TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution.

R-loops are three-stranded, RNA-DNA hybrid, nucleic acid structures produced due to inappropriate processing of newly transcribed RNA or transcription-replication collision (TRC). Although R-loops are important for many cellular processes, their accumulation causes genomic instability and malignant diseases, so these structures are tightly regulated. It was recently reported that R-loop accumulation is resolved by methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation under physiological conditions. However, it remains unclear how R-loops in the genome are recognized and induce resolution signals. Here, we demonstrate that tonicity-responsive enhancer binding protein (TonEBP) recognizes R-loops generated by DNA damaging agents such as ultraviolet (UV) or camptothecin (CPT). Single-molecule imaging and biochemical assays reveal that TonEBP preferentially binds a R-loop via both 3D collision and 1D diffusion along DNA in vitro. In addition, we find that TonEBP recruits METTL3 to R-loops through the Rel homology domain (RHD) for m6A RNA methylation. We also show that TonEBP recruits RNaseH1 to R-loops through a METTL3 interaction. Consistent with this, TonEBP or METTL3 depletion increases R-loops and reduces cell survival in the presence of UV or CPT. Collectively, our results reveal an R-loop resolution pathway by TonEBP and m6A RNA methylation by METTL3 and provide new insights into R-loop resolution processes.

The effect of using vapocoolant spray for pain reduction in arteriovenous fistula cannulation among patients undergoing hemodialysis: A randomized control trial.

AIM: The aim of this study was to assess the effects of alkane vapocoolant spray in reducing pain during arteriovenous access cannulation in adult patients undergoing hemodialysis. BACKGROUND: Developing and applying various approaches for pain relief remain important responsibility for nurses. METHODS: This study was designed as an experimental study with a cross-over design. Thirty-eight patients on hemodialysis volunteered to undergo cannulation of their arteriovenous access, after the application of vapocoolant or placebo spray or no intervention. Subjective and objective pain levels were assessed, along with various physiological parameters pre- and post-cannulation. RESULTS: Statistically significant between-group differences were observed in subjective pain at the venous (F = 4.97, p = 0.009) and arterial (F = 6.91, p = 0.001) puncture sites. The mean arterial site subjective pain scores were 4.45 ± 1.31 (no treatment), 4.04 ± 1.82 (placebo), and 2.98 ± 1.53 (vapocoolant spray). Significant between-group differences were observed in objective pain scores during arteriovenous fistula puncture (F = 5.13, p = 0.007). The mean objective pain scores after arteriovenous fistula puncture were 3.25 ± 2.66 (no treatment), 2.17 ± 1.76 (placebo), and 1.78 ± 1.66 (vapocoolant spray). Post-hoc test results indicated vapocoolant spray application was associated with significantly lower pain scores than no treatment or placebo. Patient blood pressure and heart rate recordings did not differ among the interventions. CONCLUSION: Vapocoolant application was significantly more effective than the placebo or no treatment in reducing the pain of cannulation in adult patients undergoing hemodialysis.

Functional cross talk between the Fanconi anemia and ATRX/DAXX histone chaperone pathways promotes replication fork recovery.

Fanconi anemia (FA) is a chromosome instability syndrome characterized by increased cancer predisposition. Specifically, the FA pathway functions to protect genome stability during DNA replication. The central FA pathway protein, FANCD2, locates to stalled replication forks and recruits homologous recombination (HR) factors such as CtBP interacting protein (CtIP) to promote replication fork restart while suppressing new origin firing. Here, we identify alpha-thalassemia retardation syndrome X-linked (ATRX) as a novel physical and functional interaction partner of FANCD2. ATRX is a chromatin remodeler that forms a complex with Death domain-associated protein 6 (DAXX) to deposit the histone variant H3.3 into specific genomic regions. Intriguingly, ATRX was recently implicated in replication fork recovery; however, the underlying mechanism(s) remained incompletely understood. Our findings demonstrate that ATRX forms a constitutive protein complex with FANCD2 and protects FANCD2 from proteasomal degradation. ATRX and FANCD2 localize to stalled replication forks where they cooperate to recruit CtIP and promote MRE11 exonuclease-dependent fork restart while suppressing the firing of new replication origins. Remarkably, replication restart requires the concerted histone H3 chaperone activities of ATRX/DAXX and FANCD2, demonstrating that coordinated histone H3 variant deposition is a crucial event during the reinitiation of replicative DNA synthesis. Lastly, ATRX also cooperates with FANCD2 to promote the HR-dependent repair of directly induced DNA double-stranded breaks. We propose that ATRX is a novel functional partner of FANCD2 to promote histone deposition-dependent HR mechanisms in S-phase.

