ZFAND5/ZNF216 is an activator of the 26S proteasome that stimulates overall protein degradation.

ZFAND5/ZNF216, a member of the zinc finger AN1-type domain family, is abundant in heart and brain, but is induced in skeletal muscle during atrophy (although not in proteotoxic stress). Because mice lacking ZFAND5 exhibit decreased atrophy, a role in stimulating protein breakdown seemed likely. Addition of recombinant ZFAND5 to purified 26S proteasomes stimulated hydrolysis of ubiquitinated proteins, short peptides, and ATP. Mutating its C-terminal AN1 domain abolished the stimulation of proteasomal peptidase activity. Mutating its N-terminal zinc finger A20 domain, which binds ubiquitin chains, prevented the enhanced degradation of ubiquitinated proteins without affecting peptidase activity. Mouse embryonic fibroblast (MEF) cells lacking ZFAND5 had lower rates of protein degradation and proteasomal activity than WT MEFs. ZFAND5 addition to cell lysates stimulated proteasomal activity and protein degradation. Unlike other proteasome regulators, ZFAND5 enhances multiple 26S activities and overall cellular protein breakdown.

A Ferric-Peroxo Intermediate in the Oxidation of Heme by IsdI.

The canonical heme oxygenases (HOs) catalyze heme oxidation via a heme-bound hydroperoxo intermediate that is stabilized by a water cluster at the active site of the enzyme. In contrast, the hydrophobic active site of IsdI, a heme-degrading enzyme from Staphylococcus aureus, lacks a water cluster and is expected to oxidize heme by an alternative mechanism. Reaction of the IsdI-heme complex with either H2O2 or m-chloroperoxybenzoic acid fails to produce a specific oxidized heme iron intermediate, suggesting that ferric-hydroperoxo or ferryl derivatives of IsdI are not involved in the catalytic mechanism of this enzyme. IsdI lacks a proton-donating group in the distal heme pocket, so the possible involvement of a ferric-peroxo intermediate has been evaluated. Density functional theory (DFT) calculations indicate that heme oxidation involving a ferric-peroxo intermediate is energetically accessible, whereas the energy barrier for a reaction involving a ferric-hydroperoxo intermediate is too great in the absence of a proton donor. We propose that IsdI catalyzes heme oxidation through nucleophilic attack by the heme-bound peroxo species. This proposal is consistent with our previous demonstration by nuclear magnetic resonance spectroscopy that heme ruffling increases the susceptibility of the meso-carbon of heme to nucleophilic attack.

Effect of rebamipide on gastric bleeding and ulcerogenic responses induced by aspirin plus clopidogrel under stimulation of acid secretion in rats.

BACKGROUND AND AIM: We examined the prophylactic effect of rebamipide on gastric bleeding induced by the perfusion of aspirin (acetylsalicylic acid [ASA]) plus clopidogrel under the stimulation of acid secretion in rats. METHODS: Under urethane anesthesia, acid secretion was stimulated by the i.v. infusion of histamine (8 mg/kg/h), and the stomach was perfused with 25 mmol/L ASA at a rate of 0.4 mL/min. Gastric bleeding was evaluated as the concentration of hemoglobin in the perfusate. Clopidogrel (30 mg/kg) was given p.o. 24 h before the perfusion. Rebamipide (3-30 mg/kg) or other antiulcer drugs were given i.d. before the ASA perfusion. RESULTS: Slight gastric bleeding or damage was observed with the perfusion of ASA under the stimulation of acid secretion, whereas these responses were significantly increased in the presence of clopidogrel. Both omeprazole and famotidine inhibited acid secretion and prevented these responses to ASA plus clopidogrel. Rebamipide had no effect on acid secretion, but dose-dependently prevented gastric bleeding in response to ASA plus clopidogrel, with the degree of inhibition being almost equivalent to that of the antisecretory drugs, and the same effects were obtained with the gastroprotective drugs, irsogladine and teprenone. These agents also reduced the severity of gastric lesions, although the effects were less than those of the antisecretory drugs. CONCLUSIONS: These results suggest that the antiplatelet drug, clopidogrel, increases gastric bleeding induced by ASA under the stimulation of acid secretion, and the gastroprotective drug, rebamipide, is effective in preventing the gastric bleeding induced under such conditions, similar to antisecretory drugs.

Electronic properties of the highly ruffled heme bound to the heme degrading enzyme IsdI.

