The activity of a putative polyisoprenol-linked sugar translocase (Wzx) involved in Escherichia coli O antigen assembly is independent of the chemical structure of the O repeat

During O antigen lipopolysaccharide (LPS) synthesis in bacteria, transmembrane migration of undecaprenylpyrophosphate (Und-P-P)-bound O antigen subunits occurs before their polymerization and ligation to the rest of the LPS molecule. Despite the general nature of the translocation process, putative O-antigen translocases display a low level of amino acid sequence similarity. In this work, we investigated whether complete O antigen subunits are required for translocation. We demonstrate that a single sugar, GlcNAc, can be incorporated to LPS of Escherichia coli K-12. This incorporation required the functions of two O antigen synthesis genes, wecA (UDP-GlcNAc:Und-P GlcNAc-1-P transferase) and wzx (O-antigen translocase). Complementation experiments with putative O-antigen translocases from E. coli O7 and Salmonella enterica indicated that translocation of O antigen subunits is independent of the chemical structure of the saccharide moiety. Furthermore, complementation with putative translocases involved in synthesis of exopolysaccharides demonstrated that these proteins could not participate in O antigen assembly. Our data indicate that recognition of a complete Und-P-P-bound O antigen subunit is not required for translocation and suggest a model for O antigen synthesis involving recognition of Und-P-P-linked sugars by a putative complex made of Wzx translocase and other proteins involved in the processing of O antigen.

Role of the rfe gene in the biosynthesis of the Escherichia coli O7-specific lipopolysaccharide and other O-specific polysaccharides containing N-acetylglucosamine

We report that rfe mutants of wild-type strains of Escherichia coli O7, O18, O75, and O111 did not express O-specific polysaccharide unless the rfe mutation was complemented by a cloned rfe gene supplied in a plasmid. The O polysaccharides in these strains are known to have N-acetylglucosamine (GlcNAc) in their O repeats. In addition, in vitro transferase assays with bacterial membranes from either the O7 wild-type strain or its isogenic rfe mutant showed that GlcNAc is the first carbohydrate added onto the lipid acceptor in the assembly of the O7 repeat and that this function is inhibited by tunicamycin. Our results indicate that the rfe gene product is a general requirement for the synthesis of O polysaccharides containing GlcNAc.

Identification, expression, and DNA sequence of the GDP-mannose biosynthesis genes encoded by the O7 rfb gene cluster of strain VW187 (Escherichia coli O7:K1)

The O7-specific lipopolysaccharide (LPS) in strains of Escherichia coli consists of a repeating unit made of galactose, mannose, rhamnose, 4-acetamido-2,6-dideoxyglucose, and N-acetylglucosamine. We have recently cloned and characterized genetically the O7-specific LPS biosynthesis region (rfbEcO7) of the E. coli O7:K1 strain VW187 (C. L. Marolda, J. Welsh, L. Dafoe, and M. A. Valvano, J. Bacteriol. 172:3590-3599, 1990). In this study, we localized the gnd gene encoding gluconate-6-phosphate dehydrogenase at one end of the rfbEcO7 gene cluster and sequenced that end of the cluster. Three open reading frames (ORF) encoding polypeptides of 275, 464, and 453 amino acids were identified upstream of gndEcO7, all transcribed toward the gnd gene. ORF275 had 45% similarity at the protein level with ORF16.5, which occupies a similar position in the Salmonella enterica LT2 rfb region, and presumably encodes a nucleotide sugar transferase. The polypeptides encoded by ORFs 464 and 453 were expressed under the control of the ptac promoter and visualized in Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels and by maxicell analysis. ORF464 expressed GDP-mannose pyrophosphorylase and ORF453 encoded a phosphomannomutase, the enzymes for the biosynthesis pathway of GDP-mannose, one of the nucleotide sugar precursors for the formation of the O7 repeating unit. They were designated rfbMEcO7 and rfbKEcO7, respectively. The RfbMEcO7 polypeptide was homologous to the corresponding protein in S. enterica LT2, XanB of Xanthomonas campestris, and AlgA of Pseudomonas aeruginosa, all GDP-mannose pyrophosphorylases. RfbKEcO7 was very similar to CpsG of S. enterica LT2, an enzyme presumably involved in the biosynthesis of the capsular polysaccharide colanic acid, but quite different from the corresponding RfbK protein of S. enterica LT2.

