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Class switch recombination (CSR) in B lymphocytes is initiated by introduction of multiple DNA double-strand breaks (DSBs) into switch (S) regions that flank immunoglobulin heavy chain (IgH) constant region exons. CSR is completed by joining a DSB in the donor S mu to a DSB in a downstream acceptor S region (e. g., S gamma 1) by end-joining. In normal cells, many CSR junctions are mediated by classical nonhomologous end-joining (C-NHEJ), which employs the Ku70/80 complex for DSB recognition and XRCC4/DNA ligase 4 for ligation. Alternative end-joining (A-EJ) mediates CSR, at reduced levels, in the absence of C-NHEJ, even in combined absence of Ku70 and ligase 4, demonstrating an A-EJ pathway totally distinct from C-NHEJ. Multiple DSBs are introduced into S mu during CSR, with some being rejoined or joined to each other to generate internal switch deletions (ISDs). In addition, S-region DSBs can be joined to other chromosomes to generate translocations, the level of which is increased by absence of a single C-NHEJ component (e. g., XRCC4). We asked whether ISD and S-region translocations occur in the complete absence of C-NHEJ (e. g., in Ku70/ligase 4 double-deficient B cells). We found, unexpectedly, that B-cell activation for CSR generates substantial ISD in both S mu and S gamma 1 and that ISD in both is greatly increased by the absence of C-NHEJ. IgH chromosomal translocations to the c-myc oncogene also are augmented in the combined absence of Ku70 and ligase 4. We discuss the implications of these findings for A-EJ in normal and abnormal DSB repair.

Background: Specialized hypothalamic systems that increase food intake might also increase ethanol intake. To test this possibility, morphine and receptor-specific opioid agonists were microinjected in the paraventricular nucleus (PVN) of rats that had learned to drink ethanol. To cross-validate the results, naloxone methiodide (m-naloxone), an opioid antagonist, was microinjected with the expectation that it would have the opposite effect of morphine and the specific opioid agonists. Methods: Sprague-Dawley rats were trained, without sugar, to drink 4 or 7% ethanol and were then implanted with chronic brain cannulas aimed at the PVN. After recovery, those drinking 7% ethanol, with food and water available, were injected with 2 doses each of morphine or m-naloxone. To test for receptor specificity, 2 doses each of the mu-receptor agonist [d-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin (DAMGO), delta-receptor agonist d-Ala-Gly-Phe-Met-NH2 (DALA), or kappa-receptor agonist U-50,488H were injected. DAMGO was also tested in rats drinking 4% ethanol without food or water available. As an anatomical control for drug reflux, injections were made 2 mm dorsal to the PVN. Results: A main result was a significant increase in ethanol intake induced by PVN injection of morphine. The opposite effect was produced by m-naloxone. The effects of morphine and m-naloxone were exclusively on intake of ethanol, even though food and water were freely available. In the analysis with specific receptor agonists, PVN injection of the delta-agonist DALA significantly increased 7% ethanol intake without affecting food or water intake. This is in contrast to the kappa-agonist U-50,488H, which decreased ethanol intake, and the mu-agonist DAMGO, which had no effect on ethanol intake in the presence or absence of food and water. In the anatomical control location 2 mm dorsal to the PVN, no drug caused any significant changes in ethanol, food, or water intake, providing evidence that the active site was close to the cannula tip. Conclusions: The delta-opioid receptor agonist in the PVN increased ethanol intake in strong preference over food and water, while the kappa-opioid agonist suppressed ethanol intake. Prior studies show that learning to drink ethanol stimulates PVN expression and production of the peptides enkephalin and dynorphin, which are endogenous agonists for the delta- and kappa-receptors, respectively. These results suggest that enkephalin via the delta-opioid system can function locally within a positive feedback circuit to cause ethanol intake to escalate and ultimately contribute to the abuse of ethanol. This is in contrast to dynorphin via the kappa-opioid system, which may act to counter this escalation. Naltrexone therapy for alcoholism may act, in part, by blocking the enkephalin-triggered positive feedback cycle.