IsdI, a heme-degrading protein from Staphylococcus aureus, binds heme in a manner that distorts the normally planar heme prosthetic group to an extent greater than that observed so far for any other heme-binding protein. To understand better the relationship between this distinct structural characteristic and the functional properties of IsdI, spectroscopic, electrochemical, and crystallographic results are reported that provide evidence that this heme ruffling is essential to the catalytic activity of the protein and eliminates the need for the water cluster in the distal heme pocket that is essential for the activity of classical heme oxygenases. The lack of heme orientational disorder in (1)H-NMR spectra of the protein argues that the catalytic formation of ß- and δ-biliverdin in nearly equal yield results from the ability of the protein to attack opposite sides of the heme ring rather than from binding of the heme substrate in two alternative orientations.

Inactivation of the heme degrading enzyme IsdI by an active site substitution that diminishes heme ruffling.

IsdG and IsdI are paralogous heme degrading enzymes from the bacterium Staphylococcus aureus. Heme bound by these enzymes is extensively ruffled such that the meso-carbons at the sites of oxidation are distorted toward bound oxygen. In contrast, the canonical heme oxygenase family degrades heme that is bound with minimal distortion. Trp-66 is a conserved heme pocket residue in IsdI implicated in heme ruffling. IsdI variants with Trp-66 replaced with residues having less bulky aromatic and alkyl side chains were characterized with respect to catalytic activity, heme ruffling, and electrochemical properties. The heme degradation activity of the W66Y and W66F variants was approximately half that of the wild-type enzyme, whereas the W66L and W66A variants were inactive. A crystal structure and NMR spectroscopic analysis of the W66Y variant reveals that heme binds to this enzyme with less heme ruffling than observed for wild-type IsdI. The reduction potential of this variant (-96 ± 7 mV versus standard hydrogen electrode) is similar to that of wild-type IsdI (-89 ± 7 mV), so we attribute the diminished activity of this variant to the diminished heme ruffling observed for heme bound to this enzyme and conclude that Trp-66 is required for optimal catalytic activity.

Single molecule force spectroscopy reveals that iron is released from the active site of rubredoxin by a stochastic mechanism.

Metal centers in metalloproteins involve multiple metal-ligand bonds. The release of metal ions from metalloproteins can have significant biological consequences, so understanding of the mechanisms by which metal ion dissociates has broad implications. By definition, the release of metal ions from metalloproteins involves the disruption of multiple metal-ligand bonds, and this process is often accompanied by unfolding of the protein. Detailed pathways for metal ion release from metalloproteins have been difficult to elucidate by classical ensemble techniques. Here, we combine single molecule force spectroscopy and protein engineering techniques to investigate the mechanical dissociation mechanism of iron from the active site of the simplest iron-sulfur protein, rubredoxin, at the single molecule level. Our results reveal that the mechanical rupture of this simplest iron center is stochastic and follows multiple, complex pathways that include concurrent rupture of multiple ferric-thiolate bonds as well as sequential rupture of ferric-thiolate bonds that lead to the formation of intermediate species. Our results uncover the surprising complexity of the rupture process of the seemingly simple iron center in rubredoxin and provide the first unambiguous experimental evidence concerning the detailed mechanism of mechanical disruption of a metal center in its native protein environment in aqueous solution. This study opens up a new avenue to investigating the rupture mechanism of metal centers in metalloproteins with unprecedented resolution by using single molecule force spectroscopy techniques.

Hydrogen bond strength modulates the mechanical strength of ferric-thiolate bonds in rubredoxin.

It has long been recognized that hydrogen bonds formed by protein backbone amides with cysteinyl S(γ) atoms play important roles in modulating the functional and structural properties of the iron-sulfur centers in proteins. Here we use single molecule atomic force microscopy, cyclic voltammetry, and protein engineering techniques to investigate directly how the strength of N-H···S(γ) hydrogen bonds in the secondary coordination sphere affects the mechanical stability of Fe(III)-thiolate bonds of rubredoxin. Our results show that the mechanical stability of Fe(III)-thiolate bonds in rubredoxin correlates with the strength of N-H···S(γ) hydrogen bonds as reflected by the midpoint reduction potential, providing direct evidence that N-H···S(γ) hydrogen bonds play important roles in modulating the mechanical and kinetic properties of the Fe(III)-thiolate bonds of iron-sulfur proteins and corroborating the important roles of the protein environment in tuning the properties of metal-thiolate bonds.

BAG3 and Hsc70 interact with actin capping protein CapZ to maintain myofibrillar integrity under mechanical stress.