A population-based association study of SNPs of GSTP1, MnSOD, GPX2 and Barrett's esophagus and esophageal adenocarcinoma

Oxidative stress appears to be important in the pathogenesis of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). Single-nucleotide polymorphisms (SNPs) of antioxidant enzyme genes may play a part in determining individual susceptibility to these diseases. The Factors Influencing the Barrett's Adenocarcinoma Relationship (FINBAR) study is a population-based, case-control study of BE and EAC in Ireland. DNA from EAC (n = 207), BE (> or =3 cm BE at endoscopy with specialized intestinal metaplasia on biopsy, n = 189) and normal population controls (n = 223) were analyzed. Several SNPs spanning the genes for glutathione S-transferase P1 (GSTP1), manganese superoxide dismutase (MnSOD) and glutathione peroxidase 2 (GPX2) were genotyped using multiplex polymerase chain reaction and SNaPshottrade mark. The chi(2) test was used to compare genotype and allele frequencies between case and control subjects. Linkage disequilibrium between SNPs was quantified using Lewontin's D' value and haplotype frequency estimates obtained using Haploview. Eleven SNPs were genotyped (six for GSTP1, three for MnSOD and two for GPX2); all were in Hardy-Weinberg equilibrium. None was significantly associated with EAC or BE even before Bonferroni correction. Odds ratios for EAC for individual SNPs ranged from 0.68 [95% confidence interval (CI) 0.43-1.08] to 1.25 (95% CI 0.73-2.16), and for BE from 0.84 (95% CI 0.52-1.30) to 1.30 (95% CI 0.85-1.97). SNPs in all three genes were in strong linkage disequilibrium (D' > 0.887) but haplotype analysis did not show any significant association with EAC or BE. SNPs involving the GSTP1, MnSOD and GPX2 genes were not associated with BE or EAC. Further studies aimed at identifying susceptibility genes should focus on different antioxidant genes or different pathways.

Predictive markers for colorectal cancer:current status and future prospects

Colorectal cancer (CRC) is the second leading cause of cancer death in the United States. Although there is clear evidence of the benefit of chemotherapy in adjuvant and metastatic settings, its use continues to be suboptimal because of intrinsic or acquired drug resistance. 5-Fluorouracil continues to be the mainstay of CRC therapy, and combinations with newer chemotherapeutic agents such as irinotecan and oxaliplatin have resulted in improved response rates and survival. The role of other agents including cyclooxygenase-2 inhibitors, epidermal growth factor receptor, and farnsyl transferase inhibitors remains to be elucidated. Despite these improvements, many patients undergo chemotherapy without benefit. Increased understanding of the biology of CRC has led to the identification of prognostic markers that may help identify patients who will benefit from chemotherapy. Furthermore, studies have also begun to identify markers that predict whether a tumor will respond to a particular chemotherapy. The ultimate goal of this research is to prospectively identify patients who should receive chemotherapy and, thus, to tailor treatment to the molecular profile of the tumor and patient. Such an approach has the potential to dramatically improve response rates. This review highlights potentially important prognostic and predictive factors in CRC and discusses the potential for their use in the treatment of this disease.

