Three distinct effects of SV40 T-antigen gene transfection on cellular differentiation

SV40 large T-antigen-induced transformation has been reported to block differentiation, but the mechanism(s) of this effect has not been established. The results presented here show that stable transfection of the SV40 T-antigen gene, via the pSV3neo plasmid, has at least three distinct effects on 3T3T adipocyte differentiation. Cells first show a decreased ability to undergo predifferentiation growth arrest, which is a prerequisite for in vitro 3T3T adipocyte differentiation. However, if predifferentiation growth arrest is accomplished by use of stringent differentiation-inducing culture conditions, adipocyte differentiation can occur with high frequency. The pSV3neo-transfected cell clones also show other modifications of the adipocyte differentiation process, including changes in nonterminal (reversible) and terminal (irreversible) steps of adipocyte differentiation. When compared to nontransfected 3T3T cells, the cell clones containing pSV3neo require markedly reduced growth factor concentrations to restimulate proliferation of nonterminally differentiated adipocytes and the terminal step of differentiation is also blocked. These results suggest that integration of the T-antigen gene, through pSV3neo transfection, has multiple effects on the cellular mechanisms of differentiation. It does not block the differentiation process per se; rather it appears to make cells highly sensitive to proliferation signals, thereby making differentiation more difficult.     

Thyroid-specific enhancer-binding protein/thyroid transcription factor 1 is not required for the initial specification of the thyroid and lung primordia.

Targeted disruption of the homeobox gene T/ebp (Ttf1) in mice results in ablation of the thyroid and pituitary, and severe deformities in development of the lung and hypothalamus. T/ebp is expressed in the thyroid, lung, and ventral forebrain during normal embryogenesis. Examination of thyroid development in T/ebp homozygous null mutant embryos revealed that the thyroid rudiment is initially formed but is eliminated through apoptosis. Absence of T/EBP expression in the lung primordium does not activate apoptosis since a lung tissue, albeit dysmorphic, is nevertheless formed in T/ebp-/- embryos. These results demonstrate that T/EBP is not required for the initial specification of thyroid or lung primordia, but is absolutely essential for the development and morphogenesis of these organs.     

α-Bisabolol-6-desoxy-α-altropyranoside from Carthamus turkistanikus

A sesquiterpene glycoside has been isolated from the aerial parts of Carthamus turkistanikus and identified as α-bisabolol-β-desoxy-p-altropyra     

Mouse knockout of the cholesterogenic cytochrome P450 lanosterol 14alpha-demethylase (Cyp51) resembles Antley-Bixler syndrome

Antley-Bixler syndrome (ABS) represents a group of heterogeneous disorders characterized by skeletal, cardiac, and urogenital abnormalities that have frequently been associated with mutations in fibroblast growth factor receptor 2 or cytochrome P450 reductase genes. In some ABS patients, reduced activity of the cholesterogenic cytochrome P450 CYP51A1, an ortholog of the mouse CYP51, and accumulation of lanosterol and 24,25-dihydrolanosterol has been reported, but the role of CYP51A1 in the ABS etiology has remained obscure. To test whether Cyp51 could be involved in generating an ABS-like phenotype, a mouse knock-out model was developed that exhibited several prenatal ABS-like features leading to lethality at embryonic day 15. Cyp51(-/-) mice had no functional Cyp51 mRNA and no immunodetectable CYP51 protein. The two CYP51 enzyme substrates (lanosterol and 24,25-dihydrolanosterol) were markedly accumulated. Cholesterol precursors downstream of the CYP51 enzymatic step were not detected, indicating that the targeting in this study blocked de novo cholesterol synthesis. This was reflected in the up-regulation of 10 cholesterol synthesis genes, with the exception of 7-dehydrocholesterol reductase. Lethality was ascribed to heart failure due to hypoplasia, ventricle septum, and epicardial and vasculogenesis defects, suggesting that Cyp51 deficiency was involved in heart development and coronary vessel formation. As the most likely downstream molecular mechanisms, alterations were identified in the sonic hedgehog and retinoic acid signaling pathways. Cyp51 knock-out mice provide evidence that Cyp51 is essential for embryogenesis and present a potential animal model for studying ABS syndrome in humans.     