RATIONALE: A homozygous disruption or genetic mutation of the bag3 gene, a member of the Bcl-2-associated athanogene (BAG) family proteins, causes cardiomyopathy and myofibrillar myopathy that is characterized by myofibril and Z-disc disruption. However, the detailed disease mechanism is not yet fully understood. OBJECTIVE: bag3(-/-) mice exhibit differences in the extent of muscle degeneration between muscle groups with muscles experiencing the most usage degenerating at an accelerated rate. Usage-dependent muscle degeneration suggests a role for BAG3 in supporting cytoskeletal connections between the Z-disc and myofibrils under mechanical stress. The mechanism by which myofibrillar structure is maintained under mechanical stress remains unclear. The purpose of the study is to clarify the detailed molecular mechanism of BAG3-mediated muscle maintenance under mechanical stress. METHODS AND RESULTS: To address the question of whether bag3 gene knockdown induces myofibrillar disorganization caused by mechanical stress, in vitro mechanical stretch experiments using rat neonatal cardiomyocytes and a short hairpin RNA-mediated gene knockdown system of the bag3 gene were performed. As expected, mechanical stretch rapidly disrupts myofibril structures in bag3 knockdown cardiomyocytes. BAG3 regulates the structural stability of F-actin through the actin capping protein, CapZß1, by promoting association between Hsc70 and CapZß1. BAG3 facilitates the distribution of CapZß1 to the proper location, and dysfunction of BAG3 induces CapZ ubiquitin-proteasome-mediated degradation. Inhibition of CapZß1 function by overexpressing CapZß2 increased myofibril vulnerability and fragmentation under mechanical stress. On the other hand, overexpression of CapZß1 inhibits myofibrillar disruption in bag3 knockdown cells under mechanical stress. As a result, heart muscle isolated from bag3(-/-) mice exhibited myofibrillar degeneration and lost contractile activity after caffeine contraction. CONCLUSIONS: These results suggest novel roles for BAG3 and Hsc70 in stabilizing myofibril structure and inhibiting myofibrillar degeneration in response to mechanical stress. These proteins are possible targets for further research to identify therapies for myofibrillar myopathy or other degenerative diseases.

N-terminal short fragment of TUP1 confers resistance to 5-bromodeoxyuridine in the yeast Saccharomyces cerevisiae.

Small molecules that exhibit biological activity have contributed to the understanding of the molecular mechanisms of various biological phenomena. 5-Bromodeoxyuridine (BrdU) is a thymidine analogue that modulates various biological phenomena such as cellular differentiation and cellular senescence in cultured mammalian cells. Although BrdU is thought to function through changing chromatin structure and gene expression, its precise molecular mechanisms are not understood. To study the molecular mechanism for the action of BrdU, we have employed the yeast Saccharomyces cerevisiae as a model system, and screened multi-copy suppressor genes that confer resistance to BrdU. Our genetic screen has revealed that expression of the N-terminal short fragment of TUP1, and also disruption of HDA1 or HOS1, histone deacetylases that interact with TUP1, conferred resistance to BrdU. These results suggest the implication of the chromatin proteins in the function of BrdU, and would provide novel clues to answer the old question of how BrdU modulates various biological phenomena.

Effect of pedestrian physique differences on head injury prediction in car-to-pedestrian accidents using deep learning.

OBJECTIVE: The aim of this study is to identify the effects of pedestrian physique differences on head injury prediction in car-to-pedestrian accidents via deep learning. METHODS: A series of parametric studies was carried out using a family car finite element model and MADYMO pedestrian models (AM50, AF05, 6YO). The car model was developed and tuned by 11 impact tests. The initial gaits for the pedestrian models were obtained from volunteer experiments to reproduce 420 pre-crash reactions. Furthermore, by factoring the pedestrian models (3 types), pedestrian directions (2 each), impact positions (3 each), and car velocities (6 levels) with the pre-crash parameters, a total of 45,360 car-to-pedestrian impact simulations were performed. After the simulations, image datasets were created by labeling the pedestrian collision images with head injury criteria of 15 ms (HIC) and dividing the images into training and test data based on model type. Next, deep learning was conducted using the training dataset to obtain trained models. Finally, the effects of pedestrian physique differences on head injury predictions were investigated based on the accuracy of each trained model for test data. RESULTS: The results indicate that the head impact area and the amount of pedestrian information in the image differ depending on the pedestrian models. In head injury prediction with deep learning, AF05 showed the highest prediction accuracy (93.25%), followed by AM50 (90.61%) and 6YO (88.29%). These results using deep learning show that pedestrian physique differences affect the head injury prediction accuracies by 2.32-4.96 points. CONCLUSIONS: Based on the prediction results of the trained models that learned the relationships between the pedestrian collision images and HIC from simulations, we demonstrated the desirable performance of deep learning methods in head injury prediction for adult men, women with small physique, and children. Furthermore, our results confirmed the effect of pedestrian physique differences on the injury prediction accuracy.