Effect of tea polyphenols and tea pigments on the inhibition of precancerous liver lesions in rats

The objective of this study was to investigate the inhibitory effect of tea components, tea polyphenols and tea pigments, on precancerous liver lesions in rats. A rat liver precancerous lesion model was established by multiple low-dosage N-nitrosodiethylamine (NDEA) injections, followed by intraperitoneal CCl4 injection and partial hepatectomy (PH). Tea pigments (0.1%) or tea polyphenols (0.1%) were given to Wistar rats in drinking water during the eight weeks of the experiment. The number and area of glutathione S-transferase Pi-positive foci in the rat liver were used as biomarkers of precancerous liver lesions. Western and Northern blot techniques were used to detect rat liver GST-Pi expression at the protein and mRNA levels. At the end of the experiment tea polyphenols and tea pigments significantly decreased the number and area of GST-Pi-positive foci that were overexpressed in the NDEA-CCl4-PH-treated rats compared with the positive control group. The results also showed that GST-Pi mRNA and protein expression increased significantly in the NDEA-CCl4-PH-treated group, which is consistent with the changing of GST-Pi-positive foci. Tea pigments and tea polyphenols had an inhibitory effect on the overexpression of GST-Pi mRNA and protein in NDEA-CCl4-PH-treated rats. These results suggest that tea pigments and tea polyphenols are effective in preventing the occurrence and progression of precancerous liver lesions in rats.

Substrates modified by advanced glycation end-products cause dysfunction and death in retinal pericytes by reducing survival signals mediated by platelet-derived growth factor

AIMS/HYPOTHESIS: Premature death of retinal pericytes is a pathophysiological hallmark of diabetic retinopathy. Among the mechanisms proposed for pericyte death is exposure to AGE, which accumulate during diabetes. The current study used an in vitro model, whereby retinal pericytes were exposed to AGE-modified substrate and the mechanisms underlying pericyte death explored. METHODS: Pericytes were isolated from bovine retinal capillaries and propagated on AGE-modified basement membrane (BM) extract or non-modified native BM. The extent of AGE modification was analysed. Proliferative responses of retinal pericytes propagated on AGE-modified BM were investigated using a 5-bromo-2-deoxy-uridine-based assay. The effect of extrinsically added platelet-derived growth factor (PDGF) isoforms on these proliferative responses was also analysed alongside mRNA expression of the PDGF receptors. Apoptotic death of retinal pericytes grown on AGE-modified BM was investigated using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling labelling, mitochondrial membrane depolarisation and by morphological assessment. We also measured both the ability of PDGF to reverse Akt dephosphorylation that was mediated by AGE-modified BM, and increased pericyte apoptosis. RESULTS: Retinal pericytes exposed to AGE-modified BM showed reduced proliferative responses in comparison to controls (p

The identification of p130cas-binding proteins and their role in cellular transformation

Adaptor proteins play an important role in signal transduction by regulating the establishment and maintenance of functionally important protein complexes. A recently described member of this group of proteins is p130cas (CAS), which contains numerous sequence motifs predicted to be involved in mediating protein-protein interactions. We propose that adaptor molecules like CAS may help determine the response of a cell to a particular signal by interacting with specific subsets of cellular proteins. To test this hypothesis, we have identified potential binding partners of CAS that may play a rote in cellular transformation by the oncoproteins v-SRC and/or v-CRK. We show that individual domains of CAS associate with specific subsets of proteins in vitro, and that many of these interactions are dependent on the state of tyrosine-phosphorylation of CAS. Sequences necessary for interacting with the focal adhesion kinase pp125FAK (FAK), v-SRC and v-CRK have been mapped to distinct regions of CAS. In addition, the identification of a number of putative CAS-binding partners that are present in crk-transformed cell extracts but undetectable in normal and src-transformed cell extracts supports a model in which unique protein complexes are formed in response to different signals.

RNAi dynamics in Juvenile Fasciola spp. Liver flukes reveals the persistence of gene silencing in vitro

Background: Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi) has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (ds)RNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain), validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL) and B (FheCatB) cysteine proteases, and a σ-class glutathione transferase (FheσGST).

Methodology/Principal Findings: Robust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200–320 nt) dsRNAs or 27 nt short interfering (si)RNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent) and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively.

Conclusions/Significance: In spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the development of multiple-throughput phenotypic screens for control target validation. RNAi persistence in fluke encourages in vivo studies on gene function using worms exposed to RNAi-triggers prior to infection.

