CtBP1/BARS is an activator of phospholipase D1 necessary for agonist-induced macropinocytosis

Vesicular trafficking such as macropinocytosis is a dynamic process that requires coordinated interactions between specialized proteins and lipids. A recent report suggests the involvement of CtBP1/BARS in epidermal growth factor (EGF)-induced macropinocytosis. Detailed mechanisms as to how lipid remodelling is regulated during macropinocytosis are still undefined. Here, we show that CtBP1/BARS is a physiological activator of PLD1 required in agonist-induced macropinocytosis. EGF-induced macropinocytosis was specifically blocked by 1-butanol but not by 2-butanol. In addition, stimulation of cells by serum or EGF resulted in the association of CtBP1/BARS with PLD1. Finally, CtBP1/BARS activated PLD1 in a synergistic manner with other PLD activators, including ADP-ribosylation factors as demonstrated by in vitro and intact cell systems. The present results shed light on the molecular basis of how the ‘fission protein' CtBP1/BARS controls vesicular trafficking events including macropinocytosis

Prostate transglutaminase (TGase-4) antagonizes the anti-tumour action of MDA-7/IL-24 in prostate cancer

Background Transglutamiase-4 (TGase-4), also known as prostate transglutaminase, belongs to the TGase family and is uniquely expressed in the prostate gland. The functions of this interesting protein are not clearly defined. In the present study, we have investigated an unexpected link between TGase-4 and the melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24), a cytokine known to regulate the growth and apoptosis of certain cancer and immune cells. Methods Frozen sections of normal and malignant human prostate tissues and human prostate cancer (PCa) cell lines PC-3 and CA-HPV-10, cell lines expressing low and high levels of TGase-4, and recombinant MDA-7/IL-24 (rhMDA-7/IL-24) were used. Expression construct for human TGase-4 was generated using a mammalian expression vector with full length human TGase-4 isolated from normal human prostate tissues. PC-3 cells were transfected with expression construct or control plasmid. Stably transfected cells for control transfection and TGase-4 over expression were created. Similarly, expression of TGase-4 in CA-HPV-10 cells were knocked down by way of ribozyme transgenes. Single and double immunofluorescence microscopy was used for localization and co-localization of TGase-4 and MDA-7/IL-24 in PCa tissues and cells with antibodies to TGase-4; MDA-7/IL-24; IL-20alpha; IL-20beta and IL-22R. Cell-matrix adhesion, attachment and migration were by electric cell substrate impedance sensing and growth by in vitro cell growth assay. A panel of small molecule inhibitors, including Akt, was used to determine signal pathways involving TGase-4 and MDA-7/IL-24. Results We initially noted that MDA-7 resulted in inhibition of cell adhesion, growth and migration of human PCa PC-3 cells which did not express TGase-4. However, after the cells over-expressed TGase-4 by way of transfection, the TGase-4 expressing cells lost their adhesion, growth and migratory inhibitory response to MDA-7. On the other hand, CA-HPV-10 cells, a cell type naturally expressing high levels of TGase-4, had a contrasting response to MDA-7 when compared with PC-3 cells. Inhibitor to Akt reversed the inhibitory effect of MDA-7, only in PC-3 control cells, but not the TGase-4 expressing PC-3 cells. In human prostate tissues, TGase-4 was found to have a good degree of co-localization with one of the MDA-7 receptor complexes, IL-20Ra. Conclusion The presence of TGase-4 has a biological impact on a prostate cancer cell's response to MDA-7. TGase-4, via mechanism(s) yet to be identified, blocked the action of MDA-7 in prostate cancer cells. This has an important implication when considering the use of MDA-7 as a potential anticancer cytokine in prostate cancer therapies

Reproductive factors and breast cancer risk according to joint estrogen and progesterone receptor status: a meta-analysis of epidemiological studies

