Inhibitory role of E2F-1 in the regulation of tumor suppressor p53 during DNA damage response

Appropriate regulation of DNA damage response is pivotal for maintaining genome stability. p53 as well as E2F-1 plays a critical role during DNA damage response, however, the physiological significance of their interaction has been elusive. In the present study, we found that E2F-1 has an inhibitory effect on p53 during adriamycin (ADR)-mediated DNA damage response. Upon ADR exposure, p53 and E2F-1 were markedly induced at protein and mRNA levels in p53-procifient U2OS and HCT116 cells, and formed a stable complex as examined by co-immunoprecipitation experiments. Of note, chromatin immunoprecipitation (ChIP) experiments revealed that ADR-mediated induction coincides with the efficient recruitment of p53 and E2F-1 onto the promoters of p53-target genes, such as p21(WAF1) and BAX. Subsequent RT-PCR and luciferase reporter assays demonstrated that E2F-1 strongly attenuates p53-dependent transactivation of p53-target genes. Importantly, siRNA-mediated knockdown of E2F-1 stimulated apoptosis in response to ADR, which was associated with an accelerated response of p21(WAF1) and BAX. Collectively, our present findings suggest that E2F-1 participates in p53-mediated DNA damage response and might have a checkpoint function to limit overactive p53.     

Recognition of heteropolysaccharide alginate by periplasmic solute-binding proteins of a bacterial ABC transporter

Alginate is a heteropolysaccharide that consists of β-D-mannuronate (M) and α-L-guluronate (G). The Gram-negative bacterium Sphingomonas sp. A1 directly incorporates alginate into the cytoplasm through the periplasmic solute-binding protein (AlgQ1 and AlgQ2)-dependent ABC transporter (AlgM1-AlgM2/AlgS-AlgS). Two binding proteins with at least four subsites strongly recognize the nonreducing terminal residue of alginate at subsite 1. Here, we show the broad substrate preference of strain A1 solute-binding proteins for M and G present in alginate and demonstrate the structural determinants in binding proteins for heteropolysaccharide recognition through X-ray crystallography of four AlgQ1 structures in complex with saturated and unsaturated alginate oligosaccharides. Alginates with different M/G ratios were assimilated by strain A1 cells and bound to AlgQ1 and AlgQ2. Crystal structures of oligosaccharide-bound forms revealed that in addition to interaction between AlgQ1 and unsaturated oligosaccharides, the binding protein binds through hydrogen bonds to the C4 hydroxyl group of the saturated nonreducing terminal residue at subsite 1. The M residue of saturated oligosaccharides is predominantly accommodated at subsite 1 because of the strict binding of Ser-273 to the carboxyl group of the residue. In unsaturated trisaccharide (ΔGGG or ΔMMM)-bound AlgQ1, the protein interacts appropriately with substrate hydroxyl groups at subsites 2 and 3 to accommodate M or G, while substrate carboxyl groups are strictly recognized by the specific residues Tyr-129 at subsite 2 and Lys-22 at subsite 3. Because of this substrate recognition mechanism, strain A1 solute-binding proteins can bind heteropolysaccharide alginate with different M/G ratios.     

Infectious endogenous retroviruses in cats and emergence of recombinant viruses

Endogenous retroviruses (ERVs) comprise a significant percentage of the mammalian genome, and it is poorly understood whether they will remain as inactive genomes or emerge as infectious retroviruses. Although several types of ERVs are present in domestic cats, infectious ERVs have not been demonstrated. Here, we report a previously uncharacterized class of endogenous gammaretroviruses, termed ERV-DCs, that is present and hereditary in the domestic cat genome. We have characterized a subset of ERV-DC proviral clones, which are numbered according to their genomic insertions. One of these, ERV-DC10, located in the q12-q21 region on chromosome C1, is an infectious gammaretrovirus capable of infecting a broad range of cells, including human. Our studies indicate that ERV-DC10 entered the genome of domestic cats in the recent past and appeared to translocate to or reintegrate at a distinct locus as infectious ERV-DC18. Insertional polymorphism analysis revealed that 92 of 244 domestic cats had ERV-DC10 on a homozygous or heterozygous locus. ERV-DC-like sequences were found in primate and rodent genomes, suggesting that these ERVs, and recombinant viruses such as RD-114 and BaEV, originated from an ancestor of ERV-DC. We also found that a novel recombinant virus, feline leukemia virus subgroup D (FeLV-D), was generated by ERV-DC env transduction into feline leukemia virus in domestic cats. Our results indicate that ERV-DCs behave as donors and/or acceptors in the generation of infectious, recombinant viruses. The presence of such infectious endogenous retroviruses, which could be harmful or beneficial to the host, may affect veterinary medicine and public health.     

