Ecologists undertaking stable isotopic analyses of animal diets require trophic enrichment factors (TEFs) for the specific animal tissues that they are studying. Such basic data are available for a small number of species, so values from trophically or phylogenetically similar species are often substituted for missing values. By feeding a controlled diet to captive European badgers (Meles meles) we determined TEFs for carbon and nitrogen in blood serum. TEFs for nitrogen and carbon in blood serum were +3.0±0.4‰ and +0.4±0.1‰ respectively. The TEFs for serum in badgers are notably different from those published for the red fox (Vulpes vulpes). There is currently no data for TEFs in the serum of other mustelid species. Our data show that species sharing similar niches (red fox) do not provide adequate proxy values for TEFs of badgers. Our findings emphasise the importance of having species-specific data when undertaking trophic studies using stable isotope analysis.
We report a now 6-year-old African-American male with both Alagille syndrome and pediatric sarcoidosis. With a prior JAG1 mutation positive diagnosis of Alagille syndrome, he presented to the hospital with a subacute, predominantly respiratory febrile condition, eventually diagnosed as sarcoidosis. A liver biopsy revealed paucity of bile ducts and scattered epithelioid granulomas, while a skin biopsy showed granulomatous angiitis, a manifestation of sarcoidosis not yet reported in a pediatric patient. He has subsequently been treated with corticosteroids, mycophenolate mofetil, and infliximab with clinical response. Alagille syndrome and sarcoidosis have not yet been reported in the medical literature in the same patient to the best of our knowledge. We briefly review these two seemingly unrelated conditions and propose a possible common pathogenic mechanism.
Sarcoidosis; Alagille syndrome; Granulomatous angiitis; Granuloma; JAG1; NOTCH1; T lymphocyte
The relationship between health-related quality of life (HRQoL) in people with Parkinson’s disease and their caregivers is little understood and any effects on caregiver strain remain unclear. This paper examines these relationships in an Australian sample.
Using the generic EuroQol (EQ-5D) and disease-specific Parkinson’s Disease Questionnaire-39 Item (PDQ-39), HRQoL was evaluated in a sample of 97 people with PD and their caregivers. Caregiver strain was assessed using the Modified Caregiver Strain Index. Associations were evaluated between: (i) caregiver and care-recipient HRQoL; (ii) caregiver HRQoL and caregiver strain, and; (iii) between caregiver strain and care-recipient HRQoL.
No statistically significant relationships were found between caregiver and care-recipient HRQoL, or between caregiver HRQoL and caregiver strain. Although this Australian sample of caregivers experienced relatively good HRQoL and moderately low strain, a significant correlation was found between HRQoL of people with PD and caregiver strain (rho 0.43, p < .001).
Poor HRQoL in people with PD is associated with higher strain in caregivers. Therapy interventions may target problems reported as most troublesome by people with PD, with potential to reduce strain on the caregiver.
The quinoxaline core is considered a privileged scaffold as it is found in a variety of biologically relevant molecules. Here we report the synthesis of a quinoxalin-6-amine library, screening against a panel of cancer cell lines and a structure activity relationship (SAR). This resulted in the identification of a bisfuranylquinoxalineurea analog (7c) that has low micromolar potency against the panel of cancer cell lines. We also show that cells treated with quinoxalineurea 7c results in caspase 3/7 activation, PARP cleavage and Mcl-1 dependent apoptosis.