INTRODUCTION: Although reproductive factors have been known for decades to be associated with breast cancer risk, it is unclear to what extent these associations differ by estrogen and progesterone receptor (ER/PR) status. This report presents the first meta-analysis of results from epidemiological studies that have investigated parity, age at first birth, breastfeeding, and age at menarche in relation to ER(+)PR(+ )and ER(-)PR(- )cancer risk. MATERIALS AND METHODS: We calculated summary relative risks (RRs) and corresponding 95% confidence intervals (CIs) using a fixed effects model. RESULTS: Each birth reduced the risk of ER(+)PR(+ )cancer by 11% (RR per birth = 0.89, 95% CI = 0.84–0.94), and women who were in the highest age at first birth category had, on average, 27% higher risk of ER(+)PR(+ )cancer compared with women who were in the youngest age at first birth category (RR = 1.27, 95% CI = 1.07–1.50). Neither parity nor age at first birth was associated with the risk of ER(-)PR(- )cancer (RR per birth = 0.99, 95% CI = 0.94–1.05; RR of oldest versus youngest age at first birth category = 1.01, 95% CI = 0.85–1.20). Breastfeeding and late age at menarche decreased the risk of both receptor subtypes of breast cancer. The protective effect of late age at menarche was statistically significantly greater for ER(+)PR(+ )than ER(-)PR(- )cancer (RR = 0.72 for ER(+)PR(+ )cancer; RR = 0.84 for ER(-)PR(- )cancer, p for homogeneity = 0.006). CONCLUSION: Our findings suggest that breastfeeding (and age at menarche) may act through different hormonal mechanisms than do parity and age at first birth

Proteomic Analysis of Human Skin Treated with Larval Schistosome Peptidases Reveals Distinct Invasion Strategies among Species of Blood Flukes

Schistosome parasites are a major cause of disease in the developing world, but the mechanism by which these parasites first infect their host has been studied at the molecular level only for S. mansoni. In this paper, we have mined recent genome annotations of S. mansoni and S. japonicum, a zoonotic schistosome species, to identify differential expansion of peptidase gene families that may be involved in parasite invasion and subsequent migration through skin. Having identified a serine peptidase gene family in S. mansoni and a cysteine peptidase gene family in S. japonicum, we then used a comparative proteomic approach to identify potential substrates of representative members of both classes of enzymes from S. mansoni in human skin. The results of this study suggest that while these species evolved to use different classes of peptidases in host invasion, both are capable of cleaving components of the epidermis and dermal extracellular matrix, as well as proteins involved in the host immune response against the migrating parasite

A Model Analysis of Arterial Oxygen Desaturation during Apnea in Preterm Infants

Rapid arterial O2 desaturation during apnea in the preterm infant has obvious clinical implications but to date no adequate explanation for why it exists. Understanding the factors influencing the rate of arterial O2 desaturation during apnea () is complicated by the non-linear O2 dissociation curve, falling pulmonary O2 uptake, and by the fact that O2 desaturation is biphasic, exhibiting a rapid phase (stage 1) followed by a slower phase when severe desaturation develops (stage 2). Using a mathematical model incorporating pulmonary uptake dynamics, we found that elevated metabolic O2 consumption accelerates throughout the entire desaturation process. By contrast, the remaining factors have a restricted temporal influence: low pre-apneic alveolar causes an early onset of desaturation, but thereafter has little impact; reduced lung volume, hemoglobin content or cardiac output, accelerates during stage 1, and finally, total blood O2 capacity (blood volume and hemoglobin content) alone determines during stage 2. Preterm infants with elevated metabolic rate, respiratory depression, low lung volume, impaired cardiac reserve, anemia, or hypovolemia, are at risk for rapid and profound apneic hypoxemia. Our insights provide a basic physiological framework that may guide clinical interpretation and design of interventions for preventing sudden apneic hypoxemia

Potential Associations between Severity of Infection and the Presence of Virulence-Associated Genes in Clinical Strains of Staphylococcus aureus

BACKGROUND: The clinical spectrum of Staphylococcus aureus infection ranges from asymptomatic nasal carriage to osteomyelitis, infective endocarditis (IE) and death. In this study, we evaluate potential association between the presence of specific genes in a collection of prospectively characterized S. aureus clinical isolates and clinical outcome. METHODOLOGY/PRINCIPAL FINDINGS: Two hundred thirty-nine S. aureus isolates (121 methicillin-resistant S. aureus [MRSA] and 118 methicillin-susceptible S. aureus [MSSA]) were screened by array comparative genomic hybridization (aCGH) to identify genes implicated in complicated infections. After adjustment for multiple tests, 226 genes were significantly associated with severity of infection. Of these 226 genes, 185 were not in the SCCmec element. Within the 185 non-SCCmec genes, 171 were less common and 14 more common in the complicated infection group. Among the 41 genes in the SCCmec element, 37 were more common and 4 were less common in the complicated group. A total of 51 of the 2014 sequences evaluated, 14 non-SCCmec and 37 SCCmec, were identified as genes of interest. CONCLUSIONS/SIGNIFICANCE: Of the 171 genes less common in complicated infections, 152 are of unknown function and may contribute to attenuation of virulence. The 14 non-SCCmec genes more common in complicated infections include bacteriophage-encoded genes such as regulatory factors and autolysins with potential roles in tissue adhesion or biofilm formation