The stress phytohormone ABA inhibits the developmental transition taking the mature embryo in the dry seed towards a young seedling. ABA also induces the accumulation of the basic leucine zipper (bZIP) transcription factor ABA-insensitive 5 (ABI5) which, apart from blocking endosperm rupture, also protects the embryo by stimulating the expression of late embryogenesis abundant (LEA) genes that conferred osmotolerance during seed maturation. It is unknown whether ABA recruits additional embryonic pathways to control early seedling growth and fitness. Here we identify gia3 (growth insensitive to ABA3), a recessive locus in Arabidopsis mediating cotyledon cellular maturation and ABA-dependent repression of cotyledon expansion and greening. Microarray studies showed that expression of the essential mid-embryogenesis gene Maternal Embryo Effect 26 (MEE26) is induced by ABA during early seedling growth in wild-type (WT) or abi5 plants but not in gia3 mutants. However, we also show that the GIA3 locus controls ABA-dependent gene expression responses that partially overlap with those controlled by ABI5. Thus, the gia3 locus identifies an additional arm of ABA signaling, distinct from that controlled by ABI5, which recruits MEE26 expression and maintains cotyledon embryonic identity. Fine mapping localized the gia3 locus within a 1Mb interval of chromosome 3, containing a large DNA insertion of a duplicated region of chromosome 2. It remains unknown at present whether gia3 phenotypes are the result of single or multiple genetic alterations.

Hepatitis C virus (HCV) is a liver-tropic pathogen with severe health consequences for infected individuals. Chronic HCV infection can progress to cirrhosis and hepatocellular carcinoma and is a leading indicator for liver transplantation. The HCV core protein is an essential component of the infectious virus particle, but many aspects of its role remain undefined. The C-terminal region of the core protein acts as a signal sequence for the E1 glycoprotein and undergoes dual processing events during infectious virus assembly. The exact C terminus of the mature, virion-associated core protein is not known. Here, we performed genetic analyses to map the essential determinants of the HCV core C-terminal region, as well as to define the minimal length of the protein that can function for infectious virus production in trans.

IFN-gamma R1 deficiency is a genetic etiology of Mendelian susceptibility to mycobacterial diseases, and includes two forms of complete recessive deficiency, with or without cell surface expression, and two forms of partial deficiency, dominant or recessive. We report here a novel form of partial and recessive Interferon gamma receptor 1 (IFN-gamma R1) deficiency, which is almost as severe as complete deficiency. The patient is homozygous for a mutation of the initiation codon (M1K). No detectable expression and function of IFN-gamma R1 were found in the patient's fibroblasts. However, IFN-gamma R1 expression was found to be impaired, but not abolished, on the EBV-transformed B cells, which could respond weakly to IFN-gamma. The mechanism underlying this weak expression involves leaky translation initiation at both non-AUG codons and the third AUG codon at position 19. It results in the residual expression of IFN-gamma R1 protein of normal molecular weight and function. The residual IFN-gamma signaling documented in this novel form of partial IFN-gamma R1 deficiency was not ubiquitous and was milder than that seen in other forms of partial IFN-gamma R1 deficiency, accounting for the more severe clinical phenotype of the patient, which was almost as severe as that of patients with complete deficiency.

The classical nonhomologous end-joining (C-NHEJ) DNA double-strand break (DSB) repair pathway employs the Ku70/80 complex (Ku) for DSB recognition and the XRCC4/DNA ligase 4 (Lig4) complex for ligation. During IgH class switch recombination (CSR) in B lymphocytes, switch (S) region DSBs are joined by C-NHEJ to form junctions either with short microhomologies (MHs; "MH-mediated" joins) or no homologies ("direct" joins). In the absence of XRCC4 or Lig4, substantial CSR occurs via "alternative" end-joining (A-EJ) that generates largely MH-mediated joins. Because upstream C-NHEJ components remain in XRCC4- or Lig4-deficient B cells, residual CSR might be catalyzed by C-NHEJ using a different ligase. To address this, we have assayed for CSR in B cells deficient for Ku70, Ku80, or both Ku70 and Lig4. Ku70- or Ku80-deficient B cells have reduced, but still substantial, CSR. Strikingly, B cells deficient for both Ku plus Lig4 undergo CSR similarly to Ku-deficient B cells, firmly demonstrating that an A-EJ pathway distinct from C-NHEJ can catalyze CSR end-joining. Ku-deficient or Ku-plus Lig4-deficient B cells are also biased toward MH-mediated CSR joins; but, in contrast to XRCC4- or Lig4-deficient B cells, generate substantial numbers of direct CSR joins. Our findings suggest that more than one form of A-EJ can function in CSR.