Quinoxaline urea; Antiproliferative; Mcl-1 dependent apoptosis
To evaluate the consequences of expression of the protein encoded by PAX3-FOXO1 (P3F) in the pediatric malignancy alveolar rhabdomyosarcoma (A-RMS), we developed and evaluated a genetically defined in vitro model of A-RMS tumorigenesis. The expression of P3F in cooperation with simian virus 40 (SV40) Large-T (LT) antigen in murine C3H10T1/2 fibroblasts led to robust malignant transformation. Using 2 dimensional difference gel electrophoresis (2D-DIGE) we compared proteomes from lysates from cells that express P3F + LT versus from cells that express LT alone. Analysis of 2D gel spot patterns by DeCyder™ image analysis software indicated 93 spots that were different in abundance. Peptide mass fingerprint analysis of the 93 spots by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified 37 non-redundant proteins. 2D DIGE analysis of cell culture media conditioned by cells transduced by P3F + LT versus by LT alone found 29 spots in the P3F + LT cells leading to the identification of 11 non-redundant proteins. A substantial number of proteins with potential roles in tumorigenesis and myogenesis were detected, most of which have not been identified in previous wide-scale expression studies of RMS experimental models or tumors. We validated the 2D gel image analysis findings by western blot analysis and immunohistochemistry (IHC). Thus, the 2D DIGE proteomics methodology described here provided an important discovery approach to the study of RMS biology and complements the findings of previous mRNA expression studies.
We present a new method for inferring hidden Markov models from noisy time sequences without the necessity of assuming a model architecture, thus allowing for the detection of degenerate states. This is based on the statistical prediction techniques developed by Crutchfield et al. and generates so called causal state models, equivalent in structure to hidden Markov models. The new method is applicable to any continuous data which clusters around discrete values and exhibits multiple transitions between these values such as tethered particle motion data or Fluorescence Resonance Energy Transfer (FRET) spectra. The algorithms developed have been shown to perform well on simulated data, demonstrating the ability to recover the model used to generate the data under high noise, sparse data conditions and the ability to infer the existence of degenerate states. They have also been applied to new experimental FRET data of Holliday Junction dynamics, extracting the expected two state model and providing values for the transition rates in good agreement with previous results and with results obtained using existing maximum likelihood based methods. The method differs markedly from previous Markov-model reconstructions in being able to uncover truly hidden states.
Background & Aims
Premature neonates are predisposed to necrotizing enterocolitis (NEC), an idiopathic, inflammatory bowel necrosis. We investigated the hypothesis that NEC occurs in the preterm intestine due to incomplete ‘non-inflammatory’ differentiation of intestinal macrophages, which increases the risk of a severe mucosal inflammatory response to bacterial products.
We compared inflammatory properties of human/murine fetal, neonatal, and adult intestinal macrophages. To investigate gut-specific macrophage differentiation, we next treated monocyte-derived macrophages with conditioned media from ex planted human fetal and adult intestinal tissues. Transforming growth factor-beta (TGF-β) expression and bioactivity were measured in fetal/adult intestine and in NEC. Finally, we used wild-type and transgenic mice to investigate the effects of deficient TGF-β signaling on NEC-like inflammatory mucosal injury.
Intestinal macrophages in the human preterm intestine (fetus/premature neonate), but not in full-term neonates and adults, expressed inflammatory cytokines. Macrophage cytokine production was suppressed in the developing intestine by TGF-β, particularly the TGF-β2 isoform. NEC was associated with decreased tissue expression of TGF-β2 and decreased TGF-β bioactivity. In mice, disruption of TGF-β signaling worsened NEC-like inflammatory mucosal injury, whereas enteral supplementation with recombinant TGF-β2 was protective.
Intestinal macrophages progressively acquire a non-inflammatory profile during gestational development. TGF-β, particularly the TGF-β2 isoform, suppresses macrophage inflammatory responses in the developing intestine and protects against inflammatory mucosal injury. Enterally-administered TGF-β2 protected mice from experimental NEC-like injury.
necrotizing enterocolitis; macrophage; newborn; inflammation; TGF-β
Face perception remains one of the most intensively researched areas in psychology and allied disciplines, and there has been much debate regarding the early origins and experiential determinants of face processing. This article reviews studies, the majority of which have appeared in the past decade, that discuss possible mechanisms underlying face perception at birth and document the prominent role of experience in shaping infants’ face-processing abilities. In the first months of life, infants develop a preference for female and own-race faces and become better able to recognize and categorize own-race and own-species faces. This perceptual narrowing and shaping of the “face space” forms a foundation for later face expertise in childhood and adulthood and testifies to the remarkable plasticity of the developing visual system.
infancy; face perception; neural plasticity; own-race effect; own-species effect; gender preferences; perceptual narrowing
The ER–inner nuclear membrane trafficking of 15 integral membrane proteins followed by FRAP shows distinct ATP- and Ran-dependent translocation mechanisms.
