Pancreatic cancer is one of the most lethal malignancies, with a prominent desmoplastic reaction as the defining hallmark of the disease. The past several decades have seen dramatic progress in understanding of pancreatic cancer pathogenesis, including the identification of precursor lesions, sequential transformation from normal pancreas to invasive pancreatic cancer and corresponding signature genetic events, and the biological impact of those alterations on malignant behaviors. However, the current therapeutic strategies for epithelial tumor cells, which have exhibited potent antitumor activity in cell culture and animal models, have failed to have significant effects in the clinic. The desmoplastic stroma surrounding pancreatic cancer cells, which accounts for about 90% of a tumor’s mass, clearly is not a passive scaffold for cancer cells but an active contributor to carcinogenesis. Improved understanding of the dynamic interaction between cancer cells and their stroma will be important to designing new, effective therapeutic strategies for pancreatic cancer. This review focuses on the origination of stromal molecular and cellular components in pancreatic tumors, their biological effects on pancreatic cancer cells, and the orchestration between these two components.
Cytokines; growth factors; transcriptional factors; therapy; immunology
High-resolution 2D 13C-13C NMR correlation spectra of uniformly 13C-labeled molecules in solution are obtained by homonuclear 13C-decoupling along both dimensions by the application of indirect covariance NMR to constant-time NMR spectra. The spectra are optimally suited for chemical structure elucidation and molecular identification of the components of complex mixtures, such as ones from uniformly 13C-labeled cell cultures.
Neonatal seizures pose a high risk for adverse outcome in survived infants. While the prognostic value of amplitude-integrated electroencephalogram (aEEG) is well established in neonates with encephalopathy and asphyxia, neonatal seizure studies focusing on the direct correlation between early aEEG measurement and subsequent neurologic outcome are scarce. In this study, the prognostic value of aEEG features was systematically analyzed in 143 full-term neonates to identify prognostic indicators of neurodevelopmental outcome. Neonatal aEEG features of background pattern, cyclicity, and seizure activity, as well as the etiology of neonatal seizures, were significantly associated with neurodevelopmental outcome at one year of age. aEEG background pattern was highly associated with neurologic outcomes (χ2 = 116.9), followed by aEEG cyclicity (χ2 = 87.2) and seizure etiology (χ2 = 79.3). Multiple linear regression showed that the four predictors explained 71.2% of the variation in neurological outcome, with standardized β coefficients of 0.44, 0.24, 0.22, and 0.14 for the predictors of aEEG background pattern, cyclicity, etiology, and aEEG seizure activity, respectively. This clinically applicable scoring system based on etiology and three aEEG indices would allow pediatricians to assess the risk for neurodevelopmental impairment and facilitate an early intervention in newborns developing seizures.
High altitude acclimatization is a series of physiological responses taking places when subjects go to altitude. Many factors could influence these processes, such as altitude, ascending speed and individual characteristics. In this study, based on a repeated measurement design of three sequential measurements at baseline, acute phase and chronic phase, we evaluated the effect of BMI, smoking and drinking on a number of physiological responses in high altitude acclimatization by using mixed model and partial least square path model on a sample of 755 Han Chinese young males. We found that subjects with higher BMI responses were reluctant to hypoxia. The effect of smoking was not significant at acute phase. But at chronic phase, red blood cell volume increased less while respiratory function increased more for smoking subjects compared with nonsmokers. For drinking subjects, red blood cell volume increased less than nondrinkers at both acute and chronic phases, while blood pressures increased more than nondrinkers at acute phase and respiratory function, red blood cell volume and oxygen saturation increased more than nondrinkers at chronic phase. The heavy and long-term effect of smoking, drinking and other factors in high altitude acclimatization needed to be further studied.
A customized metabolomics NMR database, TOCCATA, is introduced, which uses 13C chemical shift information for the reliable identification of metabolites, their spin systems and isomeric states. TOCCATA, whose information was derived from information of the BMRB and HMDB databases and the literature, currently contains 463 compounds and 801 spin systems and it can be used through a publicly accessible web server at http://spinportal.magnet.fsu.edu/toccata/webquery.html. TOCCATA allows the identification of metabolites in the sub-mM concentration range from 13C-13C TOCSY experiments of complex mixtures, which is demonstrated for an E.coli cell lysate, a carbohydrate mixture, and an amino acid mixture, all of which were uniformly 13C-labeled.
