Targeted deletion of alkylpurine-DNA-N-glycosylase in mice eliminates repair of 1,N6-ethenoadenine and hypoxanthine but not of 3,N4-ethenocytosine or 8-oxoguanine

It has previously been reported that 1,N6-ethenoadenine (ɛA), deaminated adenine (hypoxanthine, Hx), and 7,8-dihydro-8-oxoguanine (8-oxoG), but not 3,N4-ethenocytosine (ɛC), are released from DNA in vitro by the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG). To assess the potential contribution of APNG to the repair of each of these mutagenic lesions in vivo, we have used cell-free extracts of tissues from APNG-null mutant mice and wild-type controls. The ability of these extracts to cleave defined oligomers containing a single modified base was determined. The results showed that both testes and liver cells of these knockout mice completely lacked activity toward oligonucleotides containing ɛA and Hx, but retained wild-type levels of activity for ɛC and 8-oxoG. These findings indicate that (i) the previously identified ɛA-DNA glycosylase and Hx-DNA glycosylase activities are functions of APNG; (ii) the two structurally closely related mutagenic adducts ɛA and ɛC are repaired by separate gene products; and (iii) APNG does not contribute detectably to the repair of 8-oxoG.

The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: Peptoid molecular transporters

Certain proteins contain subunits that enable their active translocation across the plasma membrane into cells. In the specific case of HIV-1, this subunit is the basic domain Tat49–57 (RKKRRQRRR). To establish the optimal structural requirements for this translocation process, and thereby to develop improved molecular transporters that could deliver agents into cells, a series of analogues of Tat49–57 were prepared and their cellular uptake into Jurkat cells was determined by flow cytometry. All truncated and alanine-substituted analogues exhibited diminished cellular uptake, suggesting that the cationic residues of Tat49–57 play a principal role in its uptake. Charge alone, however, is insufficient for transport as oligomers of several cationic amino acids (histidine, lysine, and ornithine) are less effective than Tat49–57 in cellular uptake. In contrast, a 9-mer of l-arginine (R9) was 20-fold more efficient than Tat49–57 at cellular uptake as determined by Michaelis–Menton kinetic analysis. The d-arginine oligomer (r9) exhibited an even greater uptake rate enhancement (>100-fold). Collectively, these studies suggest that the guanidinium groups of Tat49–57 play a greater role in facilitating cellular uptake than either charge or backbone structure. Based on this analysis, we designed and synthesized a class of polyguanidine peptoid derivatives. Remarkably, the subset of peptoid analogues containing a six-methylene spacer between the guanidine head group and backbone (N-hxg), exhibited significantly enhanced cellular uptake compared to Tat49–57 and even to r9. Overall, a transporter has been developed that is superior to Tat49–57, protease resistent, and more readily and economically prepared.

Cost effectiveness of shortening screening interval or extending age range of NHS breast screening programme: computer simulation study

Objective: To compare the cost effectiveness of two possible modifications to the current UK screening programme: shortening the screening interval from three to two years and extending the age of invitation to a final screen from 64 to 69.

Surface expression and function of p75/AIRM-1 or CD33 in acute myeloid leukemias: Engagement of CD33 induces apoptosis of leukemic cells

p75/AIRM-1 is a recently identified inhibitory receptor expressed by natural killer and myeloid cells displaying high homology with CD33. Crosslinking of p75/AIRM-1 or CD33 has been shown to sharply inhibit the in vitro proliferation of both normal myeloid cells and chronic myeloid leukemias. In this study, we analyzed acute myeloid leukemic cells for the expression of p75/AIRM-1. p75/AIRM-1 marked the M5 (11/12) and M4 (2/2) but not the M1, M2, and M3 subtypes according to the French–American–British classification. Cell samples from 12 acute myeloid leukemias were cultured in the presence of granulocyte/macrophage colony-stimulating factor. Addition to these cultures of anti-CD33 antibody resulted in ≈70% inhibition of cell proliferation as assessed by [3H]thymidine uptake or by the recovery of viable cells. Anti-p75/AIRM-1 antibody exerted a strong inhibitory effect only in two cases characterized by a high in vitro proliferation rate. After crosslinking of CD33 (but not of p75/AIRM-1), leukemic cells bound Annexin V and displayed changes in their light-scattering properties and nucleosomal DNA fragmentation, thus providing evidence for the occurrence of apoptotic cell death. Remarkably, when anti-CD33 antibody was used in combination with concentrations of etoposide insufficient to induce apoptosis when used alone, a synergistic effect could be detected in the induction of leukemic cell death. These studies provide the rationale for new therapeutic approaches in myeloid leukemias by using both chemotherapy and apoptosis-inducing mAbs.