The nuclear envelope contains >100 transmembrane proteins that continuously exchange with the endoplasmic reticulum and move within the nuclear membranes. To better understand the organization and dynamics of this system, we compared the trafficking of 15 integral nuclear envelope proteins using FRAP. A surprising 30-fold range of mobilities was observed. The dynamic behavior of several of these proteins was also analyzed after depletion of ATP and/or Ran, two functions implicated in endoplasmic reticulum–inner nuclear membrane translocation. This revealed that ATP- and Ran-dependent translocation mechanisms are distinct and not used by all inner nuclear membrane proteins. The Ran-dependent mechanism requires the phenylalanine-glycine (FG)-nucleoporin Nup35, which is consistent with use of the nuclear pore complex peripheral channels. Intriguingly, the addition of FGs to membrane proteins reduces FRAP recovery times, and this also depends on Nup35. Modeling of three proteins that were unaffected by either ATP or Ran depletion indicates that the wide range in mobilities could be explained by differences in binding affinities in the inner nuclear membrane.
Repo-Man targets protein phosphatase 1 γ (PP1γ) to chromatin at anaphase onset and regulates chromosome structure during mitotic exit. Here, we show that a Repo-Man:PP1 complex forms in anaphase following dephosphorylation of Repo-Man. Upon activation, the complex localizes to chromosomes and causes the dephosphorylation of histone H3 (Thr3, Ser10, and Ser28). In anaphase, Repo-Man has both catalytic and structural functions that are mediated by two separate domains. A C-terminal domain localizes Repo-Man to bulk chromatin in early anaphase. There, it targets PP1 for the dephosphorylation of histone H3 and possibly other chromosomal substrates. An N-terminal domain localizes Repo-Man to the chromosome periphery later in anaphase. There, it is responsible for the recruitment of nuclear components such as Importin β and Nup153 in a PP1-independent manner. These observations identify Repo-Man as a key factor that coordinates chromatin remodeling and early events of nuclear envelope reformation during mitotic exit.
► Repo-Man/PP1 complex is activated in anaphase by Repo-Man dephosphorylation ► Repo-Man/PP1 dephosphorylates histone H3 at Thr3, Ser10, and Ser28 ► Repo-Man binds Importin β in anaphase and targets it to the chromosome periphery ► Repo-Man has both catalytic and structural functions during mitotic exit
Although physical therapy and falls prevention education are argued to reduce falls and disability in people with idiopathic Parkinson's disease, this has not yet been confirmed with a large scale randomised controlled clinical trial. The study will investigate the effects on falls, mobility and quality of life of (i) movement strategy training combined with falls prevention education, (ii) progressive resistance strength training combined with falls prevention education, (iii) a generic life-skills social program (control group).
People with idiopathic Parkinson's disease who live at home will be recruited and randomly allocated to one of three groups. Each person shall receive therapy in an out-patient setting in groups of 3-4. Each group shall be scheduled to meet once per week for 2 hours for 8 consecutive weeks. All participants will also have a structured 2 hour home practice program for each week during the 8 week intervention phase. Assessments will occur before therapy, after the 8 week therapy program, and at 3 and 12 months after the intervention. A falls calendar will be kept by each participant for 12 months after outpatient therapy.
Consistent with the recommendations of the Prevention of Falls Network Europe group, three falls variables will be used as the primary outcome measures: the number of fallers, the number of multiple fallers and the falls rate. In addition to quantifying falls, we shall measure mobility, activity limitations and quality of life as secondary outcomes.