The FGFR4-G388R Polymorphism Promotes Mitochondrial STAT3 Serine Phosphorylation to Facilitate Pituitary Growth Hormone Cell Tumorigenesis

Pituitary tumors are common intracranial neoplasms, yet few germline abnormalities have been implicated in their pathogenesis. Here we show that a single nucleotide germline polymorphism (SNP) substituting an arginine (R) for glycine (G) in the FGFR4 transmembrane domain can alter pituitary cell growth and hormone production. Compared with FGFR4-G388 mammosomatotroph cells that support prolactin (PRL) production, FGFR4-R388 cells express predominantly growth hormone (GH). Growth promoting effects of FGFR4-R388 as evidenced by enhanced colony formation was ascribed to Src activation and mitochondrial serine phosphorylation of STAT3 (pS-STAT3). In contrast, diminished pY-STAT3 mediated by FGFR4-R388 relieved GH inhibition leading to hormone excess. Using a knock-in mouse model, we demonstrate the ability of FGFR4-R385 to promote GH pituitary tumorigenesis. In patients with acromegaly, pituitary tumor size correlated with hormone excess in the presence of the FGFR4-R388 but not the FGFR4-G388 allele. Our findings establish a new role for the FGFR4-G388R polymorphism in pituitary oncogenesis, providing a rationale for targeting Src and STAT3 in the personalized treatment of associated disorders

Staphylococcus aureus Keratinocyte Invasion Is Dependent upon Multiple High-Affinity Fibronectin-Binding Repeats within FnBPA

Staphylococcus aureus is a commensal organism and a frequent cause of skin and soft tissue infections, which can progress to serious invasive disease. This bacterium uses its fibronectin binding proteins (FnBPs) to invade host cells and it has been hypothesised that this provides a protected niche from host antimicrobial defences, allows access to deeper tissues and provides a reservoir for persistent or recurring infections. FnBPs contain multiple tandem fibronectin-binding repeats (FnBRs) which bind fibronectin with varying affinity but it is unclear what selects for this configuration. Since both colonisation and skin infection are dependent upon the interaction of S. aureus with keratinocytes we hypothesised that this might select for FnBP function and thus composition of the FnBR region. Initial experiments revealed that S. aureus attachment to keratinocytes is rapid but does not require FnBRs. By contrast, invasion of keratinocytes was dependent upon the FnBR region and occurred via similar cellular processes to those described for endothelial cells. Despite this, keratinocyte invasion was relatively inefficient and appeared to include a lag phase, most likely due to very weak expression of α5β1 integrins. Molecular dissection of the role of the FnBR region revealed that efficient invasion of keratinocytes was dependent on the presence of at least three high-affinity (but not low-affinity) FnBRs. Over-expression of a single high-affinity or three low-affinity repeats promoted invasion but not to the same levels as S. aureus expressing an FnBPA variant containing three high-affinity repeats. In summary, invasion of keratinocytes by S. aureus requires multiple high-affinity FnBRs within FnBPA, and given the importance of the interaction between these cell types and S. aureus for both colonisation and infection, may have provided the selective pressure for the multiple binding repeats within FnBPA

A comprehensive analysis of common genetic variation in prolactin (PRL) and PRL receptor (PRLR) genes in relation to plasma prolactin levels and breast cancer risk: the Multiethnic Cohort