Spore photoproduct lyase (SPL) repairs a special thymine dimer 5-thyminyl-5,6-dihydrothymine, which is commonly called spore photoproduct or SP at the bacterial early germination phase. SP is the exclusive DNA photo-damage product in bacterial endospores; its generation and swift repair by SPL are responsible for the spores’ extremely high UV resistance. The early in vivo studies suggested that SPL utilizes a direct reversal strategy to repair the SP in the absence of light. The research in the past decade further established SPL as a radical SAM enzyme, which utilizes a tri-cysteine CXXXCXXC motif to harbor a [4Fe-4S] cluster. At the 1+ oxidation state, the cluster provides an electron to the S-adenosylmethionine (SAM), which binds to the cluster in a bidentate manner as the fourth and fifth ligands, to reductively cleave the C-S bond associated with the sulfonium ion in SAM, generating a reactive 5′-deoxyadenosyl (5′-dA) radical. This 5′-dA radical abstracts the proR hydrogen atom from the C6 carbon of SP to initiate the repair process; the resulting SP radical subsequently fragments to generate a putative thymine methyl radical, which accepts a back-donated H atom to yield the repaired TpT. SAM is suggested to be regenerated at the end of each catalytic cycle; and only a catalytic amount of SAM is needed in the SPL reaction. The H atom source for the back donation step is suggested to be a cysteine residue (C141 in B. subtilis SPL), and the H-atom transfer reaction leaves a thiyl radical behind on the protein. This thiyl radical thus must participate in the SAM regeneration process; however how the thiyl radical abstracts an H atom from the 5′-dA to regenerate SAM is unknown. This paper reviews and discusses the history and the latest progress in the mechanistic elucidation of SPL. Despite some recent breakthroughs, more questions are raised in the mechanistic understanding of this intriguing DNA repair enzyme.
Sepsis in older children and adults modifies immune system function. We compared serotype-specific antibody responses to heptavalent pneumococcal conjugate vaccine (PCV7) in very low birth weight infants (<1500g,VLBW) with and without blood stream infection (BSI) during their birth hospitalization.
Patients and Methods
Retrospective analysis of prospectively collected data for the Neonatal Research Network study of PCV7 responses among VLBWs. Infants received PCV7 at 2, 4, and 6 months after birth with blood drawn 4–6 weeks after 3rd dose. Serotype antibodies were compared between infants with or without a history of BSI. Regression models were constructed with birth-weight groups and other confounding factors identified in the primary study.
244 infants completed the vaccine series and had serum antibody available; 82 had BSI. After adjustment, BSI was not associated with reduced odds of serum antibody ≥0.35μg/mL.
BSI was not associated with reduced odds of WHO-defined protective PCV7 responses in VLBWs.
VLBW; immune response; vaccine; sepsis; blood stream infection
The aim of this study was to test whether a low dose of interferon-α-2b (IFN-α2b) enhances the clinical outcome of docetaxel (DXT) in patients with castration-resistant prostate cancer (CRPC). A prospective controlled trial of 40 CRPC patients receiving 5 mg of prednisone twice daily was conducted, where patients were randomly assigned to be administered 75 mg/m2 DXT plus 3 mIU/m2 IFN-α2b (group A, n=20) or 75 mg/m2 DXT alone (group B, n=20). The prostate-specific antigen (PSA) response, tumor response, progression-free survival (PFS) and overall survival (OS) were evaluated. There was no statistically significant difference in PSA response rate between groups A and B (65 vs. 47.4%, P=0.341). The tumor response rate in group A was significantly greater compared with that in group B (55 vs. 21.1%, P=0.048). The median PFS was longer in group A compared with that in group B (10 vs. 8 months, P=0.043). There was no statistically significant difference in median OS between the two groups (19 vs. 17 months, P=0.348), but one patient displayed a complete tumor response in group A. In groups A and B, transient grade 3 to 4 neutropenia was observed in nine and six patients, grade 3 to 4 anemia was observed in three and five patients, and grade 3 to 4 general fatigue was observed in four and one patient(s), respectively. The proportion of patients with grade 3 to 4 toxicity was not statistically different between the two groups. A low dosage of IFN-α2b may improve the antitumor activity of DXT with an acceptable toxicity profile in patients with CRPC.
castration-resistant prostate cancer; docetaxel; interferon-α-2b
Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP—labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells.
Cells have developed a variety of mechanisms for repairing DNA molecules when breaks occur in one or both of the DNA strands. However, we know relatively little about the causes of these breaks, which often occur naturally, or even about how common they are. Learning more about the most common forms of DNA breakage is important because the genomic changes caused by these breaks are driving forces behind both cancer and evolution, including the evolution of drug resistance in bacteria.