This study has the potential to determine whether outpatient movement strategy training combined with falls prevention education or progressive resistance strength training combined with falls prevention education are effective for reducing falls and improving mobility and life quality in people with Parkinson's disease who live at home.
Australia and New Zealand Clinical Trials Register (ANZCTR): ACTRN12606000344594
Adults from Eastern (e.g., China) and Western (e.g., USA) cultural groups display pronounced differences in a range of visual processing tasks. For example, the eye movement strategies used for information extraction during a variety of face processing tasks (e.g., identification and facial expressions of emotion categorization) differs across cultural groups. Currently, many of the differences reported in previous studies have asserted that culture itself is responsible for shaping the way we process visual information, yet this has never been directly investigated. In the current study, we assessed the relative contribution of genetic and cultural factors by testing face processing in a population of British Born Chinese adults using face recognition and expression classification tasks. Contrary to predictions made by the cultural differences framework, the majority of British Born Chinese adults deployed “Eastern” eye movement strategies, while approximately 25% of participants displayed “Western” strategies. Furthermore, the cultural eye movement strategies used by individuals were consistent across recognition and expression tasks. These findings suggest that “culture” alone cannot straightforwardly account for diversity in eye movement patterns. Instead a more complex understanding of how the environment and individual experiences can influence the mechanisms that govern visual processing is required.
culture; eye movements; face processing; individual differences
Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore
Here, centromeric histone marks on a human artificial chromosome are found to resemble the chromatin landscape in transcribed genes, and selective manipulation shows them to govern the incorporation of the centromere-specifying CENP-A histone variant.
Kinetochores assemble on distinct ‘centrochromatin' containing the histone H3 variant CENP-A and interspersed nucleosomes dimethylated on H3K4 (H3K4me2). Little is known about how the chromatin environment at active centromeres governs centromeric structure and function. Here, we report that centrochromatin resembles K4–K36 domains found in the body of some actively transcribed housekeeping genes. By tethering the lysine-specific demethylase 1 (LSD1), we specifically depleted H3K4me2, a modification thought to have a role in transcriptional memory, from the kinetochore of a synthetic human artificial chromosome (HAC). H3K4me2 depletion caused kinetochores to suffer a rapid loss of transcription of the underlying α-satellite DNA and to no longer efficiently recruit HJURP, the CENP-A chaperone. Kinetochores depleted of H3K4me2 remained functional in the short term, but were defective in incorporation of CENP-A, and were gradually inactivated. Our data provide a functional link between the centromeric chromatin, α-satellite transcription, maintenance of CENP-A levels and kinetochore stability.
CENP-A; centromere; chromatin; kinetochore; non-coding RNA
Many systems regulating cell polarity involve stable landmarks defined by internal cues [1–5]. In the rod-shaped fission yeast Schizosaccharomyces pombe, microtubules regulate polarized vegetative growth via a landmark involving the protein Tea1 [6–9]. Tea1 is delivered to cell tips as packets of molecules associated with growing microtubule ends  and anchored at the plasma membrane via a mechanism involving interaction with the membrane protein Mod5 [11, 12]. Tea1 and Mod5 are highly concentrated in clusters at cell tips in a mutually dependent manner, but how the Tea1-Mod5 interaction contributes mechanistically to generating a stable landmark is not understood. Here, we use live-cell imaging, FRAP, and computational modeling to dissect dynamics of the Tea1-Mod5 interaction. Surprisingly, we find that Tea1 and Mod5 exhibit distinctly different turnover rates at cell tips. Our data and modeling suggest that rather than acting simply as a Tea1 receptor or as a molecular “glue” to retain Tea1, Mod5 functions catalytically to stimulate incorporation of Tea1 into a stable tip-associated cluster network. The model also suggests an emergent self-focusing property of the Tea1-Mod5 cluster network, which can increase the fidelity of polarized growth.
