A structural model of 20S immunoproteasomes: effect of LMP2 codon 60 polymorphism on expression, activity, intracellular localisation and insight into the regulatory mechanisms

The immunoproteasome subunit low molecular weight protein 2 (LMP2) codon 60 polymorphism has been associated with autoimmune diseases. It has also been demonstrated to influence susceptibility to TNF-alpha-induced apoptosis in blood cells and proteasome activity in aged human brain. In the present study, an in silico model of immunoproteasome was used to examine the effect of the R60H polymorphism in the LMP2 subunit. The investigation of immunoproteasome expression, activity and intracellular localisation in an in vitro cellular model, namely lymphoblastoid cell lines, showed no major variations in functionality and amount, while a significant difference in antibody affinity was apparent. These data were integrated with previous results obtained in different tissues and combined with a structural model of the LMP2 polymorphism. Accordingly, we identified three prospective mechanisms that could explain the biological data for the polymorphism, such as modulation of the binding affinity of a putative non-catalytic modifier site on the external surface of the immunoproteasome core, or the modification of any channel between alpha and beta rings.     

STAT1 regulates IFN-alpha beta- and IFN-gamma-dependent control of infection with Chlamydia pneumoniae by nonhemopoietic cells

STAT1 mediates signaling in response to IFN-alpha, -beta, and -gamma, cytokines required for protective immunity against several viral, bacterial, and eukaryotic pathogens. The protective role of STAT1 in the control of intranasal infection with the obligate intracellular bacterium Chlamydia pneumoniae was analyzed. IFN-gamma-/- or IFN-gamma receptor (R)-/- mice were highly susceptible to infection with C. pneumoniae. We found that STAT1-/- mice were even more susceptible to C. pneumoniae than IFN-gamma-/- or IFN-gammaR-/- mice. Phosphorylation of STAT1 was detected in the lungs of C. pneumoniae-infected wild-type, IFN-gammaR-/-, and IFN-alphabetaR-/- mice, but not in mice lacking both IFN-alphabetaR and IFN-gammaR. In line with this, IFN-alphabetaR-/-/IFN-gammaR-/- mice showed increased susceptibility to infection compared with IFN-gammaR-/- mice. However, C. pneumoniae-infected IFN-alphabetaR-/- or IFN regulatory factor 3-/- mice showed no increased susceptibility and similar IFN-gamma expression compared with wild-type mice. CD4+ or CD8+ cells released IFN-gamma in vivo and conferred protection against C. pneumoniae in a STAT1-independent manner. In contrast, STAT1 mediated a nonredundant protective role of nonhemopoietic cells but not of hemopoietic cells. Nonhemopoietic cells accounted for the expression of STAT1-mediated indoleamine 2, 3-dioxygenase and the p47 GTPase LRG-47, but not inducible NO synthase mRNA. In summary, we demonstrate that STAT1 mediates a cooperative effect of IFN-alphabeta and IFN-gamma on nonhemopoietic cells, resulting in protection against C. pneumoniae.     

鼠类超声波通讯的社会原因和功能

鼠类的超声通讯已有报道,并且在实验室深入研究了小鼠和大鼠接受和释放这些叫声的能力。由于对不同刺激的反应和出自不同的目的,幼鼠和成年鼠都释放超声。发出者的年龄/性/品系以及自然和社会环境的不同,导致这些超声的频率和持续时间也不同。在社会隔离和操作处理时,未成年个体可释放超声波。作为成熟程度以及对环境信号和药物处理时反应的指标,这些叫声得到了广泛研究,表明超声在调节出生后早期的母幼联系中起相应的作用。随着幼鼠的长大及体温调节和感觉能力的增强,发生的数量大大减少。然而,成年小鼠和大鼠在不同的社会条件下,释放出的超声有种间差异:大鼠的超声与正面和负面的社会作用有关,而在小鼠中,这些叫声主要与领域有关.     

Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration

Insertional mutagenesis represents a major hurdle to gene therapy and necessitates sensitive preclinical genotoxicity assays. Cdkn2a-/- mice are susceptible to a broad range of cancer-triggering genetic lesions. We exploited hematopoietic stem cells from these tumor-prone mice to assess the oncogenicity of prototypical retroviral and lentiviral vectors. We transduced hematopoietic stem cells in matched clinically relevant conditions, and compared integration site selection and tumor development in transplanted mice. Retroviral vectors triggered dose-dependent acceleration of tumor onset contingent on long terminal repeat activity. Insertions at oncogenes and cell-cycle genes were enriched in early-onset tumors, indicating cooperation in tumorigenesis. In contrast, tumorigenesis was unaffected by lentiviral vectors and did not enrich for specific integrants, despite the higher integration load and robust expression of lentiviral vectors in all hematopoietic lineages. Our results validate a much-needed platform to assess vector safety and provide direct evidence that prototypical lentiviral vectors have low oncogenic potential, highlighting a major rationale for application to gene therapy.     

5-benzylidene-hydantoins as new EGFR inhibitors with antiproliferative activity

A series of 1,5-disubstituted hydantoins, whose structure was designed to interact at the ATP-binding site of EGFR, was synthesized and evaluated for inhibition of EGFR kinase activity and antiproliferative action. Some of these compounds, characterized by a 1-phenethyl and a 5-(E)-benzylidene substituent, inhibited EGFR autophosphorylation and polyGAT phosphorylation, and also inhibited the growth and proliferation of human A431 cells, which overexpress EGFR. These compounds can therefore be regarded as examples of a new scaffold for tyrosine kinase inhibitors.     

Dibasic non-imidazole histamine H3 receptor antagonists with a rigid biphenyl scaffold

A class of rigid, dibasic, non-imidazole H3 antagonists was developed, starting from a series of previously described flexible compounds. The original polymethylene chain between two tertiary amine groups was replaced by a rigid scaffold, composed by a phenyl ring or a biphenyl fragment. Modulation of the distance between the two amine groups, and of their alkyl substituents, was driven by superposition of molecular models and docking into a receptor model, resulting in the identification of 1,1'-[biphenyl-4,4'-diylbis(methylene)]bis-piperidine (5) as a subtype-selective H3 antagonist with high binding affinity (pKi=9.47) at human H3 histamine receptor.     

Notch1b and neuregulin are required for specification of central cardiac conduction tissue

Normal heart function is critically dependent on the timing and coordination provided by a complex network of specialized cells: the cardiac conduction system. We have employed functional assays in zebrafish to explore early steps in the patterning of the conduction system that previously have been inaccessible. We demonstrate that a ring of atrioventricular conduction tissue develops at 40 hours post-fertilization in the zebrafish heart. Analysis of the mutant cloche reveals a requirement for endocardial signals in the formation of this tissue. The differentiation of these specialized cells, unlike that of adjacent endocardial cushions and valves, is not dependent on blood flow or cardiac contraction. Finally, both neuregulin and notch1b are necessary for the development of atrioventricular conduction tissue. These results are the first demonstration of the endocardial signals required for patterning central ;slow' conduction tissue, and they reveal the operation of distinct local endocardial-myocardial interactions within the developing heart tube.