Individuality and ethnicity eclipse a short-term dietary intervention in shaping microbiomes and viromes

Many diseases linked with ethnic health disparities associate with changes in microbial communities in the United States, but the causes and persistence of ethnicity-associated microbiome variation are not understood. For instance, microbiome studies that strictly control for diet across ethnically diverse populations are lacking. Here, we performed multiomic profiling over a 9-day period that included a 4-day controlled vegetarian diet intervention in a defined geographic location across 36 healthy Black and White females of similar age, weight, habitual diets, and health status. We demonstrate that individuality and ethnicity account for roughly 70% to 88% and 2% to 10% of taxonomic variation, respectively, eclipsing the effects a short-term diet intervention in shaping gut and oral microbiomes and gut viromes. Persistent variation between ethnicities occurs for microbial and viral taxa and various metagenomic functions, including several gut KEGG orthologs, oral carbohydrate active enzyme categories, cluster of orthologous groups of proteins, and antibiotic-resistant gene categories. In contrast to the gut and oral microbiome data, the urine and plasma metabolites tend to decouple from ethnicity and more strongly associate with diet. These longitudinal, multiomic profiles paired with a dietary intervention illuminate previously unrecognized associations of ethnicity with metagenomic and viromic features across body sites and cohorts within a single geographic location, highlighting the importance of accounting for human microbiome variation in research, health determinants, and eventual therapies.Trial Registration: ClinicalTrials.gov ClinicalTrials.gov Identifier: {"type":"clinical-trial","attrs":{"text":"NCT03314194","term_id":"NCT03314194"}}NCT03314194.

Many diseases linked with ethnic health disparities associate with changes in microbial communities in the United States, but the causes and persistence of ethnicity-associated microbiome variation are not understood. This study reveals that individuality and ethnicity eclipse a short-term dietary intervention in shaping microbiomes and viromes.     

Each Member of the Poly-r(C)-binding Protein 1 (PCBP) Family Exhibits Iron Chaperone Activity toward Ferritin

The mechanisms through which iron-dependent enzymes receive their metal cofactors are largely unknown. Poly r(C)-binding protein 1 (PCBP1) is an iron chaperone for ferritin; both PCBP1 and its paralog PCBP2 are required for iron delivery to the prolyl hydroxylase that regulates HIF1. Here we show that PCBP2 is also an iron chaperone for ferritin. Co-expression of PCBP2 and human ferritins in yeast activated the iron deficiency response and increased iron deposition into ferritin. Depletion of PCBP2 in Huh7 cells diminished iron incorporation into ferritin. Both PCBP1 and PCBP2 were co-immunoprecipitated with ferritin in HEK293 cells, and expression of both PCBPs was required for ferritin complex formation in cells. PCBP1 and -2 exhibited high affinity binding to ferritin in vitro. Mammalian genomes encode 4 PCBPs, including the minimally expressed PCBPs 3 and 4. Expression of PCBP3 and -4 in yeast activated the iron deficiency response, but only PCBP3 exhibited strong interactions with ferritin. Expression of PCBP1 and ferritin in an iron-sensitive, ccc1 yeast strain intensified the toxic effects of iron, whereas expression of PCBP4 protected the cells from iron toxicity. Thus, PCBP1 and -2 form a complex for iron delivery to ferritin, and all PCBPs may share iron chaperone activity.     

On Black Feminist Thought: thinking oppression and resistance through intersectional paradigm

Located in African American women's everyday and historical experiences of oppression and resistance, black feminist epistemology and critical social theory, Patricia Hill Collins raises the intellectual level in all these arenas. Developed through a dynamic interaction with black women's everyday struggles, black feminist thought is important not only for its contribution to critical social theories and methodologies, but also for providing important knowledge for the use of social justice movements. It uses intersectional analysis to shed light on the relationships between the structural, symbolic and everyday aspects of domination and individual and collective struggles in various domains of social life. Collins offers an interpretive framework for understanding the experiences of African American women. However, the significance of black feminist thought reaches far beyond US and black American communities. This article is a reading of Collins’s concept of intersectionality, the relationship between oppression and resistance, and the politics of empowerment.     