Shee et al. have developed a new method for detecting double-strand breaks in both bacterial and mammalian cells. The method involved combining a natural virus protein called Gam with a fluorescent protein called GFP (short for green fluorescent protein) to make a fusion protein called GamGFP. Gam was chosen because it binds only to double-strand breaks, traps double-strand breaks, and does not bind to any proteins. Genetic engineering techniques were used to introduce GamGFP into cells, with DNA breaks in these cells showing up as fluorescent spots when viewed under a microscope.
Shee et al. used this approach to detect double-strand breaks in both Escherichia coli cells and mammalian cells, and to measure the rate of spontaneous DNA breakage in E. coli. The number of double-strand breaks in E. coli was proportional to the number of times the cells had divided, which provides support for DNA replication-dependent models of spontaneous DNA breakage.
The GamGFP method also provided various insights into DNA breaks in mouse and human cells. In particular, Shee et al. found evidence for a mechanism of DNA breakage that appears to be specific to primates. This mechanism involves an enzyme that is only found in the innate immune system of primates removing an amine group from a cytosine. In future, this approach might allow the trapping, mapping and quantification of DNA breaks in all kinds of cells, and the highly specific way GamGFP binds to breaks could make it the preferred tool for studying DNA breakage in mammalian cells.
DNA double-strand breaks; endogenous DNA damage; GFP; fluorescent-protein fusions; spontaneous DNA breaks; synthetic biology; E. coli; Human; Mouse
Salmonella enterica serotype Typhimurium human blood strains isolated from outside Africa are rarely sequenced. Here, we report the draft genome sequences of two S. Typhimurium clinical strains isolated in the same year, one from blood and another from stool, in order to gain insights into the genetic basis leading to invasive diseases.
Several single nucleotide polymorphisms (SNPs) of the Glutamate metabotrophic receptor 7 gene (GRM7) have recently been identified by the genome-wide association study (GWAS) as potentially playing a role in susceptibility to age-related hearing impairment (ARHI), however this has not been validated in the Han Chinese population. The aim of this study was to determine if these SNPs are also associated with ARHI in an elderly male Han Chinese population. In this case-control candidate genes association study, a total of 982 men with ARHI and 324 normal-hearing controls subjects were studied. Using K-means cluster analysis, four audiogram shape subtypes of ARHI were identified in the case group: ‘‘flat shape (FL)’’, ‘‘sloping shape (SL)’’, ‘‘2-4 kHz abrupt loss (AL) shape’’ and ‘‘8 kHz dip (8D) shape’’. Results suggested that the SNP rs11928865 (A>T) of GRM7 was significantly associated with ARHI after adjusting for non-genetic factors (p= 0.000472, OR= 1.599, 95%CI= 1.229~2.081). Furthermore, frequency of TT genotype (rs11928865) were significant higher in the SL subgroup and AL subgroup with compared to controls group (p= 9.41E-05, OR= 1.945, 95%CI= 1.393~2.715; p= 0.000109, OR= 1.915, 95%CI= 1.378~2.661 adjusted, respectively) after Bonferroni correction. However, there wasn’t significant difference in the frequency of the TT genotype between cases in the FL subgroup or the 8D subgroup with when compared with controls. Results of the current study suggest that, in an elderly male Han Chinese population, GRM7 SNP rs11928865 (TT) occurs more frequently in ARHI patients with SL and AL phenotype patterns.
Increased de novo lipogenesis is one of the major metabolic events in cancer. In human hepatocellular carcinoma (HCC), de novo lipogenesis has been found to be increased and associated with the activation of AKT/mTOR signaling. In mice, overexpression of an activated form of AKT results in increased lipogenesis and hepatic steatosis, ultimately leading to liver tumor development. Hepatocarcinogenesis is dramatically accelerated when AKT is co-expressed with an oncogenic form of N-Ras. SCD1, the major isoform of stearoyl-CoA desaturases, catalyzing the conversion of saturated fatty acids (SFA) into monounsaturated fatty acids (MUFA), is a key enzyme involved in de novo lipogenesis. While many studies demonstrated the requirement of SCD1 for tumor cell growth in vitro, whether SCD1 is necessary for tumor development in vivo has not been previously investigated. Here, we show that genetic ablation of SCD1 neither inhibits lipogenesis and hepatic steatosis in AKT-overexpressing mice nor affects liver tumor development in mice co-expressing AKT and Ras oncogenes. Molecular analysis showed that SCD2 was strongly upregulated in liver tumors from AKT/Ras injected SCD1-/- mice. Noticeably, concomitant silencing of SCD1 and SCD2 genes was highly detrimental for the growth of AKT/Ras cells in vitro. Altogether, our study provides the evidence, for the first time, that SCD1 expression is dispensable for AKT/mTOR-dependent hepatic steatosis and AKT/Ras-induced hepatocarcinogenesis in mice. Complete inhibition of stearoyl-CoA desaturase activity may be required to efficiently suppress liver tumor development.