Efficacy of FGF-2 in Periodontal Regeneration in a Case of Severe Intrabony Defect and Furcation Involvement With 15-Month Follow-Up.

INTRODUCTION: Several studies have demonstrated that basic fibroblast growth factor (FGF-2) is one of the most effective growth factors for periodontal regeneration. The Ministry of Health, Labor and Welfare in Japan have approved 0.3% human recombinant FGF-2 for periodontal regeneration, and it has been commercially available since 2016. In this case report, a patient was treated with this periodontal regenerative medicine and demonstrated success at 15-month follow-up, as confirmed by dental X-ray and on cone-beam computed tomography (CBCT). CASE PRESENTATION: A 42-year-old woman with a one by two walled intrabony defect and Class III furcation involvement in tooth #19, and Class II furcation involvement in tooth #18 (lingual) underwent periodontal regenerative surgery with FGF-2 without any bone graft materials. Favorable clinical and radiographic outcomes were noted 15 months after the procedure. The vertical bone defect in tooth #19 showed a clinical attachment level gain of 8 mm. Moreover, CBCT analysis revealed considerable new bone formation in the Class II furcation involvement in tooth #18 and limited bone formation in the Class III furcation involvement in tooth #19. CONCLUSIONS: This case report indicates that FGF-2 showed a positive outcome in terms of periodontal regeneration in a case of one by two wall intrabony defects with Class III furcation involvement. A complete recovery of Class II furcation involvement was observed without artificial bone graft materials.

A phase III, 52-week, open-label study to evaluate the safety and efficacy of 5% sofpironium bromide (BBI-4000) gel in Japanese patients with primary axillary hyperhidrosis.

A long-term study was conducted in Japanese patients with primary axillary hyperhidrosis who completed the preceding 6-week phase III, confirmatory study of 5% sofpironium bromide gel (hereinafter referred to as sofpironium) to evaluate the safety and efficacy of 52-week treatment with sofpironium. In the long-term study, 185 patients who completed the confirmatory study (94 and 91 patients in the vehicle and sofpironium groups, respectively) started to receive sofpironium (switching and extension groups, respectively), and all these patients were included in both the full analysis set (FAS) and the safety analysis set (SAF). In the FAS, there were more females than males (73.0% vs. 27.0%), and median age was 38.0 years. A total of 161 patients (86 and 75 patients in the switching and extension groups, respectively) completed the study at week 52. The proportions of patients with hyperhidrosis disease severity score of 1 or 2 and a 50% or more reduction in total gravimetric weight of sweat were 57.4% in the switching group and 58.2% in the extension group at week 52. The proportions of patients who achieved this efficacy end-point in the long-term study were similar to that (53.9%) in the sofpironium group in the confirmatory study. In the SAF, the incidences of adverse events (AEs) were 80.9% in the switching group and 83.5% in the extension group, and the incidences of adverse drug reactions were 39.4% and 45.1%, respectively. AEs that occurred in at least 20% of patients in both treatment groups were application site dermatitis (25.5% and 33.0%, respectively) and nasopharyngitis (31.9% and 23.1%, respectively). Reported AEs were generally mild, and there were no deaths. Serious AEs occurred in three patients, but none were considered related to the study drug. In this study, the efficacy of sofpironium was maintained during 52-week treatment, and no new safety risk was observed.

A phase 3, multicenter, randomized, double-blind, vehicle-controlled, parallel-group study of 5% sofpironium bromide (BBI-4000) gel in Japanese patients with primary axillary hyperhidrosis.