<p>Abstract</p> <p>Background</p> <p>Studies in animals and humans clearly indicate a role for prolactin (PRL) in breast epithelial proliferation, differentiation, and tumorigenesis. Prospective epidemiological studies have also shown that women with higher circulating PRL levels have an increase in risk of breast cancer, suggesting that variability in PRL may also be important in determining a woman's risk.</p> <p>Methods</p> <p>We evaluated genetic variation in the PRL and PRL receptor (PRLR) genes as predictors of plasma PRL levels and breast cancer risk among African-American, Native Hawaiian, Japanese-American, Latina, and White women in the Multiethnic Cohort Study (MEC). We selected single nucleotide polymorphisms (SNPs) from both the public (dbSNP) and private (Celera) databases to construct high density SNP maps that included up to 20 kilobases (kb) upstream of the transcription initiation site and 10 kb downstream of the last exon of each gene, for a total coverage of 59 kb in PRL and 210 kb in PRLR. We genotyped 80 SNPs in PRL and 173 SNPs in PRLR in a multiethnic panel of 349 unaffected subjects to characterize linkage disequilibrium (LD) and haplotype patterns. We sequenced the coding regions of PRL and PRLR in 95 advanced breast cancer cases (19 of each racial/ethnic group) to uncover putative functional variation. A total of 33 and 60 haplotype "tag" SNPs (tagSNPs) that allowed for high predictability (R_h²≥ 0.70) of the common haplotypes in PRL and PRLR, respectively, were then genotyped in a multiethnic breast cancer case-control study of 1,615 invasive breast cancer cases and 1,962 controls in the MEC. We also assessed the association of common genetic variation with circulating PRL levels in 362 postmenopausal controls without a history of hormone therapy use at blood draw. Because of the large number of comparisons being performed we used a relatively stringent type I error criteria (p < 0.0005) for evaluating the significance of any single association to correct for performing approximately 100 independent tests, close to the number of tagSNPs genotyped for both genes.</p> <p>Results</p> <p>We observed no significant associations between PRL and PRLR haplotypes or individual SNPs in relation to breast cancer risk. A nominally significant association was noted between prolactin levels and a tagSNP (tagSNP 44, rs2244502) in intron 1 of PRL. This SNP showed approximately a 50% increase in levels between minor allele homozygotes vs. major allele homozygotes. However, this association was not significant (p = 0.002) using our type I error criteria to correct for multiple testing, nor was this SNP associated with breast cancer risk (p = 0.58).</p> <p>Conclusion</p> <p>In this comprehensive analysis covering 59 kb of the PRL locus and 210 kb of the PRLR locus, we found no significant association between common variation in these candidate genes and breast cancer risk or plasma PRL levels. The LD characterization of PRL and PRLR in this multiethnic population provide a framework for studying these genes in relation to other disease outcomes that have been associated with PRL, as well as for larger studies of plasma PRL levels.</p

Epistatic Relationships between sarA and agr in Staphylococcus aureus Biofilm Formation

Background: The accessory gene regulator (agr) and staphylococcal accessory regulator (sarA) play opposing roles in Staphylococcus aureus biofilm formation. There is mounting evidence to suggest that these opposing roles are therapeutically relevant in that mutation of agr results in increased biofilm formation and decreased antibiotic susceptibility while mutation of sarA has the opposite effect. To the extent that induction of agr or inhibition of sarA could potentially be used to limit biofilm formation, this makes it important to understand the epistatic relationships between these two loci. Methodology/Principal Findings: We generated isogenic sarA and agr mutants in clinical isolates of S. aureus and assessed the relative impact on biofilm formation. Mutation of agr resulted in an increased capacity to forma biofilmin the 8325-4 laboratory strain RN6390 but had little impact in clinical isolates S. aureus. In contrast, mutation of sarA resulted in a reduced capacity to form a biofilm in all clinical isolates irrespective of the functional status of agr. This suggests that the regulatory role of sarA in biofilm formation is independent of the interaction between sarA and agr and that sarA is epistatic to agr in this context. This was confirmed by demonstrating that restoration of sarA function restored the ability to form a biofilm even in the corresponding agr mutants. Mutation of sarA in clinical isolates also resulted in increased production of extracellular proteases and extracellular nucleases, both of which contributed to the biofilm-deficient phenotype of sarA mutants. However, studies comparing different strains with and without proteases inhibitors and/or mutation of the nuclease genes demonstrated that the agr-independent, sarA-mediated repression of extracellular proteases plays a primary role in this regard. Conclusions and Significance: The results we report suggest that inhibitors of sarA-mediated regulation could be used to limit biofilm formation in S. aureus and that the efficacy of such inhibitors would not be limited by spontaneous mutation of agr in the human host