► Interacting polarity proteins Tea1 and Mod5 have different mobilities at cell tips ► Modeling suggests Tea1 assembles into polymeric cluster networks ► Mod5 acts catalytically in formation of Tea1 cluster networks ► Self-focusing property of cluster networks increases fidelity of polarized growth
The Schizosaccharomyces pombe glucose/cyclic AMP (cAMP) signaling pathway includes the Gpa2-Git5-Git11 heterotrimeric G protein, whose Gpa2 Gα subunit directly binds to and activates adenylate cyclase in response to signaling from the Git3 G protein-coupled receptor. To study intrinsic and extrinsic regulation of Gpa2, we developed a plasmid-based screen to identify mutationally activated gpa2 alleles that bypass the loss of the Git5-Git11 Gβγ dimer to repress transcription of the glucose-regulated fbp1+ gene. Fifteen independently isolated mutations alter 11 different Gpa2 residues, with all but one conferring a receptor-independent activated phenotype upon integration into the gpa2+ chromosomal locus. Biochemical characterization of three activated Gpa2 proteins demonstrated an increased GDP-GTP exchange rate that would explain the mechanism of activation. Interestingly, the amino acid altered in the Gpa2(V90A) exchange rate mutant protein is in a region of Gpa2 with no obvious role in Gα function, thus extending our understanding of Gα protein structure-function relationships.
Epithelial ovarian carcinoma (EOC) is a leading cause of death from gynecologic malignancy, due mainly to the prevalence of undetected metastatic disease. The process of cell invasion during intra-peritoneal anchoring of metastatic lesions requires concerted regulation of many processes, including modulation of adhesion to the extracellular matrix and localized invasion. Exploratory cDNA microarray analysis of early response genes (altered after 4 hours of 3-dimensional collagen culture) coupled with confirmatory real-time RT-PCR, multiple three-dimensional cell culture matrices, Western blot, immunostaining, adhesion, migration, and invasion assays were used to identify modulators of adhesion pertinent to EOC progression and metastasis. cDNA microarray analysis indicated a dramatic downregulation of connective tissue growth factor (CTGF) in EOC cells placed in invasion-mimicking conditions (3-dimensional type I collagen). Examination of human EOC specimens revealed that CTGF expression was absent in 46% of the tested samples (n=41), but was present in 100% of normal ovarian epithelium samples (n=7). Reduced CTGF expression occurs in many types of cells and may be a general phenomenon displayed by cells encountering a 3D environment. CTGF levels were inversely correlated with invasion such that downregulation of CTGF increased, while its upregulation reduced, collagen invasion. Cells adhered preferentially to a surface comprised of both collagen I and CTGF relative to either component alone using α6β1 and α3β1 integrins. Together these data suggest that downregulation of CTGF in EOC cells may be important for cell invasion through modulation of cell-matrix adhesion.
ovarian carcinoma; metastasis; invasion; adhesion; connective tissue growth factor
A favored hypothesis to explain the pathology underlying nuclear envelopathies is that mutations in nuclear envelope proteins alter genome/chromatin organization and thus gene expression. To identify nuclear envelope proteins that play roles in genome organization, we analyzed nuclear envelopes from resting and phytohemagglutinin-activated leukocytes because leukocytes have a particularly high density of peripheral chromatin that undergoes significant reorganization upon such activation. Thus, nuclear envelopes were isolated from leukocytes in the two states and analyzed by multidimensional protein identification technology using an approach that used expected contaminating membranes as subtractive fractions. A total of 3351 proteins were identified between both nuclear envelope data sets among which were 87 putative nuclear envelope transmembrane proteins (NETs) that were not identified in a previous proteomics analysis of liver nuclear envelopes. Nuclear envelope localization was confirmed for 11 new NETs using tagged fusion proteins and antibodies on spleen cryosections. 27% of the new proteins identified were unique to one or the other of the two leukocyte states. Differences in expression between activated and resting leukocytes were confirmed for some NETs by RT-PCR, and most of these proteins appear to only be expressed in certain types of blood cells. Several known proteins identified in both data sets have functions in chromatin organization and gene regulation. To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects. One screen found two NETs that can recruit a specific gene locus to the nuclear periphery, and the second found a different NET that promotes chromatin condensation. The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.