Point mutation of the autophosphorylation site or in the nuclear location signal causes protein kinase A RII beta regulatory subunit to lose its ability to revert transformed fibroblasts.

The RII beta regulatory subunit of cAMP-dependent protein kinase (PKA) contains an autophosphorylation site and a nuclear location signal, KKRK. We approached the structure-function analysis of RII beta by using site-directed mutagenesis. Ser114 (the autophosphorylation site) of human RII beta was replaced with Ala (RII beta-P) or Arg264 of KKRK was replaced with Met (RII beta-K). ras-transformed NIH 3T3 (DT) cells were transfected with expression vectors for RII beta, RII beta-P, and RII beta-K, and the effects on PKA isozyme distribution and transformation properties were analyzed. DT cells contained PKA-I and PKA-II isozymes in a 1:2 ratio. Over-expression of wild-type or mutant RII beta resulted in an increase in PKA-II and the elimination of PKA-I. Only wild-type RII beta cells demonstrated inhibition of both anchorage-dependent and -independent growth and phenotypic change. The growth inhibitory effect of RII beta overexpression was not due to suppression of ras expression but was correlated with nuclear accumulation of RII beta. DT cells demonstrated growth inhibition and phenotypic change upon treatment with 8-Cl-cAMP. RII beta-P or RII beta-K cells failed to respond to 8-Cl-cAMP. These data suggest that autophosphorylation and nuclear location signal sequences are integral parts of the growth regulatory mechanism of RII beta.

Major histocompatibility complex binding affinity of an antigenic determinant is crucial for the differential secretion of interleukin 4/5 or interferon gamma by T cells.

Differential activation of CD4+ T-cell precursors in vivo leads to the development of effectors with unique patterns of lymphokine secretion. To investigate whether the differential pattern of lymphokine secretion is influenced by factors associated with either the display and/or recognition of the ligand, we have used a set of ligands with various class II binding affinities but unchanged T-cell specificity. The ligand that exhibited approximately 10,000-fold higher binding to I-Au considerably increased the frequency of interferon gamma-producing but not interleukin (IL) 4- or IL-5-secreting cells in vivo. Using an established ligand-specific, CD4+ T-cell clone secreting only IL-4, we also demonstrated that stimulation with the highest affinity ligand resulted in interferon gamma production in vitro. In contrast, ligands that demonstrated relatively lower class II binding induced only IL-4 secretion. These data suggest that the major histocompatibility complex binding affinity of antigenic determinants, leading to differential interactions at the T cell-antigen-presenting cell interface, can be crucial for the differential development of cytokine patterns in T cells.

The conceptual table: a strategy for the development of a theoretical framework of a field research or knowledge

This is a guide to develop a theoretical framework for any field of knowledge. It is a rational and organized to put everything that is known or has been written about an issue or a problem way.

Monography of the family Unionidae, or Naiades of Lamarck (fresh water bivalve shells,) of North America, : illustrated by figures drawn on stone from nature. / by T.A. Conrad ...

1

An elegie on the deplored decease of that truly pious and religious matron, Mrs. Sara Leveret: relict of the late hon[or]able John Leveret, Esq., sometime governor of his then Majesty's colony of the Massethusets [sic] Bay in New England : manuscript, 1704

Autograph copy, signed.

Member State BITS after the Treaty of Lisbon: solid foundation or first victims of EU investment policy? Research Paper in Law, 02/2012

[Introduction.] This paper discusses the uncertain future of Member State BITs with third countries in the light of the developing EU investment policy. The question will be examined on the basis of the proposed Regulation establishing transitional arrangements for bilateral investment agreements between Member States and third countries presented by the Commission on 7 July 20101 and the European Parliament’s Position adopted at first reading on 10 May 2011.2 The proposed Regulation and the Commission Communication of the same day are meant to be the “first steps in the development of an EU international investment policy”.3 The first chapters present the legal framework relevant for this question and its evolution to better understand the particular challenges of this transition process. The second chapter examines the relationship of EU law and investment law, with a brief introduction of the notion of investment law and the scope of the EU’s new investment competence. The third chapter outlines the legal framework for the continuation and termination of treaties under international and EU law. The fourth chapter concerns BITs, first covering the particular nature of BITs and then the CJEU’s judgments in the BIT Cases of 2009. The fifth chapter consists of a step by step analysis of the different provisions of the proposed Regulation.