Replication of the association of GLT6D1 with aggressive periodontitis in a Sudanese population

BACKGROUND: Susceptibility to aggressive periodontitis (AgP) is influenced by genetic as well as environmental factors. Studies linking gene variants to AgP have been mainly centred in developed countries with limited data from Africa.
AIM: To investigate whether previously reported candidate gene associations with AgP could be replicated in a population from Sudan.


METHODS: The investigation was a case-control design. Cases with AgP (n = 132) and controls (n = 136) were identified from patients attending the Periodontal Department in Khartoum Dental Hospital. Genotyping was performed using the Sequenom MassARRAY iPLEX platform. Analysis focused on gene variants with a minor allele frequency (MAF) > 25% in the Sudanese subjects that had previously been reported to be associated with AgP.


RESULTS: One candidate gene rs1537415 (GLT6D1) was significantly associated with AgP, OR = 1.50 (95% CI 1.04-2.17), p = 0.0295 (increasing to p = 0.09 after correction for multiple testing). The association strengthened to OR = 1.56 (95% CI 1.15-2.16), p = 0.0042 when the controls were supplemented with data from the Hap map for the Yoruba in Ibadan (n = 147) and remained significant (p = 0.013) after correction for multiple testing.


CONCLUSION: The study independently replicated the finding that rs1537415, a variant in glycosyl transferase gene GLT6D1, is associated with AgP and provided the first report of genetic associations with AgP in a Sudanese population.

Inflammatory and cytotoxic effects of acrolein, nicotine, acetylaldehyde and cigarette smoke extract on human nasal epithelial cells

Background: Cigarette smoke induces a pro-inflammatory response in airway epithelial cells but it is not clear which of the various chemicals contained within cigarette smoke (CS) should be regarded as predominantly responsible for these effects. We hypothesised that acrolein, nicotine and acetylaldehyde, important chemicals contained within volatile cigarette smoke in terms of inducing inflammation and causing addiction, have immunomodulatory effects in primary nasal epithelial cell cultures (PNECs).

Methods: PNECs from 19 healthy subjects were grown in submerged cultures and were incubated with acrolein, nicotine or acetylaldehyde prior to stimulation with Pseudomonas aeruginosa lipopolysaccharide (PA LPS). Experiments were repeated using cigarette smoke extract (CSE) for comparison. IL-8 was measured by ELISA, activation of NF-κB by ELISA and Western blotting, and caspase-3 activity by Western blotting. Apoptosis was evaluated using Annexin-V staining and the terminal transferase-mediated dUTP nick end-labeling (TUNEL) method.

Results: CSE was pro-inflammatory after a 24 h exposure and 42% of cells were apoptotic or necrotic after this exposure time. Acrolein was pro-inflammatory for the PNEC cultures (30 μM exposure for 4 h inducing a 2.0 fold increase in IL-8 release) and also increased IL-8 release after stimulation with PA LPS. In contrast, nicotine had anti-inflammatory properties (0.6 fold IL-8 release after 50 μM exposure to nicotine for 24 h), and acetylaldehyde was without effect. Acrolein and nicotine had cellular stimulatory and anti-inflammatory effects respectively, as determined by NF-κB activation. Both chemicals increased levels of cleaved caspase 3 and induced cell death.

Conclusions: Acrolein is pro-inflammatory and nicotine anti-inflammatory in PNEC cultures. CSE induces cell death predominantly by apoptotic mechanisms. 

Discovery of DNA hypermethylation using a DHPLC screening strategy

Promoter hypermethylation is recognized as a hallmark of human cancer, in addition to conventional mechanisms of gene inactivation. As such, many new technologies have been developed over the past two decades to uncover novel targets of methylation and decipher complex epigenetic patterns. However, many of these are either labor intensive or provide limited data, confined to oligonucleotide hybridization sequences or enzyme cleavage sites and cannot be easily applied to screening large sets of sequences or samples. We present an application of denaturing high performance liquid chromatography (DHPLC), which relies on bisulfite modification of genomic DNA, for methylation screening. We validated DHPLC as a methylation screening tool using GSTP1, a well known target of methylation in prostate cancer. We developed an in silico approach to identify potential targets of promoter hypermethylation in prostate cancer. Using DHPLC, we screened two of these targets LGALS3 and SMAD4 for methylation. We show that DHPLC has an application as a fast, sensitive, quantitative and cost effective method for screening novel targets or DNA samples for DNA methylation.