Long chain acyl-CoA synthetase 1 (ACSL1) contributes 50 to 90% of total ACSL activity in liver, adipose tissue, and heart and appears to direct the use of long chain fatty acids for energy. Although the functional importance of ACSL1 is becoming clear, little is understood about its post-translational regulation. In order to investigate the post-translational modifications of ACSL1 under different physiological conditions, we overexpressed ACSL1 in hepatocytes, brown adipocytes, and 3T3-L1 differentiated adipocytes, treated these cells with different hormones, and analyzed the resulting phosphorylated and acetylated amino acids by mass spectrometry. We then compared these results to the post-translational modifications observed in vivo in liver and brown adipose tissue after mice were fasted or exposed to a cold environment. We identified universal N-terminal acetylation, 15 acetylated lysines, and 25 phosphorylation sites on ACSL1. Several unique acetylation and phosphorylation sites occurred under conditions in which fatty acid β-oxidation is normally enhanced. Thirteen of the acetylated lysines had not previously been identified, and none of the phosphorylation sites had been previously identified. Site-directed mutagenesis was used to introduce mutations at three potential acetylation and phosphorylation sites believed to be important for ACSL1 function. At the ATP/AMP binding site and at a highly conserved site near the C terminus, modifications of Ser278 or Lys676, respectively, totally inhibited ACSL1 activity. In contrast, mutations of Lys285 that mimicked acetylation (Lys285Ala and Lys285Gln) reduced ACSL activity, whereas full activity was retained by Lys285Arg, suggesting that acetylation of Lys285 would be likely to decrease ACSL1 activity. These results indicate that ACSL1 is highly modified post-translationally. Several of these modifications would be expected to alter enzymatic function, but others may affect protein stability or protein-protein interactions.
Fatty acid metabolism; Beta-oxidation; Post-translational modification; Triacylglycerol synthesis; phosphorylation; Acetylation
5-thyminyl-5,6-dihydrothymine (also called spore photoproduct or SP) is the exclusive DNA photo-damage product in bacterial endospores. It is repaired by a radical SAM (S-adenosylmethionine) enzyme, the spore photoproduct lyase (SPL), at the bacterial early germination phase. Our previous studies proved that SPL utilizes the 5′-dA• generated by SAM cleavage reaction to abstract the H6proR atom to initiate the SP repair process. The resulting thymine allylic radical was suggested to take an H atom from an unknown protein source, most likely the cysteine 141. Here we show that C141 can be readily alkylated in the native SPL by iodoacetamide treatment, suggesting that it is accessible to the TpT radical. SP repair by the SPL C141A mutant yields TpTSO2− and TpT simultaneously from the very beginning of the reaction; no lag phase is observed for the TpTSO2− formation. Should any other protein residue serve as the H donor, its presence would result in TpT as the major product at least for the first enzyme turnover. These observations provide strong evidence to support C141 as the direct H atom donor. Moreover, due to the lack of this intrinsic H donor, the C141A mutant produces TpT via an unprecedented thymine cation radical reduction (proton coupled electron transfer) process, contrasting to the H atom transfer mechanism in the WT SPL reaction. The C141A mutant repairs SP at a rate which is ~3-fold slower than the WT enzyme. Formation of TpTSO2− and TpT exhibit a Vmax deuterium kinetic isotope effect (KIE) of 1.7 ± 0.2 respectively, which is smaller than the DVmax KIE of 2.8 ± 0.3 determined in the WT SPL reaction. These findings suggest that removing the intrinsic H atom donor disturbs the rate-limiting process in the enzyme catalysis. As expected, the pre-reduced C141A mutant only supports ~ 0.4 turnover, which is in sharp contrast to the > 5 turnovers exhibited by the WT SPL reaction, suggesting that the enzyme catalytic cycle (SAM regeneration) is disrupted by this single mutation.
