Cholesteryl-6-O-acyl-alpha-D-glucopyranoside of Helicobacter pylori relate to relative lysophospholipid content

The presence of cholesteryl glucosides and high levels of lysophospholipids are elements making the cell wall of Helicobacter pylori unique. In this study, we have investigated the relationship between lysophospholipid content and cholesteryl glucoside composition of variants of 6 clinical isolates. The samples were characterized by diverse outer membrane phospholipase A activity measured as lysophospholipid content of the cell wall. A pldA negative mutant was also included in the study. Thin-layer chromatography showed that cholesteryl glucosides were present in all samples. However, the distribution of cholesteryl-6-O-acyl-alpha-D-glucopyranoside, cholesteryl-alpha-D-glucopyranoside and cholesteryl-6-O-phosphatidyl-alpha-D-glucopyranoside varied according to lysophospholipid content. Cholesteryl-6-O-acyl-alpha-D-glucopyranoside was exclusively observed in the isolates/variants with an intact pldA and where a significant amount of lysophospholipids could be demonstrated. High lysophospholipid content destabilizes membranes. The balance between cholesteryl-6-O-acyl-alpha-D-glucopyranoside, cholesteryl-alpha-D-glucopyranoside and cholesteryl-6-O-phosphatidyl-alpha-D-glucopyranoside in H. pylori is probably important for the stability of the membrane when the lysophospholipid content varies.     

Direct real-time PCR quantification of Campylobacter jejuni in chicken fecal and cecal samples by integrated cell concentration and DNA purification

Campylobacter jejuni is a major cause of diarrheal disease and food-borne gastroenteritis. The main reservoir of C. jejuni in poultry is the cecum, with an estimated content of 6 to 8 log10 CFU/g. If a flock is infected with C. jejuni, the majority of the birds in that flock will harbor the bacterium. Diagnostics at the flock level could thus be an important control point. The aim of the work presented here was to develop a complete quantitative PCR-based detection assay for C. jejuni obtained directly from cecal contents and fecal samples. We applied an approach in which the same paramagnetic beads were used both for cell isolation and for DNA purification. This integrated approach enabled both fully automated and quantitative sample preparation and a DNA extraction method. We developed a complete quantitative diagnostic assay through the combination of the sample preparation approach and real-time 5'-nuclease PCR. The assay was evaluated both by spiking the samples with C. jejuni and through the detection of C. jejuni in naturally colonized chickens. Detection limits between 2 and 25 CFU per PCR and a quantitative range of >4 log10 were obtained for spiked fecal and cecal samples. Thirty-one different poultry flocks were screened for naturally colonized chickens. A total of 262 (204 fecal and 58 cecal) samples were analyzed. Nineteen of the flocks were Campylobacter positive, whereas 12 were negative. Two of the flocks contained Campylobacter species other than C. jejuni. There was a large difference in the C. jejuni content, ranging from 4 to 8 log10 CFU/g of fecal or cecal material, for the different flocks tested. Some issues that have not yet promoted much attention are the prequantitative differences in the ability of C. jejuni to colonize poultry and the importance of these differences for causing human disease through food contamination. Understanding the colonization kinetics in poultry is therefore of great importance for controlling human infections by this bacterium.     

Biased distribution of DNA uptake sequences towards genome maintenance genes

Repeated sequence signatures are characteristic features of all genomic DNA. We have made a rigorous search for repeat genomic sequences in the human pathogens Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae and found that by far the most frequent 9–10mers residing within coding regions are the DNA uptake sequences (DUS) required for natural genetic transformation. More importantly, we found a significantly higher density of DUS within genes involved in DNA repair, recombination, restriction-modification and replication than in any other annotated gene group in these organisms. Pasteurella multocida also displayed high frequencies of a putative DUS identical to that previously identified in H.influenzae and with a skewed distribution towards genome maintenance genes, indicating that this bacterium might be transformation competent under certain conditions. These results imply that the high frequency of DUS in genome maintenance genes is conserved among phylogenetically divergent species and thus are of significant biological importance. Increased DUS density is expected to enhance DNA uptake and the over-representation of DUS in genome maintenance genes might reflect facilitated recovery of genome preserving functions. For example, transient and beneficial increase in genome instability can be allowed during pathogenesis simply through loss of antimutator genes, since these DUS-containing sequences will be preferentially recovered. Furthermore, uptake of such genes could provide a mechanism for facilitated recovery from DNA damage after genotoxic stress.     

