DNA Break Mapping Reveals Topoisomerase II Activity Genome-Wide

Genomic DNA is under constant assault by endogenous and exogenous DNA damaging agents. DNA breakage can represent a major threat to genome integrity but can also be necessary for genome function. Here we present approaches to map DNA double-strand breaks (DSBs) and single-strand breaks (SSBs) at the genome-wide scale by two methods called DSB- and SSB-Seq, respectively. We tested these methods in human colon cancer cells and validated the results using the Topoisomerase II (Top2)-poisoning agent etoposide (ETO). Our results show that the combination of ETO treatment with break-mapping techniques is a powerful method to elaborate the pattern of Top2 enzymatic activity across the genome.     

Leptin is a physiologically important regulator of food intake

OBJECTIVE: These studies were designed to test the hypothesis that endogenous leptin, acting within the brain plays a physiologically important role in the control of food intake in lean rats. DESIGN: Antibodies directed against mouse leptin were raised in rabbits. The purified IgG fractions prepared from pre-immune and immune sera were injected into the right lateral ventricle of lean Sprague-Dawley rats and obese Zucker fatty fa/fa rats. Changes in food intake were measured over the following 20 h period. RESULTS: The anti-leptin antibodies recognized a major epitope in the C-terminal region of the leptin molecule. The antibodies bound both mouse and rat leptin with high affinity, but did not bind human leptin, or a selected range of other hormones and neurotransmitters known to affect food intake. In competition studies, the binding of mouse, but not human leptin to the human Ob-Rb receptor was prevented by the antibodies. This indicates that the antibodies can block the action of leptin by preventing its binding to the ob-Rb receptor. Injection of the anti-leptin antibodies into the brain of lean rats led to an increase in food intake during the first hour after injection which was not compensated during the following 19 h period. Injection of the anti-leptin antibodies did not affect food intake in Zucker fatty fa/fa rats which express an abnormal ob-Rb receptor. CONCLUSION: Endogenous leptin acting within the brain plays a physiologically important role in the control of food intake in lean rats.     

A variant human IgG1-Fc mediates improved ADCC

Ribosome display was applied to the Fc region of human immunoglobulin G (IgG1) to select for improved binding to human FcγRIIIa, the receptor expressed on human natural killer cells that mediates antibody-dependent cellular cytotoxicity (ADCC). A library of human Fcγ1 variants was generated using error-prone polymerase chain reaction, and subjected to multiple rounds of ribosome display selection against progressively decreasing concentrations of soluble human FcγRIIIa, to enrich for improved binders. Radioimmunoassay and alphascreen analyses of the aglycosylated IgG-Fc output revealed variants with improved binding to FcγRIIIa relative to wild-type IgG-Fc. Subsequent expression in human (HEK-EBNA) cells generated glycosylated IgGs with modified activity in ADCC assays. One particular variant, 125_B01 triggered enhanced ADCC (EC(50) up to four-fold reduced with increased maximal lysis) relative to wild-type antibody, having more equal levels of ADCC for each allotype (V158/F158) of FcγRIIIa. Deconvolution of individual replacements within the variant showed that improved function arose from the Phe243Leu replacement within the CH2 domain, rather than the CH3 domain replacements Thr393Ala or His433Pro. Surprisingly, the oligosaccharide profiles of 125_B01 indicated more oligosaccharide chains lacking fucose, or with bisecting N-acetylglucosamine relative to wild-type IgG1, which correlates with improved function and the replacement Phe243Leu that is a carbohydrate contact residue within the C(H)2 domain.     

All-carbon N-heterocyclic Carbene-catalyzed (3+2) Annulation using Donor-Acceptor Cyclopropanes

Donor-acceptor cyclopropanes are known to serve as dipole precursors capable of engaging in (3+2) annulations with electron-deficient π-systems. In 2013, the reaction of donor-acceptor cyclopropanes with α,β-unsaturated acyl fluorides in an all-carbon (3+2) annulation was discovered. The reaction proceeds in good yields using the IMes NHC to provide diastereomerically pure β-lactone-fused cyclopentanes bearing four contiguous stereocentres. Subsequent studies demonstrated that N-t-butyl substituted homochiral morpholinone NHCs allowed the reaction to be achieved in up to 98 % ee. In this account, a background to this reaction is introduced, along with a complete account of the strengths, limitations and challenges encountered while developing this chemistry.     