Hepatic Involvement of Diffuse Large B-Cell Lymphoma Mimicking Antinuclear Antibody-Negative Autoimmune Hepatitis Diagnosed by Liver Biopsy.

Non-Hodgkin's lymphoma (NHL) is the fifth most common hematologic disorder in the United States, and its prevalence has been rising in Western countries. Among the subtypes of NHL, diffuse large B-cell lymphoma (DLBCL) mostly involves the lymph nodes, stomach, and gastrointestinal tract, whereas hepatic involvement of DLBCL is rare. On serologic testing, elevated immunoglobulin G (IgG) levels can be observed in DLBCL; however, elevated IgG levels are mainly observed in autoimmune hepatitis. A targeted-lesion biopsy is required for the diagnosis of DLBCL. Based on a final diagnosis, the patient was treated with rituximab-based chemotherapy, including cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy (R-CHOP). Herein, we report a case of DLBCL mimicking antinuclear antibody-negative autoimmune hepatitis, which was finally diagnosed as DLBCL involving the liver, and was confirmed by liver biopsy.

FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response.

Fanconi anemia (FA) is an inherited cancer predisposition syndrome characterized by cellular hypersensitivity to DNA interstrand crosslinks (ICLs). To repair these lesions, the FA proteins act in a linear hierarchy: following ICL detection on chromatin, the FA core complex monoubiquitinates and recruits the central FANCI and FANCD2 proteins that subsequently coordinate ICL removal and repair of the ensuing DNA double-stranded break by homology-dependent repair (HDR). FANCD2 also functions during the replication stress response by mediating the restart of temporarily stalled replication forks thereby suppressing the firing of new replication origins. To address if FANCI is also involved in these FANCD2-dependent mechanisms, we generated isogenic FANCI-, FANCD2- and FANCI:FANCD2 double-null cells. We show that FANCI and FANCD2 are partially independent regarding their protein stability, nuclear localization and chromatin recruitment and contribute independently to cellular proliferation. Simultaneously, FANCD2-but not FANCI-plays a major role in HDR-mediated replication restart and in suppressing new origin firing. Consistent with this observation, deficiencies in HDR-mediated DNA DSB repair can be overcome by stabilizing RAD51 filament formation in cells lacking functional FANCD2. We propose that FANCI and FANCD2 have partially non-overlapping and possibly even opposing roles during the replication stress response.

Pacemaker-neuron-dependent disturbance of the molecular clockwork by a Drosophila CLOCK mutant homologous to the mouse Clock mutation.

Circadian clocks are composed of transcriptional/translational feedback loops (TTFLs) at the cellular level. In Drosophila TTFLs, the transcription factor dCLOCK (dCLK)/CYCLE (CYC) activates clock target gene expression, which is repressed by the physical interaction with PERIOD (PER). Here, we show that amino acids (AA) 657-707 of dCLK, a region that is homologous to the mouse Clock exon 19-encoded region, is crucial for PER binding and E-box-dependent transactivation in S2 cells. Consistently, in transgenic flies expressing dCLK with an AA657-707 deletion in the Clock (Clk(out)) genetic background (p{dClk-Δ};Clk(out)), oscillation of core clock genes' mRNAs displayed diminished amplitude compared with control flies, and the highly abundant dCLKΔ657-707 showed significantly decreased binding to PER. Behaviorally, the p{dClk-Δ};Clk(out) flies exhibited arrhythmic locomotor behavior in the photic entrainment condition but showed anticipatory activities of temperature transition and improved free-running rhythms in the temperature entrainment condition. Surprisingly, p{dClk-Δ};Clk(out) flies showed pacemaker-neuron-dependent alterations in molecular rhythms; the abundance of dCLK target clock proteins was reduced in ventral lateral neurons (LNvs) but not in dorsal neurons (DNs) in both entrainment conditions. In p{dClk-Δ};Clk(out) flies, however, strong but delayed molecular oscillations in temperature cycle-sensitive pacemaker neurons, such as DN1s and DN2s, were correlated with delayed anticipatory activities of temperature transition. Taken together, our study reveals that the LNv molecular clockwork is more sensitive than the clockwork of DNs to dysregulation of dCLK by AA657-707 deletion. Therefore, we propose that the dCLK/CYC-controlled TTFL operates differently in subsets of pacemaker neurons, which may contribute to their specific functions.