Analysis of ETV6/RUNX1 fusions for evaluating the late effects of cancer therapy in ALL (acute lymphoblastic leukemia) cured patients

Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in childhood. The improvements of therapies have increased the number of long-term survivors. However, an increased incidence of secondary neoplasias has been observed in this cohort. Our purpose was to evaluate the late effects of cancer therapy in cured patients previously treated for ALL, considering previous reports on the occurrence of gene fusions as putative markers of chromosomal instability. Twelve ALL patients (aged 5 to 16 years) and twelve healthy subjects (aged 18 to 22 years) were studied for the presence of ETV6/RUNX1 (TEL/AML1) translocations, which were detected by FISH (fluorescence in situ hybridization). The blood samples were collected months or years after completion of the therapy, and the frequencies of gene fusions in lymphocytes were compared with those obtained retrospectively for bone marrow samples at the time of diagnosis, and also for the control group. It was demonstrated that ETV6/RUNX1 gene fusion was a frequent event (0.59-1.84/100 cells) in peripheral blood lymphocytes from normal individuals and the ALL patients who underwent chemotherapy showed significantly (P = 0.0043) increased frequencies (0.62-3.96/100 cells) of the rearrangement when compared with the control groups (patients at diagnosis and healthy subjects). However, a significant difference was not found between the groups of patients at diagnosis and healthy subjects, when the two patients who were positive for the rearrangement were excluded. Therefore, increased frequencies of ETV6/RUNX1 fusions in ALL cured patients indicate the influence of previous exposure to anti-cancer drugs, and they may represent an important genetic marker for estimating the risk of relapse, or development of secondary neoplasias.     

Metabolic Factors Associated with Risk of Renal Cell Carcinoma

Previous studies have shown that obesity and hypertension are associated with increased risk of renal cell carcinoma (RCC), but less is known about the association to other metabolic factors. In the Metabolic Syndrome and Cancer project (Me-Can) data on body mass index (BMI, kg/m2), blood pressure, and circulating levels of glucose, cholesterol, and triglycerides were collected from 560,388 men and women in cohorts from Norway, Austria, and Sweden. By use of Cox proportional hazard models, hazard ratios (HR) were calculated for separate and composite metabolic exposures. During a median follow-up of 10 years, 592 men and 263 women were diagnosed with RCC. Among men, we found an increased risk of RCC for BMI, highest vs. lowest quintile, (HR = 1.51, 95% CI 1.13–2.03), systolic blood pressure, (HR = 3.40, 95% CI 1.91–6.06), diastolic blood pressure, (HR = 3.33, 95% CI 1.85–5.99), glucose, (HR = 3.75, 95% CI 1.46–9.68), triglycerides, (HR = 1.79, 95% CI 1.00–3.21) and a composite score of these metabolic factors, (HR = 2.68, 95% CI 1.75–4.11). Among women we found an increased risk of RCC for BMI, highest vs. lowest quintile, (HR = 2.21, 95% CI 1.32–3.70) and the composite score, (HR = 2.29, 95% CI 1.12–4.68). High levels of the composite score were also associated with risk of death from RCC among both men and women. No multiplicative statistical or biological interactions between metabolic factors on risk of RCC were found. High levels of BMI, blood pressure, glucose and triglycerides among men and high BMI among women were associated with increased risk of RCC.     

Using proteomics to discover novel biomarkers for fatty liver development and response to CB1R antagonist treatment in an obese mouse model

Over activity of cannabinoid receptor type 1 (CB1R) plays a key role in increasing the incidence of obesity‐induced non‐alcoholic fatty liver disease. Tissue proteome analysis has been applied to investigate the bioinformatics regarding the mode of action and therapeutic mechanism. The aim of this study was to explore the potential pathways altered with CB1R in obesity‐induced fatty liver. Male C57BL/6 mice were fed either a standard chow diet (STD) or a high‐fat diet (HFD) with or without 1‐week treatment of CB1R inverse agonist AM251 at 5 mg/kg. Then, liver tissues were harvested for 2DE analysis and protein profiles were identified by using MALDI‐MS. Results showed that eight of significantly altered protein spots at the level of changes > twofold were overlapped among the three groups, naming major urinary protein 1, ATP synthase subunit β, glucosamine‐fructose‐6‐phosphate aminotransferase 1, zine finger protein 2, s‐adenosylmethionine synthase isoform type‐1, isocitrate dehydrogenase subunit α, epoxide hydrolase 2 and 60S acidic ribosomal protein P0. These identified proteins were involved in glucose/lipid metabolic process, xenobiotic metabolic system, and ATP synthesized process in mitochondria. Based on the findings, we speculated that CB1R blockade might exert its anti‐metabolic disorder effect via improvement of mitochondrial function in hepatic steatosis in HFD condition.     

Generation of transgenic mice producing a human transthyretin variant: a possible mouse model for familial amyloidotic polyneuropathy

Type I familial amyloidotic polyneuropathy (FAP) results from the systemic deposition of a plasma transthyretin (TTR) variant with a Val----Met change at position 30. In an attempt to establish a model of this disease, we generated transgenic mice producing the variant TTR. A DNA fragment containing the mouse metallothionein-I promoter fused to the structural gene coding for the human TTR variant was microinjected into fertilized mouse eggs. Among 72 mice that developed from these eggs, ten carried the fusion gene and three of these showed significant concentrations of the variant TTR in their serum. These mice may be useful in elucidating the pathogenesis of FAP and in establishing a therapy for this intractable disorder.