Stimulation of feeding in lean but not in obese Zucker rats by a selective neuropeptide Y Y5 receptor agonist

Obese Zucker rats are characterized by a reduced hypothalamic NPY receptor density. We tested the effects of intracerebroventricular injections of human NPY (hNPY) and [D-Trp32]NPY, a weak but selective NPY Y5 receptor agonist, on food intake in lean and obese Zucker rats. The effect of a maximal dose of hNPY (10 microg) on feeding was more pronounced in lean than in obese rats. [D-Trp32]NPY (10 microg) stimulated feeding in lean but not in obese Zucker rats. It did not affect the feeding response to hNPY, excluding the activation of an inhibitory receptor. These results are in favor of a down-regulation of the NPY 'feeding' receptor in the obese rat, which is suggested to be the Y5 subtype.     

Does blockade of angiotensin II receptors offer clinical benefits over inhibition of angiotensin-converting enzyme?

Angiotensin AT1 receptor antagonists represent a new class of drugs for the treatment of hypertension. They are specific for the renin-angiotensin system, selective for the angiotensin AT1 receptor, and act independently of the angiotensin II synthetic pathway. Blockade of the renin-angiotensin system at the receptor level should therefore be more complete. The high circulating levels of angiotensin II following angiotensin AT1 receptor blockade could be beneficial in stimulating other unblocked angiotensin receptors, especially the AT2 receptor. It has been proposed that the angiotensin AT2 receptor, which is re-expressed or up-regulated during pathological circumstances, counterbalances the effect of the stimulation of the angiotensin AT1 receptor. Through this mechanism, angiotensin AT1 antagonists may be superior to ACE inhibitors in cardiac and vascular remodelling as well as in kidney insufficiency. Long-term trials are required to demonstrate the possible clinical superiority of this new class of antihypertensive agents.     

Rumination and impaired resource allocation in depression.

Depression is characterized by a range of cognitive deficits that theorists posit are due to the resource capturing properties of rumination. The present study was designed to examine the relation between rumination and resource allocation in depression. Twenty-five depressed and 25 nondepressed participants completed a modified dual-task version of the recency-probes task, which assesses the controlled allocation of cognitive resources by comparing performance across low- and high-interference conditions. In low-interference conditions, participants performed either the recency-probes task or a tracking task, which required participants to track specific stimuli across trials (i.e., no dual-task interference). In the high-interference condition, participants performed both the recency-probes task and the tracking task, which required the controlled allocation of resources to resolve dual-task interference. Depressed participants performed significantly worse than did their nondepressed counterparts in only the high-interference condition; performance of the 2 groups was comparable in the low-interference conditions. Furthermore, the degree to which depressed participants were impaired in the high-interference condition was correlated .74 with rumination. These findings suggest that an association between rumination and impairments in resource allocation underlies the cognitive difficulties experienced by depressed individuals. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Optimization of wide-area ATM and local-Area Ethernet/FDDI networkconfigurations for high-speed telemedicine communications employingNASA's ACTS

A high data rate terrestrial and satellite network was implemented to transfer medical images and data. This article describes the optimization of the workstations and switching equipment incorporated into the network. Topics discussed include tuning of the network software, the configuration of the Sun Microsystems workstations, the FORE Systems asynchronous transfer mode switches, as well as the throughput results of two telemedicine experiments undertaken by Mayo's physician staff. The technical staff was successful in achieving the data throughput needed by the telemedicine software; particularly important was the proper determination of peak throughput and TCP window sizes to ensure optimum use of the resources available on the Sun Microsystems and Hewlett Packard workstations