Spatial distribution of viruses associated with planktonic and attached microbial communities in hydrothermal environments

Viruses play important roles in marine surface ecosystems, but little is known about viral ecology and virus-mediated processes in deep-sea hydrothermal microbial communities. In this study, we examined virus-like particle (VLP) abundances in planktonic and attached microbial communities, which occur in physical and chemical gradients in both deep and shallow submarine hydrothermal environments (mixing waters between hydrothermal fluids and ambient seawater and dense microbial communities attached to chimney surface areas or macrofaunal bodies and colonies). We found that viruses were widely distributed in a variety of hydrothermal microbial habitats, with the exception of the interior parts of hydrothermal chimney structures. The VLP abundance and VLP-to-prokaryote ratio (VPR) in the planktonic habitats increased as the ratio of hydrothermal fluid to mixing water increased. On the other hand, the VLP abundance in attached microbial communities was significantly and positively correlated with the whole prokaryotic abundance; however, the VPRs were always much lower than those for the surrounding hydrothermal waters. This is the first report to show VLP abundance in the attached microbial communities of submarine hydrothermal environments, which presented VPR values significantly lower than those in planktonic microbial communities reported before. These results suggested that viral lifestyles (e.g., lysogenic prevalence) and virus interactions with prokaryotes are significantly different among the planktonic and attached microbial communities that are developing in the submarine hydrothermal environments.     

CRUMPLED LEAF (CRL) homologs of Physcomitrella patens are involved in the complete separation of dividing plastids

Plastid division is controlled by numerous nuclear genes. Arabidopsis thaliana CRUMPLED LEAF (AtCRL) is a plastid division-related gene, and the crl mutant exhibits a dwarf phenotype with abnormal cell division and a significant reduction in plastid numbers. However, the function of AtCRL is not fully understood. Here, we identified and characterized two AtCRL homologs, PpCRL1 and PpCRL2, in the moss Physcomitrella patens. PpCRL1 and PpCRL2 shared 77% amino acid identity with each other and 47% identity with AtCRL. Single PpCRL1 or -2 gene knockout (KO) mutants could not be distinguished from the wild-type mosses, but PpCRL1 and -2 double KO mutants displayed growth retardation of protonemata and gametophores and harbored approximately 10 large chloroplasts per cell. This indicates that PpCRL1 and PpCRL2 have redundant functions in chloroplast division and plant growth. Unlike the A. thaliana crl mutants, however, the PpCRL double KO mutants did not display abnormal orientation of the cell division plane. Complementation experiments showed that AtCRL partially rescued the defects in chloroplast size and number of the PpCRL double KO mutant. This suggests that PpCRL has a similar, but not identical, function to AtCRL. Time-lapse microscopic observation of the double PpCRL KO mutants revealed that some dumbbell-shaped chloroplasts failed to complete division at the late stage of plastid division; enlarged chloroplasts were thus generated. This strongly suggests that PpCRLs are involved in the complete separation of dividing chloroplasts.     

Evaluation of portable point-of-care CD4 counter with high sensitivity for detecting patients eligible for antiretroviral therapy

Background

Accurate, inexpensive point-of-care CD4+ T cell testing technologies are needed that can deliver CD4+ T cell results at lower level health centers or community outreach voluntary counseling and testing. We sought to evaluate a point-of-care CD4+ T cell counter, the Pima CD4 Test System, a portable, battery-operated bench-top instrument that is designed to use finger stick blood samples suitable for field use in conjunction with rapid HIV testing.

Methods

Duplicate measurements were performed on both capillary and venous samples using Pima CD4 analyzers, compared to the BD FACSCalibur (reference method). The mean bias was estimated by paired Student''s t-test. Bland Altman plots were used to assess agreement.

Results

206 participants were enrolled with a median CD4 count of 396 (range; 18–1500). The finger stick PIMA had a mean bias of −66.3 cells/µL (95%CI −83.4−49.2, P<0.001) compared to the FACSCalibur; the bias was smaller at lower CD4 counts (0–250 cells/µL) with a mean bias of −10.8 (95%CI −27.3−+5.6, P = 0.198), and much greater at higher CD4 cell counts (>500 cells/µL) with a mean bias of −120.6 (95%CI −162.8, −78.4, P<0.001). The sensitivity (95%CI) of the Pima CD4 analyzer was 96.3% (79.1–99.8%) for a <250 cells/ul cut-off with a negative predictive value of 99.2% (95.1–99.9%).