Extracorporeal membrane oxygenation (ECMO) is a life-saving support system used in neonates and young children with severe cardiorespiratory failure. Although ECMO has reduced mortality in these critically-ill patients, almost all patients treated with ECMO develop a systemic inflammatory response syndrome (SIRS) characterized by a ‘cytokine storm’, leukocyte activation, and multisystem organ dysfunction. We used a neonatal porcine model of ECMO to investigate whether rising plasma concentrations of inflammatory cytokines during ECMO reflect de novo synthesis of these mediators in inflamed tissues, and therefore, can be used to assess the severity of ECMO-related SIRS.
Three-week-old previously-healthy piglets were subjected to venoarterial ECMO for up to 8 hours. SIRS was assessed by histopathological analysis, measurement of neutrophil activation (flow cytometry), plasma cytokine concentrations (enzyme immunoassays), and tissue expression of inflammatory genes (polymerase chain reaction/western blots). Mast cell degranulation was investigated by measurement of plasma tryptase activity.
Porcine neonatal ECMO was associated with systemic inflammatory changes similar to those seen in human neonates. TNF-α and interleukin-8 (IL-8) concentrations rose rapidly during the first 2 hours of ECMO, faster than the tissue expression of these cytokines. ECMO was associated with increased plasma mast cell tryptase activity, indicating that increased plasma concentrations of inflammatory cytokines during ECMO may result from mast cell degranulation and associated release of preformed cytokines stored in mast cells.
TNF-α and IL-8 concentrations rose faster in plasma than in the peripheral tissues during ECMO, indicating that rising plasma levels of these cytokines immediately following the initiation of ECMO may not reflect increasing tissue synthesis of these cytokines. Mobilization of preformed cellular stores of inflammatory cytokines such as in mucosal mast cells may play an important pathophysiological role in ECMO-related SIRS.
ECMO; venoarterial; neonate; SIRS; mast cells; intestine; CRP; cytokines
Culture affects the way people move their eyes to extract information in their visual world. Adults from Eastern societies (e.g., China) display a disposition to process information holistically, whereas individuals from Western societies (e.g., Britain) process information analytically. In terms of face processing, adults from Western cultures typically fixate the eyes and mouth, while adults from Eastern cultures fixate centrally on the nose region, yet face recognition accuracy is comparable across populations. A potential explanation for the observed differences relates to social norms concerning eye gaze avoidance/engagement when interacting with conspecifics. Furthermore, it has been argued that faces represent a ‘special’ stimulus category and are processed holistically, with the whole face processed as a single unit. The extent to which the holistic eye movement strategy deployed by East Asian observers is related to holistic processing for faces is undetermined. To investigate these hypotheses, we recorded eye movements of adults from Western and Eastern cultural backgrounds while learning and recognizing visually homogeneous objects: human faces, sheep faces and greebles. Both group of observers recognized faces better than any other visual category, as predicted by the specificity of faces. However, East Asian participants deployed central fixations across all the visual categories. This cultural perceptual strategy was not specific to faces, discarding any parallel between the eye movements of Easterners with the holistic processing specific to faces. Cultural diversity in the eye movements used to extract information from visual homogenous objects is rooted in more general and fundamental mechanisms.