Magnetic resonance imaging based anatomical assessment of tongue impairment due to amyotrophic lateral sclerosis: A preliminary study.

Amyotrophic Lateral Sclerosis (ALS) is a neurological disorder, which impairs tongue function for speech and swallowing. A widely used Diffusion Tensor Imaging (DTI) analysis pipeline is employed for quantifying differences in tongue fiber myoarchitecture between controls and ALS patients. This pipeline uses both high-resolution magnetic resonance imaging (hMRI) and DTI. hMRI is used to delineate tongue muscles, while DTI provides indices to reveal fiber connectivity within and between muscles. The preliminary results using five controls and two patients show quantitative differences between the groups. This work has the potential to provide insights into the detrimental effects of ALS on speech and swallowing.

Phosphorylation of a central clock transcription factor is required for thermal but not photic entrainment.

Transcriptional/translational feedback loops drive daily cycles of expression in clock genes and clock-controlled genes, which ultimately underlie many of the overt circadian rhythms manifested by organisms. Moreover, phosphorylation of clock proteins plays crucial roles in the temporal regulation of clock protein activity, stability and subcellular localization. dCLOCK (dCLK), the master transcription factor driving cyclical gene expression and the rate-limiting component in the Drosophila circadian clock, undergoes daily changes in phosphorylation. However, the physiological role of dCLK phosphorylation is not clear. Using a Drosophila tissue culture system, we identified multiple phosphorylation sites on dCLK. Expression of a mutated version of dCLK where all the mapped phospho-sites were switched to alanine (dCLK-15A) rescues the arrythmicity of Clk(out) flies, yet with an approximately 1.5 hr shorter period. The dCLK-15A protein attains substantially higher levels in flies compared to the control situation, and also appears to have enhanced transcriptional activity, consistent with the observed higher peak values and amplitudes in the mRNA rhythms of several core clock genes. Surprisingly, the clock-controlled daily activity rhythm in dCLK-15A expressing flies does not synchronize properly to daily temperature cycles, although there is no defect in aligning to light/dark cycles. Our findings suggest a novel role for clock protein phosphorylation in governing the relative strengths of entraining modalities by adjusting the dynamics of circadian gene expression.

Sources of strength-training information and strength-training behavior among Japanese older adults.

The promotion of strength training is now recognized as an important component of public health initiatives for older adults. To develop successful communication strategies to increase strength-training behavior among older adults, the identification of effective communication channels to reach older adults is necessary. This study aimed to identify the information sources about strength training that were associated with strength-training behaviors among Japanese older adults. The participants were 1144 adults (60-74 years old) randomly sampled from the registry of residential addresses. A cross-sectional questionnaire survey was conducted. The independent variables were sources of strength-training information (healthcare providers, friends, families, radio, television, newspapers, newsletters, posters, books, magazines, booklets, the Internet, lectures, other sources), and the dependent variable was regular strength-training behavior. Logistic regression analysis was used to identify potential relationships. After adjusting for demographic factors and all other information sources, strength-training information from healthcare providers, friends, books and the Internet were positively related to regular strength-training behavior. The findings of the present study contribute to a better understanding of strength-training behavior and the means of successful communication directed at increasing strength training among older adults. The results suggest that healthcare providers, friends, books and the Internet are effective methods of communication for increasing strength-training behaviors among older adults.

Drosophila peptidyl-prolyl isomerase Pin1 modulates circadian rhythms via regulating levels of PERIOD.