The emerging roles of DNA methylation in the clinical management of prostate cancer

Aberrant DNA methylation is one of the hallmarks of carcinogenesis and has been recognized in cancer cells for more than 20 years. The role of DNA methylation in malignant transformation of the prostate has been intensely studied, from its contribution to the early stages of tumour development to the advanced stages of androgen independence. The most significant advances have involved the discovery of numerous targets such as GSTP1, Ras-association domain family 1A (RASSF1A) and retinoic acid receptor beta2 (RARbeta2) that become inactivated through promoter hypermethylation during the course of disease initiation and progression. This has provided the basis for translational research into methylation biomarkers for early detection and prognosis of prostate cancer. Investigations into the causes of these methylation events have yielded little definitive data. Aberrant hypomethylation and how it impacts upon prostate cancer has been less well studied. Herein we discuss the major developments in the fields of prostate cancer and DNA methylation, and how this epigenetic modification can be harnessed to address some of the key issues impeding the successful clinical management of prostate cancer.

In silico mining identifies IGFBP3 as a novel target of methylation in prostate cancer

Promoter hypermethylation is central in deregulating gene expression in cancer. Identification of novel methylation targets in specific cancers provides a basis for their use as biomarkers of disease occurrence and progression. We developed an in silico strategy to globally identify potential targets of promoter hypermethylation in prostate cancer by screening for 5' CpG islands in 631 genes that were reported as downregulated in prostate cancer. A virtual archive of 338 potential targets of methylation was produced. One candidate, IGFBP3, was selected for investigation, along with glutathione-S-transferase pi (GSTP1), a well-known methylation target in prostate cancer. Methylation of IGFBP3 was detected by quantitative methylation-specific PCR in 49/79 primary prostate adenocarcinoma and 7/14 adjacent preinvasive high-grade prostatic intraepithelial neoplasia, but in only 5/37 benign prostatic hyperplasia (P < 0.0001) and in 0/39 histologically normal adjacent prostate tissue, which implies that methylation of IGFBP3 may be involved in the early stages of prostate cancer development. Hypermethylation of IGFBP3 was only detected in samples that also demonstrated methylation of GSTP1 and was also correlated with Gleason score > or =7 (P=0.01), indicating that it has potential as a prognostic marker. In addition, pharmacological demethylation induced strong expression of IGFBP3 in LNCaP prostate cancer cells. Our concept of a methylation candidate gene bank was successful in identifying a novel target of frequent hypermethylation in early-stage prostate cancer. Evaluation of further relevant genes could contribute towards a methylation signature of this disease.

Intersectin is a negative regulator of dynamin recruitment to the synaptic endocytic zone in the central synapse

Intersectin is a multidomain dynamin-binding protein implicated in numerous functions in the nervous system, including synapse formation and endocytosis. Here, we demonstrate that during neurotransmitter release in the central synapse, intersectin, like its binding partner dynamin, is redistributed from the synaptic vesicle pool to the periactive zone. Acute perturbation of the intersectin-dynamin interaction by microinjection of either intersectin antibodies or Src homology 3 (SH3) domains inhibited endocytosis at the fission step. Although the morphological effects induced by the different reagents were similar, antibody injections resulted in a dramatic increase in dynamin immunoreactivity around coated pits and at constricted necks, whereas synapses microinjected with the GST (glutathione S-transferase)-SH3C domain displayed reduced amounts of dynamin in the neck region. Our data suggest that intersectin controls the amount of dynamin released from the synaptic vesicle cluster to the periactive zone and that it may regulate fission of clathrin-coated intermediates.