Detection of Anti-tetanus Toxoid Monoclonal Antibody by Using Modified Polycarbonate Surface

In recent years, CD surface modification methods are employed for immunoassay techniques that is called BioCD technology. In this research, first polycarbonate surface was activated with UV ozone and a hydrophilic surface was obtained. Contact angle measurements and atomic force microscopy technique confirmed the hydrophilic property of surface. After that, tetanus toxoid was immobilized on modified CD surface then specific monoclonal antibody, gold nanoparticles conjugated antibody, silver salt, and hydroquinone were added on modified CD surface. So a sandwiches complex as tetanus toxoid, tetanus toxoid monoclonal antibody, and gold nanoparticles conjugated antibody was obtained on CD surface. ATR result showed the immobilization of tetanus toxoid on modified CD surface. Localized surface plasmon resonance (LSPR) and DLS results confirmed the complex formation. Silver salt and hydroquinone were added for signal amplification. Detection limit of anti-tetanus toxoid IgG monoclonal antibody was obtained 0.005 IU/ml by LSPR and DLS techniques. The presented method increases the assay’s sensitivity. BioCD-based immunoassay for detection of anti-tetanus toxoid IgG monoclonal antibody could be applicable in development and fabrication of biomedical devices.     

Bcr and Abr Cooperate in Negatively Regulating Acute Inflammatory Responses

Bcr and Abr are GTPase-activating proteins for the small GTPase Rac. Both proteins are expressed in cells of the innate immune system, including neutrophils and macrophages. The function of Bcr has been linked to the negative regulation of neutrophil reactive oxygen species (ROS) production, but the function of Abr in the innate immune system was unknown. Here, we report that mice lacking both proteins are severely affected in two models of experimental endotoxemia, including exposure to Escherichia coli lipopolysaccharide and polymicrobial sepsis, with extensive microvascular leakage, resulting in severe pulmonary edema and hemorrhage. Additionally, in vivo-activated neutrophils of abr and bcr null mutant mice produced excessive tissue-damaging myeloperoxidase (MPO), elastase, and ROS. Moreover, the secretion of the tissue metalloproteinase MMP9 by monocytes and ROS by elicited macrophages was abnormally high. In comparison, ROS production from bone marrow monocytes was not significantly different from that of controls, and the exocytosis of neutrophil secondary and tertiary granule products, including lactoferrin, was normal. These data show that Abr and Bcr normally curb very specific functions of mature tissue innate immune cells, and that each protein has distinct as well as partly overlapping functions in the downregulation of inflammatory processes.BCR originally was discovered as a human gene on chromosome 22 that, in chronic myeloid leukemia, becomes fused to the c-ABL tyrosine kinase gene originating from chromosome 9 (18). The normal gene encodes a 160-kDa protein that contains a domain with GTPase-activating (GAP) activity toward Rho family GTPases (7, 11, 12, 32, 36). There is only one other gene in mouse and human, called ABR, that is closely homologous to BCR (17). Abr shares several domains with Bcr, which includes a Dbl homology (DH) domain and a GAP domain. Bcr has an additional N-terminal part consisting of a coiled-coil and a serine/threonine kinase domain that is not present in Abr, suggesting that each GAP has a distinct cellular function.Rho GTPases, including Rho, Rac, and Cdc42, play important roles in many functions of cells of the innate immune system (16). They cycle between active GTP and inactive GDP-bound conformations. GAP proteins catalyze the conversion of bound GTP to GDP on Rho GTPases and thus act as negative, inactivating regulators.In previous studies, we showed that both Abr and Bcr specifically act as GAPs for Rac and not for the related Cdc42 (6). To investigate the normal cellular function of these two related GAPs, we generated mice defective in the production of Abr or Bcr through gene targeting. Mice that lack both proteins have defects in the architecture of the inner ear, with the partial absence of otoconia and hair cells. Additionally, postnatal cerebellar development is abnormal, with a persistence of ectopic granule cells at the cerebellar surface. These combined abnormalities cause persistent circling and balance problems (20, 21).As reported previously, neutrophils from mice lacking Bcr produce increasing amounts of reactive oxygen species (ROS), and bcr^−/− mice injected with Escherichia coli lipopolysaccharide (LPS) are much more severely affected than are wild-type mice (39). We further explored the role of Bcr and Abr in the innate immune system with a detailed study of bone marrow-derived macrophages (BMM). Interestingly, macrophages isolated from double-knockout (abr × bcr^−/−) mice exhibited multiple defects. These include aberrant actin cytoskeletal organization and the increased colony-stimulating factor 1-stimulated chemotaxis and phagocytosis of opsonized zymosan or E. coli (6).In the current study, we examined whether the defects observed in vitro result in an observable phenotype in vivo, under inflammatory conditions. Here, we report that Abr plays a distinct role in negatively regulating the innate immune system in vivo, as well as exhibiting overlap with the function of Bcr. Mice lacking both Abr and Bcr have a severely impaired ability to resolve septic shock, showing that the activity of both proteins is required for the appropriate negative control of innate immune responses.