In animal circadian clock machinery, the phosphorylation program of PERIOD (PER) leads to the spatio-temporal regulation of diverse PER functions, which are crucial for the maintenance of ~24-hr circadian rhythmicity. The peptidyl-prolyl isomerase PIN1 modulates the diverse functions of its substrates by inducing conformational changes upon recognizing specific phosphorylated residues. Here, we show that overexpression of Drosophila pin1, dodo (dod), lengthens the locomotor behavioral period. Using Drosophila S2 cells, we demonstrate that Dod associates preferentially with phosphorylated species of PER, which delays the phosphorylation-dependent degradation of PER. Consistent with this, PER protein levels are higher in flies overexpressing dod. Taken together, we suggest that Dod plays a role in the maintenance of circadian period by regulating PER metabolism.

Airway management in a patient with severe tracheal stenosis: bilateral superficial cervical plexus block with dexmedetomidine sedation.

A 54-year-old woman with a history of severe tracheal stenosis caused by papillary thyroid cancer with tracheal invasion was admitted for an elective surgery. A bilateral superficial cervical plexus block with 0.5 % ropivacaine 14 ml (7 ml per side) under dexmedetomidine sedation was performed, followed by tracheal dissection and endotracheal tube (ETT) insertion. The patient continued spontaneous respiration without any hypoxic event, and the bispectral index was maintained at a range of 50-80. After ETT insertion, a total thyroidectomy and tracheal resection with end-to-end anastomosis were performed under general anesthesia. The patient was transferred to the surgical intensive care unit after extubation, and she recovered without any complications.

A Candidate Single Nucleotide Polymorphism in the 3' Untranslated Region of Stearoyl-CoA Desaturase Gene for Fatness Quality and the Gene Expression in Berkshire Pigs.

Fatness qualities in pigs measured by the amount of fat deposition and composition of fatty acids (FAs) in pork have considerable effect on current breeding goals. The stearoyl-CoA desaturase (SCD) gene plays a crucial role in the conversion of saturated FAs into monounsaturated FAs (MUFAs), and hence, is among the candidate genes responsible for pig fatness traits. Here, we identified a single nucleotide polymorphism (SNP, c.*2041T>C) in the 3' untranslated region by direct sequencing focused on coding and regulatory regions of porcine SCD. According to the association analysis using a hundred of Berkshire pigs, the SNP was significantly associated with FA composition (MUFAs and polyunsaturated FAs [PUFAs]), polyunsaturated to saturated (P:S) FA ratio, n-6:n-3 FA ratio, and extent of fat deposition such as intramuscular fat and marbling (p<0.05). In addition, the SNP showed a significant effect on the SCD mRNA expression levels (p = 0.041). Based on our results, we suggest that the SCD c.*2041T>C SNP plays a role in the gene regulation and affects the fatness qualities in Berkshire pigs.

[Longitudinal change in awareness levels of Japanese exercise guidelines and physical activity].

OBJECTIVES: According to the interim report "National Health Promotion in the 21st Century (Healthy Japan 21)", the Exercise and Physical Activity Guide for Health Promotion 2006 (EPAGH2006) is an im-portant resource in the field of physical activity in Japan. A previous study (Higo & Nakamura, 2008) showed that although the awareness level of EPAGH2006 was lower than for other health pro-motion policies (e.g., Healthy Japan 21), the proportion of people who walked regularly was higher in the EPAGH2006 awareness group than in the no-awareness group. This result indicates that pro-motion of EPAGH2006 would contribute to increase in the number of physically active people. The purpose of the present study was to identify longitudinal change in awareness of EPAGH2006, and to examine the relationship with levels of physical activity. METHODS: The subjects were 1,100 Japanese adults (mean [SD], 39.8 [10.1 years) recruited from among the registrants of a Japanese social research company. This longitudinal study was conducted using online questionnaires in surveys in November 2007 (T1) and December 2008 (T2). The awareness level of EPAGH2006 was assessed with 3 choices, i.e., good understanding, awareness and no-awareness. The International Physical Activity Questionnaire short Version (Craig et al., 2003; Murase et al., 2002) was used to estimate the amount of physical activity that the subjects engaged in. The Mann-Whitney test was utilized to assess inter-group differences in changes in the amount of physical activity between the understanding and no-awareness groups. RESULTS: The proportion of those who fully understood EPAGH2006 was 1.4% at T1 and 2.2%0 at T2 and did not significantly increase. The physical activity level in the understanding group at T2 was significantly decreased compared with the no-awareness groups at both T1 and T2 (p=0.013). CONCLUSION: The lack of increase in the awareness level between the two time points suggests that dissemination of EPAGH2006 has not been effective. A more strategic approach would appear to be required. However, the results also did not indicate that the awareness level of EPAGH2006 had a positive in-fluence on physical activity. Further studies, conducted when the awareness levels improve, may elucidate the relationship between the levels of EPAGH2006 awareness and physical activity.