Conclusions

The Pima CD4 finger stick test is an easy-to-use, portable, relatively fast device to test CD4+ T cell counts in the field. Issues of negatively-biased CD4 cell counts especially at higher absolute numbers will limit its utility for longitudinal immunologic response to ART. The high sensitivity and negative predictive value of the test makes it an attractive option for field use to identify patients eligible for ART, thus potentially reducing delays in linkage to care and ART initiation.     

Executive Function Deficits and Social-Behavioral Abnormality in Mice Exposed to a Low Dose of Dioxin In Utero and via Lactation

An increasing prevalence of mental health problems has been partly ascribed to abnormal brain development that is induced upon exposure to environmental chemicals. However, it has been extremely difficult to detect and assess such causality particularly at low exposure levels. To address this question, we here investigated higher brain function in mice exposed to dioxin in utero and via lactation by using our recently developed automated behavioral flexibility test and immunohistochemistry of neuronal activation markers Arc, at the 14 brain areas. Pregnant C57BL/6 mice were given orally a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at a dose of either 0, 0.6 or 3.0 µg/kg on gestation day 12.5. When the pups reached adulthood, they were group-housed in IntelliCage to assess their behavior. As a result, the offspring born to dams exposed to 0.6 µg TCDD/kg were shown to have behavioral inflexibility, compulsive repetitive behavior, and dramatically lowered competitive dominance. In these mice, immunohistochemistry of Arc exhibited the signs of hypoactivation of the medial prefrontal cortex (mPFC) and hyperactivation of the amygdala. Intriguingly, mice exposed to 3.0 µg/kg were hardly affected in both the behavioral and neuronal activation indices, indicating that the robust, non-monotonic dose-response relationship. In conclusion, this study showed for the first time that perinatal exposure to a low dose of TCDD in mice develops executive function deficits and social behavioral abnormality accompanied with the signs of imbalanced mPFC-amygdala activation.     

X-ray structures of Aerococcus viridans lactate oxidase and its complex with D-lactate at pH 4.5 show an alpha-hydroxyacid oxidation mechanism

l-Lactate oxidase (LOX) belongs to a family of flavin mononucleotide (FMN)-dependent α-hydroxy acid-oxidizing enzymes. Previously, the crystal structure of LOX (pH 8.0) from Aerococcus viridans was solved, revealing that the active site residues are located around the FMN. Here, we solved the crystal structures of the same enzyme at pH 4.5 and its complex with d-lactate at pH 4.5, in an attempt to analyze the intermediate steps. In the complex structure, the d-lactate resides in the substrate-binding site, but interestingly, an active site base, His265, flips far away from the d-lactate, as compared with its conformation in the unbound state at pH 8.0. This movement probably results from the protonation of His265 during the crystallization at pH 4.5, because the same flip is observed in the structure of the unbound state at pH 4.5. Thus, the present structure appears to mimic an intermediate after His265 abstracts a proton from the substrate. The flip of His265 triggers a large structural rearrangement, creating a new hydrogen bonding network between His265-Asp174-Lys221 and, furthermore, brings molecular oxygen in between d-lactate and His265. This mimic of the ternary complex intermediate enzyme-substrate-O₂ could explain the reductive half-reaction mechanism to release pyruvate through hydride transfer. In the mechanism of the subsequent oxidative half-reaction, His265 flips back, pushing molecular oxygen into the substrate-binding site as the second substrate, and the reverse reaction takes place to produce hydrogen peroxide. During the reaction, the flip-flop action of His265 has a dual role as an active base/acid to define the major chemical steps. Our proposed reaction mechanism appears to be a common mechanistic strategy for this family of enzymes.     

A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-kappaB activation

Nod1 and Nod2 are intracellular proteins that are involved in host recognition of specific bacterial molecules and are genetically associated with several inflammatory diseases. Nod1 and Nod2 stimulation activates NF-kappaB through RICK, a caspase-recruitment domain-containing kinase. However, the mechanism by which RICK activates NF-kappaB in response to Nod1 and Nod2 stimulation is unknown. Here we show that RICK is conjugated with lysine-63-linked polyubiquitin chains at lysine 209 (K209) located in its kinase domain upon Nod1 or Nod2 stimulation and by induced oligomerization of RICK. Polyubiquitination of RICK at K209 was essential for RICK-mediated IKK activation and cytokine/chemokine secretion. However, RICK polyubiquitination did not require the kinase activity of RICK or alter the interaction of RICK with NEMO, a regulatory subunit of IkappaB kinase (IKK). Instead, polyubiquitination of RICK was found to mediate the recruitment of TAK1, a kinase that was found to be essential for Nod1-induced signaling. Thus, RICK polyubiquitination links TAK1 to IKK complexes, a critical step in Nod1/Nod2-mediated NF-kappaB activation.