culture; eye movements; face processing
Advanced and metastatic ovarian cancer is a leading cause of death from gynecologic malignancies. A more detailed understanding of the factors controlling invasion and metastasis may lead to novel anti-metastatic therapies. To model cellular interactions that occur during intraperitoneal metastasis, comparative cDNA microarray analysis and confirmatory real time RT-PCR were employed to uncover changes in gene expression that may occur in late stage ovarian cancer in response to microenvironmental cues, particularly native three-dimensional collagen I. Gene expression in human ovarian carcinoma tissues was evaluated on the RNA and protein level using real time RT-PCR and immunohistochemistry. Cell invasion and migration were evaluated in a collagen invasion assay and a scratch wound assay. Three-dimensional collagen I culture led to differential expression of several genes. The role of actinin alpha-4 (ACTN4), a cytosketeton-associated protein implicated in regulation of cell motility, was examined in detail. ACTN4 RNA and protein expression were associated with advanced and metastatic human ovarian carcinoma. This report demonstrates that a cytoskeletal-associated protein ACTN4 is upregulated by three-dimensional collagen culture conditions, leading to increased invasion and motility of ovarian cancer cells. Expression of ACTN4 in human ovarian tumors was found to be associated with advanced stage disease and peritoneal metastases.
actinin alpha-4; invasion; metastasis; microenvironment; motility; ovarian carcinoma
We previously used a human artificial chromosome (HAC) with a synthetic kinetochore that could be targeted with chromatin modifiers fused to tetracycline repressor to show that targeting of the transcriptional repressor tTS within kinetochore chromatin disrupts kinetochore structure and function. Here we show that the transcriptional corepressor KAP1, a downstream effector of the tTS, can also inactivate the kinetochore. The disruption of kinetochore structure by KAP1 subdomains does not simply result from loss of centromeric CENP-A nucleosomes. Instead it reflects a hierarchical disruption of the outer kinetochore, with CENP-C levels falling before CENP-A levels and, in certain instances, CENP-H being lost more readily than CENP-C. These results suggest that this novel approach to kinetochore dissection may reveal new patterns of protein interactions within the kinetochore.
Doxorubicin (Dox) incorporated in nanosized polymeric micelles, SP1049C, has shown promise as monotherapy in patients with advanced esophageal carcinoma. The formulation contains amphiphilic block copolymers, Pluronics, that exhibit unique ability to chemosensitize multidrug resistant (MDR) tumors by inhibiting P-glycoprotein (Pgp) drug efflux system and enhancing pro-apoptotic signaling in cancer cells. This work evaluates whether a representative block copolymer, Pluronic P85 (P85) can also prevent development of Dox-induced MDR in leukemia cells. For in vitro studies murine lymphocytic leukemia cells (P388) were exposed to increasing concentrations of Dox with/without P85. For in vivo studies, BDF1 mice bearing P388 ascite were treated with Dox or Dox/P85. The selected P388 cell sublines and ascitic tumor-derived cells were characterized for Pgp expression and functional activity (RT-PCR, Western Blot, rhodamine 123 accumulation) as well as Dox resistance (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay). The global gene expression was determined by oligonucleotide gene microarrays. We demonstrated that P85 prevented development of MDR1 phenotype in leukemia cells in vitro and in vivo as determined by Pgp expression and functional assays of the selected cells. Cells selected with Dox in the presence of P85 in vitro and in vivo exhibited some increases in IC50 values compared to parental cells, but these values were much less than IC50 in respective cells selected with the drug alone. In addition to mdr1, P85 abolished alterations of genes implicated in apoptosis, drug metabolism, stress response, molecular transport and tumorigenesis. In conclusion, Pluronic formulation can prevent development of MDR in leukemia cells in vitro and in vivo.