To reevaluate the effect of isolated teratozoospermia on IVF and determine if there was any therapeutic benefit to isolated teratozoospermia by ICSI, since there are no widely accepted criteria for the treatment technique about isolated teratozoospermia.
A total of 441 couples with >20 million and progressive motility >30 % sperm undergoing their first IVF/ICSI cycle were included in the study between 2008 and 2010, for whom at least 8 oocytes were retrived. Isolated teratozoospermia was diagnosed in 183 of the included couples, and the rest couples (normal sperm morphology) were studied as control. Sibling oocytes were randomized to be inseminated either by ICSI or IVF. Fertilization rate, embryo quality, pregnancy rate, implantation rate and spontaneous abortion rate were assessed.
There was no difference in the percentage of eggs fertilized, implantation rate, pregnancy rate and spontaneous abortion rate between conventional IVF and ICSI regardless of the percentage of normal morphology. The day 3 embryonic morphology and rate of development were not different despite the insemination method and percentage of normal morphology.
Because isolated teratozoospermia did not influence the major indices of IVF and the unnecessary use of ICSI is time-consuming, costly and potential risks, couples with isolated teratozoospermia need not be subjected to ICSI.
In vitro fertilization(IVF); Intracytoplasmic sperm injection(ICSI); Teratozoospermia; Sperm morphology
Toll-like receptor 9 (TLR9) recognizes genomes of double-stranded DNA (dsDNA) viruses in the endosome to stimulate plasmacytoid dendritic cells (pDCs). However, how and if viruses with single-stranded DNA (ssDNA) genomes are detected by pDCs remain unclear. Here we have shown that despite the ability of purified genomic DNA to stimulate TLR9 and despite the ability to enter TLR9 endosomes, ssDNA viruses of the Parvoviridae family failed to elicit an interferon (IFN) response in pDCs.
Activation of lymphocytes can effectively produce a large amount of cytokines. The types of cytokines produced may depend on stimulating reagents and treatments. To find an optimal method to stimulate cytokine production and evaluate its effect on immunotoxicity assessments, the authors analyzed production of IL-2, IL-4, IL-6, IL-10, IL-13, IFN-γ, TNF-α, GM-CSF, RANTES and TGF-β in undiluted rat whole blood culture (incubation for 0, 2, 4, 6, 8 or 10 h) with different concentrations of PMA/ionomycin, PHA, Con A, LPS and PWM. We also evaluated the effects of cyclosporin A and azathioprine on cytokine production. The results revealed a rapid increase of IL-2, IFN-γ, TNF-α, RANTES and TGF-β secretion within 6 h after stimulation with 25 ng/mL PMA and 1 μg/mL ionomycin. The inhibition of these cytokine profiles reflected the effects of immunosuppressants on the immune system. Therefore, the results of this is study recommend the detection of cytokine profiles in undiluted whole blood stimulated 6 h with 25 ng/mL PMA and 1 μg/mL ionomycin as a powerful immunotoxicity assessment method.
immunotoxicity assessment; cytokines; whole blood; immunosuppressant
Increased CCL2 expression in prostate cancer (PCa) cells enhanced metastasis via macrophage recruitment. However, its linkage to androgen receptor (AR)-mediated PCa progression remains unclear. Here, we identified a previously unrecognized regulation: targeting AR with siRNA in PCa cells increased macrophage recruitment via CCL2 up-regulation, which might then result in enhancing PCa invasiveness. Molecular mechanism dissection revealed that targeting PCa AR with siRNA promoted PCa cell migration/invasion via CCL2-dependent STAT3 activation and epithelial–mesenchymal transition (EMT) pathways. Importantly, pharmacologic interruption of the CCL2/CCR2-STAT3 axis suppressed EMT and PCa cell migration, providing a new mechanism linking CCL2 and EMT. Simultaneously targeting PCa AR with siRNA and the CCL2/CCR2-STAT3 axis resulted in better suppression of PCa growth and metastasis in a xenograft PCa mouse model. Human PCa tissue microarray analysis suggests that increased CCL2 expression may be potentially associated with poor prognosis of PCa patients. Together, these results may provide a novel therapeutic approach to better battle PCa progression and metastasis at the castration resistant stage via the combination of targeting AR with siRNA and anti-CCL2/CCR2-STAT3 signalling.
androgen receptor; CCL2; epithelial–mesenchymal transition; prostate cancer; STAT3
Interstrand crosslinks (ICLs) represent a major challenge for DNA replication and transcription by preventing DNA strand separation. Cells deficient in ICL repair are hypersensitive to a variety of bifunctional alkylating agents and exhibit excessive genomic instability. Patients with deficient ICL repair, such as those with Fanconi anemia, are predisposed to a broad spectrum of cancers. The profound cellular toxicity of ICLs is exploited clinically in cancer chemotherapy. Therefore, understanding the mechanism of ICL repair and its impact on cancer development and treatment is very important. Studies of diseases with defective ICL repair, especially Fanconi anemia, have revealed unique ICL repair mechanisms in humans. In this review, we describe pathways and factors involved in ICL damage response and their implications in cancer development and treatment.