SDE2 integrates into the TIMELESS-TIPIN complex to protect stalled replication forks.

Protecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances its stability, thereby aiding TIM localization to replication forks and the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.

Antidepressant-like effects of ß-caryophyllene on restraint plus stress-induced depression.

Chronic stress is depressogenic by altering neurotrophic and neuroinflammatory environments of the organism. The endocannabinoid system controls cognitive and emotional responses related with stress through the interaction with endocannabinoid receptors. ß-Caryophyllene (BCP) is a CB2 agonist that exhibited anti-inflammatory, analgesic effects but minimal psychoactive effects. To test if BCP exhibits antidepressant-like action, animals were chronically restrained with additional stressors for 28 days, and BCP (25, 50, 100 mg/kg) was intraperitoneally injected once a day during the stress inflicting period. Then despair related behaviors and hippocampal expression of neurotrophic, inflammatory and cannabinoid receptor levels were measured. To test the effect of BCP on long-term depression, field potentials were measured during the application of lipopolysaccharide and low frequency stimulation. In the tail suspension test and forced swim test, chronic stress-induced despair behaviors were reduced by BCP. Also BCP improved the stress-related changes in the hippocampal expression of COX-2, BDNF, and CB2 receptor expression. In organotypic hippocampal slices, BCP reduced the lipopolysaccharide-induced intensification of the long-term depression. In conclusion, BCP improved chronic stress related behavioral and biochemical changes. These results suggest that BCP may be effective in treating depression and stress related mental illnesses.

Effects of Intraoperative Low-Dose Ketamine on Persistent Postsurgical Pain after Breast Cancer Surgery: A Prospective, Randomized, Controlled, Double-Blind Study.

BACKGROUND: Compared to acute postsurgical pain, studies regarding the role of ketamine in persistent postsurgical pain (PPSP) are limited. OBJECTIVES: The aim of this clinical trial was to test if intraoperative low-dose ketamine without postoperative infusion would reduce PPSP development after breast cancer surgery. STUDY DESIGN: We used a randomized, double-blinded, placebo study design. SETTING: This study was conducted at Pusan National University Hospital, Republic of Korea, between December 2013 and August 2016. METHODS: A total of 184 patients scheduled for breast cancer surgery were randomly assigned to either the control or ketamine group. Before skin incision, a bolus (0.5 mg/kg of ketamine or placebo), followed by a continuous infusion (0.12 mg/kg/h of ketamine or placebo), was administered until the end of the surgery. The patients were interviewed via telephone 1, 3, and 6 months after surgery. The first question was whether the patient had surgery-related pain. If answered affirmatively, questions from the Numeric Rating Scale for pain at rest (NRSr) and for coughing (NRSd) were also asked. Our primary outcome was the incidence of PPSP at 3 months after surgery. RESULTS: For PPSP analysis, 168 patients were included. The number of patients who experienced pain was significantly lower in the ketamine group at 3 months (86.9% in the control group vs 69.0% in the ketamine group, P = .005) postoperatively. However, the NRSr and NRSd did not differ between the groups throughout the follow-up. LIMITATIONS: There were no postoperative low-dose ketamine infusion groups to compare due to hospital regulations. Dosage of ketamine was too low to reduce the severity of PPSP. And by using propofol and remifentanil for anesthesia, different results can be deduced with volatile anesthetics. Data from written questionnaires would have been more specific than telephone interviews for long-term assessment. CONCLUSIONS: Though intraoperative low-dose ketamine without postoperative infusion significantly reduced the incidence of PPSP up to 3 months after breast cancer surgery, it failed to reduce clinically significant PPSP and improve patients' quality of life. KEY WORDS: Analgesia, breast cancer, chronic pain, ketamine, mastectomy, morphine, pain, postoperative, propofol.

TonEBP Regulates PCNA Polyubiquitination in Response to DNA Damage through Interaction with SHPRH and USP1.