Multidrug Resistance; P-glycoprotein; Pluronic; Poloxamer; Doxorubicin
Campylobacter jejuni is a gastrointestinal pathogen of humans but can asymptomatically colonize the avian gut. C. jejuni therefore grows at both 37°C and 42°C, the internal temperatures of humans and birds respectively. Microarray and proteomic studies on temperature regulation in C. jejuni strain 81–176 revealed the upregulation at 42°C of two proteins, Cj0414 and Cj0415, orthologous to gluconate dehydrogenase (GADH) from Pectobacterium cypripedii. 81–176 demonstrated GADH activity, converting d-gluconate to 2-keto-d-gluconate, that was higher at 42°C than at 37°C. In contrast, cj0414 and cj0415 mutants lacked GADH activity. Wild-type but not cj0415 mutant bacteria exhibited gluconate-dependent respiration. Neither strain grew in defined media with d-gluconate or 2-keto-d-gluconate as a sole carbon source, revealing that gluconate was used as an electron donor rather than as a carbon source. When administered to chicks individually or in competition with wild-type, the cj0415 mutant was impaired in establishing colonization. In contrast, there were few significant differences in colonization of BALB/c-ByJ mice in single or mixed infections. These results suggest that the ability of C. jejuni to use gluconate as an electron donor via GADH activity is an important metabolic characteristic that is required for full colonization of avian but not mammalian hosts.
Children’s recognition of familiar own-age peers was investigated. Four-, 8-, and 14-year-old Chinese children were asked to identify their classmates from photographs showing the entire face, the internal facial features only, the external facial features only, or the eyes, nose, or mouth only. Participants from all age groups were familiar with the faces used as stimuli for one academic year. The results showed that children from all age groups demonstrated an advantage for recognition of the internal facial features relative to their recognition of the external facial features. Previous observations of a shift in reliance from external to internal facial features can, thus, be attributed to experience with faces rather than to age-related changes in face processing.
Ductal adenocarcinoma of the pancreas is the fourth leading cause of cancer death and is usually diagnosed late. Intraductal papillary mucinous neoplasms are an increasingly recognized precursor to invasive ductal adenocarcinoma of the pancreas. Identifying the alterations in DNA methylation that arise during intraductal papillary mucinous neoplasm development may facilitate the development of markers that could be used to differentiate intraductal papillary mucinous neoplasms from non-neoplastic pancreatic cystic lesions. Surgically resected intraductal papillary mucinous neoplasms and adjacent ductal adenocarcinomas were microdissected from 50 patients. Normal pancreas was also obtained from 27 patients with intraductal papillary mucinous neoplasms or pancreatic adenocarcinomas and 10 patients with well-differentiated pancreatic endocrine neoplasms. Methylation-specific PCR was performed on isolated DNA for seven genes (SPARC, SARP2, TSLC1, RELN, TFPI2, CLDN5, UCHL1) known to be commonly aberrantly methylated in pancreatic ductal adenocarcinomas. The mean percentage of genes methylated in invasive ductal adenocarcinomas arising in association with an intraductal papillary mucinous neoplasm (mean±s.d., 81±17%) was significantly higher than that in noninvasive-intraductal papillary mucinous neoplasms (57±26%, P=0.007) or peritumoral normal epithelial cells (22±17%, P<0.0001). Carcinomas (intraductal papillary mucinous neoplasms with carcinoma in situ or their associated infiltrating adenocarcinoma) had significantly more methylated genes (71±19%) than low-grade (low and moderate dysplasia) intraductal papillary mucinous neoplasms (44±26%, P<0.0001). The mean percentage of genes methylated in histologically normal pancreatic ductal cells from patients with ductal neoplasia (22±17%) was significantly higher than in normal ductal cells from patients with well-differentiated pancreatic endocrine neoplasms (4±7%, P=0.002). Thus, aberrant DNA methylation increases with histologic grades of intraductal papillary mucinous neoplasm. Low-level aberrant methylation in the normal ductal cells is more prevalent in patients with ductal neoplasia than in controls without ductal neoplasms and may contribute to carcinogenesis. The detection of aberrant methylation in pancreatic cystic lesions could facilitate the diagnosis of intraductal papillary mucinous neoplasms.
intraductal papillary mucinous neoplasm; DNA methylation; pancreatic cancer; SPARC; pancreatic endocrine neoplasm