Ubiquitination plays an important role in many cellular processes and is implicated in many diseases. Experimental identification of ubiquitination sites is challenging due to rapid turnover of ubiquitinated proteins and the large size of the ubiquitin modifier. We identified 141 new ubiquitination sites using a combination of liquid chromatography, mass spectrometry, and mutant yeast strains. Investigation of the sequence biases and structural preferences around known ubiquitination sites indicated that their properties were similar to those of intrinsically disordered protein regions. Using a combined set of new and previously known ubiquitination sites, we developed a random forest predictor of ubiquitination sites, UbPred. The class-balanced accuracy of UbPred reached 72%, with the area under the ROC curve at 80%. The application of UbPred showed that high confidence Rsp5 ubiquitin ligase substrates and protein with very short half-lives were significantly enriched in the number of predicted ubiquitination sites. Proteome-wide prediction of ubiquitination sites in Saccharomyces cerevisiae indicated that highly ubiquitinated substrates were prevalent among transcription/enzyme regulators and proteins involved in cell cycle control. In the human proteome, cytoskeletal, cell cycle, regulatory, and cancer-associated proteins display higher extent of ubiquitination than proteins from other functional categories. We show that gain and loss of predicted ubiquitination sites may likely represent a molecular mechanism behind a number of disease-associated mutations. UbPred is available at http:// www.ubpred.org. Proteins 2010; 78:365-380. (C) 2009 Wiley-Liss, Inc.

Recent work reveals that the innate immune system is able to recognize self-targets and initiate an inflammatory response similar to that of pathogens. One novel example of this innate autoimmunity is ischemia/reperfusion (I/R) injury. in which reperfusion of the ischemic tissues elicits an acute inflammatory response activated by natural IgM (nIgM) binding to ischemia-specific self-antigens, which are non-muscle myosin heavy chains type II (NMHC-II) subtype A and C Subsequently, the complement lectin pathway is activated and eventually tissue injury occurs. Although earlier studies in the intestinal model showed that the classical complement pathway did not initiate I/R injury, C1q deposition was still observed in the local injured tissues by imaging analysis Moreover, the involvement of the alternative complement pathway became unclear due to conflicting reports using different knockout mice To explore the immediate downstream pathway following nIgM-ischemic antigen interaction, we isolated the nIgM-ischemic antigen immunocomplexes from the local tissue of animals treated in the intestinal I/R injury model, and examined the presence of initial molecules of three complement pathways. Our results showed that mannan-binding lectin (MBL), the early molecule of the lectin pathway, was present in the nIgM-ischemic Ag immunocomplex In addition, C1q, the initial molecule of the classical pathway was also detected on the immunocomplex However, Factor B, the early molecule in the alternative pathway, was not detected in the immunocomplex. To further examine the role of the alternative pathway in I/R injury, we utilized Factor B knockout mice in the intestinal model Our results showed that Factor B knockout mice were not protected from local tissue injury, and their complement system was activated in the local tissues by nIgM during I/R These results indicated that the lectin complement pathway operates immediately downstream of the nIgM-ischemic antigen interaction during intestinal I/R. Furthermore. the classical complement pathway also appears to interact with the of nIgM-ischemic antigen immunocomplex Finally, the alternative complement pathway is not involved in I/R injury induction in the current intestinal model (C) 2009 Elsevier Ltd All rights reserved

BENCHMRK-1 and -2 are ongoing double-blind phase III studies of raltegravir in patients experiencing failure of antiretroviral therapy with triple-class drug-resistant human immunodeficiency virus infection. At week 96 (combined data), raltegravir (400 mg twice daily) plus optimized background therapy was generally well tolerated, with superior and durable antiretroviral and immunological efficacy, compared with optimized background therapy alone.

Objective: To demonstrate that the use of highly active anti-retroviral therapy (HAART) to interrupt transmission of HIV-1 from mother to baby is effective, safe, and feasible in a remote rural region of China. Methods: Between November 2005 and May 2009, we enrolled 279 HIV-1-infected pregnant women to receive HAART to interrupt transmission of HIV-1 to their newborns across 16 counties in Yunnan. All women were started on triple combination therapy and submitted to regular blood draws to monitor CD4 T cells and viral load in their blood plasma. Infants received a single dose of nevirapine at birth and 1 or 4 weeks of zidovudine depending on the length of the mother's regimen. Exclusive formula feeding was recommended, and families were provided with 12-month supply of formula. Mothers and infant pairs were followed for 12-18 months postdelivery. Results: Of 279 enrolled HIV-infected women, 222 (79.6%) were identified and started treatment by 28 weeks of pregnancy. Viral load was undetectable at time of delivery for 62.4% (136 of 218) at delivery, with a mean 1.76 log viral load reduction between enrollment and delivery. Two of 193 babies (1.0%) who have already been tested became infected with HIV-1. Seven of 223 babies have died. By Kaplan-Meier analysis, cumulative one-year survival was 96.3%. Conclusions: The project demonstrated that HAART for all infected pregnant women is effective with a vertical transmission rate of similar to 1%. Thus, this project provides a model for China to scale up its efforts to prevent mother-to-child transmission of HIV-1.