A phase 3 study was conducted to verify the efficacy and safety of 5% sofpironium bromide (BBI-4000) gel (hereinafter referred to as sofpironium) administrated for 6 weeks in Japanese patients with primary axillary hyperhidrosis. The primary efficacy end-point was the proportion of patients who satisfied both criteria of a Hyperhidrosis Disease Severity Score (HDSS) of 1 or 2 at the end of 6-week treatment and a 50% or more reduction in total gravimetric weight of sweat at the end of treatment relative to baseline. A total of 281 patients were randomized to receive 5% sofpironium (141 patients) or vehicle (140 patients), and all patients were included in the full analysis set (FAS). In the FAS, 70.1% of patients were female, and the median age was 35.0 years. The proportion of patients who achieved the primary efficacy end-point was 53.9% in the sofpironium group and 36.4% in the vehicle group, with a statistically significant difference of 17.5% (95% confidence interval, 6.02-28.93) between these two groups (P = 0.003). The incidence of adverse events was 44.0% in the sofpironium group and 30.7% in the vehicle group, and the incidence of adverse drug reactions was 16.3% in the sofpironium group and 5.0% in the vehicle group. Reported adverse events were generally mild or moderate in severity. In the sofpironium group, common events (incidence, ≥5%) were nasopharyngitis (14.2%) and dermatitis/erythema at the application site (8.5%/5.7%), with no serious adverse events reported. This study demonstrated the efficacy and safety of 5% sofpironium.

BAG3 directly associates with guanine nucleotide exchange factor of Rap1, PDZGEF2, and regulates cell adhesion.

BAG3, a member of the Hsc70 binding co-chaperone BAG-family proteins, has critical roles in regulating actin organization, cell adhesion, cell motility and tumor metastasis. The PDZ domain containing guanine nucleotide exchange factor 2 (PDZGEF2) was cloned as a BAG3-interacting protein. PDZGEF2 induces activation of Rap1 and increases integrin-mediated cell adhesion. The PPDY motif at the C-terminus of PDZGEF2 binds to the WW domain of BAG3 in vitro and in vivo. BAG3 deletion mutant lacking the WW domain lose its cell adhesion and motility activity. Gene knockdown of PDZGEF2 leads to the loss of cell adhesion on fibronectin-coated plates while BAG3 overexpression increases cell adhesion in Cos7 cells, but not in PDZGEF2 gene knockdown cells indicating that PDZGEF2 is a critical partner for BAG3 in regulating cell adhesion.

Identification of genes that affect sensitivity to 5-bromodeoxyuridine in the yeast Saccharomyces cerevisiae.

Small molecules that exhibit biological effects have been successfully used to study various biological phenomena. 5-Bromodeoxyuridine (BrdU) is a thymidine analog that affects various biological processes, such as cellular differentiation and cellular senescence in cultured mammalian cells. Although BrdU is thought to modulate these phenomena by changing chromatin structure and gene expression, the molecular mechanisms for the action of BrdU are not understood well. To analyze the molecular mechanisms of BrdU with genetic methods, we used the yeast Saccharomyces cerevisiae as a model. Our genetic screening has revealed that a defect in MPT5/HTR1/UTH4/PUF5 led to an increased sensitivity to BrdU, and that overexpression of VHT1 or SDT1 led to resistance to BrdU. The increased sensitivity to BrdU caused by a defect in MPT5 was suppressed by a mutation in SIR2, SIR3, or SIR4, which is involved in chromatin silencing and transcriptional repression. These findings suggest that chromatin silencing proteins are involved in the modulation of the cellular phenomena by BrdU, and would provide clues to answer the old question of how BrdU affects various biological phenomena.

Application of Deep Learning Methods for Pedestrian Collision Detection Using Dashcam Videos.

The goal of this study is to clarify the usefulness of deep learning methods for pedestrian collision detection using dashcam videos for advanced automatic collision notification, focusing on pedestrians, as they make up the highest number of traffic fatalities in Japan. First, we created a dataset for deep learning from dashcam videos. A total of 78 dashcam videos of pedestrian-to-automobile accidents were collected from a video hosting website and from the Japan Automobile Research Institute (JARI). Individual frames were selected from the video data amounting to a total of 1,212 still images, which were added to our dataset with class and location information. This dataset was then divided to create training, validation, and test datasets. Next, deep learning was performed based on the training dataset to learn the features of pedestrian collision images, which are images that capture pedestrian behavior at the time of the collision. Pedestrian collision detection performance of the trained model was evaluated as the percentage of correct predictions of pedestrian collisions in image data according to varied test sets with different combinations of characteristics. Our results for the proposed method show high-precision collision detection for daytime, clear pedestrian wrap trajectory accident data, including accurate detection of pedestrian collision location information. However, nighttime, unclear accident data resulted in false detection or no detection. Reduction of exposure value and resolution was confirmed to reduce detection rate. The results of the present study suggest the possibility of pedestrian collision detection by deep learning using dashcam videos.