Interstrand crosslink; DNA repair; cancer; Fanconi anemia
Despite the wide acceptance of laparoscopic resection for treatment of abdominal tumors, only few cases of simultaneous laparoscopic removal of the spleen and the right liver have been reported to date. Littoral cell angiosarcoma (LCAS), which arises from the littoral cells lining the sinus channels of the splenic red pulp, is a rare condition, and there is limited literature on littoral cell angiosarcoma with liver metastases. We present the case of a 28-year-old woman with postoperative pathologically-proven LCAS with right liver metastases. The patient’s surgery was safely performed, and her postoperative course was uneventful until now. This case suggests that concomitant laparoscopic splenectomy (LS) and right hemihepatectomy is a suitable surgical option for selected patients.
Laparoscopic splenectomy; Laparoscopic right hemihepatectomy; Concomitant surgery; Littoral cell angiosarcoma
With the development of compressive sensing theory, image reconstruction from few-view projections has received considerable research attentions in the field of computed tomography (CT). Total-variation- (TV-) based CT image reconstruction has been shown to be experimentally capable of producing accurate reconstructions from sparse-view data. In this study, a distributed reconstruction algorithm based on TV minimization has been developed. This algorithm is very simple as it uses the alternating direction method. The proposed method can accelerate the alternating direction total variation minimization (ADTVM) algorithm without losing accuracy.
Almost all Streptococcus pneumoniae (pneumococcus) capsule serotypes employ the Wzy-dependent pathway for their capsular polysaccharide (CPS) biosynthesis. The assembly of the CPS repeating unit (RU) is the first committed step in this pathway. The wciN gene was predicted to encode a galactosyl transferase involved in the RU assembly of pneumococcus type 6B CPS. Herein, we provide the unambiguous in vitro biochemical evidence that wciN encodes an α-1,3-galactosyl transferase catalyzing the transfer of galactosyl from UDP-Gal onto the Glcα-pyrophosphate-lipid (Glcα-PP-lipid) acceptor to form Galα(1-3)Glcα-PP-lipid. A chemically synthesized acceptor (Glcα-PP-O(CH2)10CH3) was used to characterize the WciN activity. The disaccharide product, i.e. Galα (1-3)Glcα-PP-O(CH2)10CH3, was characterized by mass and NMR spectroscopy. Substrate specificity study indicated that the acceptor structural region composed of pyrophosphate and lipid moieties may play an important role in the enzyme-acceptor recognition. Furthermore, divalent metal cations were found indispensable to the WciN activity, suggesting that this glycosyltransferase (GT) belongs to the GT-A superfamily. By analyzing the activities of six WciN mutants, a DXD motif involved in the coordination of a divalent metal cation was identified. This work provides a chemical biology approach to characterize the activities of pneumococcal CPS GTs in vitro, and will help to understand the pneumococcal CPS biosynthetic pathway.
The Fanconi anemia (FA) protein network is necessary for repair of DNA interstrand crosslinks (ICLs), but its control mechanism remains unclear. Here we show that the network is regulated by a ubiquitin signaling cascade initiated by RNF8 and its partner, UBC13; and mediated by FAAP20, a component of the FA core complex. FAAP20 preferentially binds the ubiquitin product of RNF8-UBC13; and its recruitment to ICLs requires this ubiquitin-binding activity, RNF8 and UBC13. Both RNF8 and FAAP20 are required for recruitment of FA core complex and FANCD2 to ICLs, whereas RNF168 can modulate efficiency of the recruitment. RNF8 and FAAP20 are needed for efficient FANCD2 monoubiquitination, a key step of the FA network; RNF8 and FA core complex work in the same pathway to promote cellular resistance to ICLs. Thus, the RNF8-FAAP20 ubiquitin cascade is critical for recruiting FA core complex to ICLs and for normal function of the FA network.
RNF8; FAAP20; RNF168; UBC13; Fanconi Anemia; ubiquitin