Polyubiquitination of proliferating cell nuclear antigen (PCNA) regulates the error-free template-switching mechanism for the bypass of DNA lesions during DNA replication. PCNA polyubiquitination is critical for the maintenance of genomic integrity; however, the underlying mechanism is poorly understood. Here, we demonstrate that tonicity-responsive enhancer-binding protein (TonEBP) regulates PCNA polyubiquitination in response to DNA damage. TonEBP was recruited to DNA damage sites with bulky adducts and sequentially recruited E3 ubiquitin ligase SHPRH, followed by deubiquitinase USP1, to DNA damage sites, in correlation with the dynamics of PCNA polyubiquitination. Similarly, TonEBP was found to be required for replication fork protection in response to DNA damage. The Rel-homology domain of TonEBP, which encircles DNA, was essential for the interaction with SHPRH and USP1, PCNA polyubiquitination, and cell survival after DNA damage. The present findings suggest that TonEBP is an upstream regulator of PCNA polyubiquitination and of the DNA damage bypass pathway.

Pre-operative predictive factors for gallbladder cholesterol polyps using conventional diagnostic imaging.

AIM: To determine the clinical data that might be useful for differentiating benign from malignant gallbladder (GB) polyps by comparing radiological methods, including abdominal ultrasonography (US) and computed tomography (CT) scanning, with postoperative pathology findings. METHODS: Fifty-nine patients underwent laparoscopic cholecystectomy for a GB polyp of around 10 mm. They were divided into two groups, one with cholesterol polyps and the other with non-cholesterol polyps. Clinical features such as gender, age, symptoms, size and number of polyps, the presence of a GB stone, the radiologically measured maximum diameter of the polyp by US and CT scanning, and the measurements of diameter from postoperative pathology were recorded for comparative analysis. RESULTS: Fifteen of the 41 cases with cholesterol polyps (36.6%) were detected with US but not CT scanning, whereas all 18 non-cholesterol polyps were observed using both methods. In the cholesterol polyp group, the maximum measured diameter of the polyp was smaller by CT scan than by US. Consequently, the discrepancy between those two scanning measurements was greater than for the non-cholesterol polyp group. CONCLUSION: The clinical signs indicative of a cholesterol polyp include: (1) a polyp observed by US but not observable by CT scanning, (2) a smaller diameter on the CT scan compared to US, and (3) a discrepancy in its maximum diameter between US and CT measurements. In addition, US and the CT scan had low accuracy in predicting the polyp diameter compared to that determined by postoperative pathology.

Stimulation of osteoclastogenesis by enhanced levels of MIP-1alpha in BALB/c mice in vitro.

OBJECTIVES: We compared osteoclast (OC) formation in bone marrow-derived macrophages (BMM) from C57BL/6 (B/6) and BALB/c (B/c) mice. After stimulation of receptor activator of nuclear factor-kappaB ligand (RANKL), enhanced OC formation and higher level of macrophage inflammatory protein-1alpha (MIP-1alpha) were observed in the BMM from B/c mice. In this study, we determined whether MIP-1alpha is responsible for stimulated OC formation in the BMM. MATERIALS AND METHODS: OC formation was evaluated in BMM. Expression of MIP-1alpha during OC formation was analyzed at the mRNA and protein levels. Apoptosis of mature OCs was evaluated by observing the degradation of DNA. Activation of nuclear factor-kappaB (NF-kappaB) was measured by electrophoretic mobility shift assay. RESULTS: After stimulation by RANKL expression of MIP-1alpha at the mRNA and protein levels was much higher in BMM from B/c mice than in BMM from B/6 mice. Transcripts of the MIP-1alpha receptors, CCR1 and CCR5, were present at similar levels in unstimulated BMM of the two strains. Blockade of MIP-1alpha inhibited OC formation, and exogenously added MIP-1alpha stimulated it in RANKL-stimulated BMM. MIP-1alpha affected not only the early precursors but also mature OCs. It prevented apoptosis of mature OCs by activating NF-kappaB, and the effect of RANKL on survival was dependent on its ability to induce MIP-1alpha. CONCLUSIONS: MIP-1alpha, induced by RANKL during OC differentiation, increases OC formation by acting on OC progenitor cells, and prolongs survival of mature OC via signaling through NF-kappaB. The enhanced OC formation in BMM from B/c mice could be due to, at least in part, to their higher levels of MIP-1alpha.