to genetically label the mRGCs. This revealed that only a subset of mRGCs express enough immunocytochemically detectable levels of melanopsin. We also used a Cre-inducible diphtheria toxin receptor (iDTR) expression approach to express the DTR in mRGCs. mRGCs develop normally, but can be acutely ablated upon diphtheria toxin administration. The mRGC-ablated mice exhibited normal outer retinal function. However, they completely lacked non-image forming visual responses such as circadian photoentrainment, light modulation of activity, and PLR. These results point to the mRGCs as the site of functional integration of the rod/cone and melanopsin phototransduction pathways and as the primary anatomical site for the divergence of image-forming and non-image forming photoresponses in mammals.