Electron transfer from cytochrome c to cupredoxins.

Electron transfer (ET) through and between proteins is a fundamental biological process. The activation energy for an ET reaction depends upon the Gibbs energy change upon ET (DeltaG(0)) and the reorganization energy. Here, we characterized ET from Pseudomonas aeruginosa cytochrome c(551) (PA) and its designed mutants to cupredoxins, Silene pratensis plastocyanin (PC) and Acidithiobacillus ferrooxidans rusticyanin (RC), through measurement of pseudo-first-order ET rate constants (k(obs)). The influence of the DeltaG (0) value for ET from PA to PC or RC on the k(obs) value was examined using a series of designed PA proteins exhibiting a variety of E (m) values, which afford the DeltaG (0) variation range of 58-399 meV. The plots of the k(obs) values obtained against the DeltaG(0) values for both PA-PC and PA-RC redox pairs could be fitted well with a single Marcus equation. We have shown that the ET activity of cytochrome c can be controlled by tuning the E(m) value of the protein through the substitution of amino acid residues located in hydrophobic-core regions relatively far from the redox center. These findings provide novel insights into the molecular design of cytochrome c, which could be utilized for controlling its ET activity by means of protein engineering.

Correlation between the stability and redox potential of three homologous cytochromes c from two thermophiles and one mesophile.

The stability of the oxidized and reduced forms of three homologous cytochromes c from two thermophiles and one mesophile was systematically monitored by means of Soret absorption measurements in the presence of various concentrations of a denaturant, guanidine thiocyanate, at pH 7.0 at 25 degrees C. Thermophilic Hydrogenobacter thermophilus cytochrome c(552) was the most stable in both redox states, followed by moderately thermophilic Hydrogenophilus thermoluteolus cytochrome c(552), and then mesophilic Pseudomonas aeruginosa cytochrome c(551). Further stability and electrochemical analysis of the three proteins and the reciprocal variants, which exhibited a different hydrophobic interaction with the heme, showed that the one with the higher stability in both redox states had the lower redox potential. Consequently, these cytochromes c probably adapted to the cellular environments of the original bacteria with correlated stability and redox potential constraints, which are in part regulated by the hydrophobicity around the heme.

Bag1 is essential for differentiation and survival of hematopoietic and neuronal cells.

Bag1 is a cochaperone for the heat-shock protein Hsp70 that interacts with C-Raf, B-Raf, Akt, Bcl-2, steroid hormone receptors and other proteins. Here we use targeted gene disruption in mice to show that Bag1 has an essential role in the survival of differentiating neurons and hematopoietic cells. Cells of the fetal liver and developing nervous system in Bag1-/- mice underwent massive apoptosis. Lack of Bag1 did not disturb the primary function of Akt or Raf, as phosphorylation of the forkhead transcription factor FKHR and activation of extracellular signal-regulated kinase (Erk)-1/2 were not affected. However, the defect was associated with the disturbance of a tripartite complex formed by Akt, B-Raf and Bag1, in addition to the absence of Bad phosphorylation at Ser136. We also observed reduced expression of members of the inhibitor of apoptosis (IAP) family. Our data show that Bag1 is a physiological mediator of extracellular survival signals linked to the cellular mechanisms that prevent apoptosis in hematopoietic and neuronal progenitor cells.

The requirement of Artemis in double-strand break repair depends on the type of DNA damage.

Artemis is a recently identified factor involved in V(D)J recombination and nonhomologous end joining (NHEJ) of DNA double-strand break (DSB) repair. Here, we performed targeted disruption of the Artemis gene (ARTEMIS) in the human pre-B cell line Nalm-6. Unexpectedly, we found that cells lacking Artemis exhibit increased sensitivity to low doses, but not high doses, of ionizing radiation. We also show that ARTEMIS-deficient cells are hypersensitive to the topoisomerase II inhibitor etoposide, but to a much lesser extent than cells lacking DNA ligase IV, a critical component of NHEJ. Unlike DNA ligase IV-deficient cells, ARTEMIS-deficient cells were not hypersensitive to ICRF-193, a topoisomerase II inhibitor that does not stabilize topoisomerase II-DNA cleavable complexes. Collectively, our results suggest that Artemis only partially participates in the NHEJ pathway to repair DSBs in human somatic cells.