An evaluation of the care of displaced elderly evacuated during the San Bernardino mountain firestorms of Fall 2003

This project reflects on the observations and experiences that occurred during the firestorms in Southern California in October and November 2003. It specifically focused on elders during this disaster as a vulnerable population. Recommendations are made for the role of the nurse in preparation and planning for evacuation of the elderly

Immunological analysis of plasminogen activators from normal and transformed hamster cells...

Rabbits were immunized against the plasminogen activator released by SV4- virus-transformed hamster embryo cells. The resulting antiplasminogen activator immunoglobulin (APA-IgG) inhibited the enzymatic activity of the plasminogen activator produced by SV40-transformed hamster cells, and the plasmin-catalyzed release of these cells from the tissue culture dish. APA-IgG was not cytotoxic for these cells even in the presence of complement and did not inhibit their release of plasminogen activator. APA-IgG formed a single precipitin line in immunodiffusion plates using highly purified plasminogen activator as antigen. APA-IgG inhibited the plasminogen activator produced by newborn hamster lung cells and by an established diploid line (DON) of hamster lung cells, but did not inhibit plasminogen activators produced by normal or transformed hamster kidney cells or by cells of other species (mouse and human). We derive three major conclusions from these data: (a) There are several immunologically distinguishable forms (isozymes) of plasminogen activators in normal hamster tissues. (b) The plasminogen activators produced by normal hamster lung cells and by SV40 virus-transformed hamster embryo cells share antigenic determinants and are presumably the same isozyme. (c) The plasminogen activators produced by different hamster tumor cells do not share antigenic determinants and are presumably different isozymes

Expression in Escherichia coli of a cloned DNA sequence encoding the pre-S2 region of hepatitis B virus

A DNA sequence encoding the entire pre-S2 region (amino acids 120-174; serotype ayw) of human hepatitis B virus envelope protein has been inserted into the lacZ gene of the plasmid pSKS105 yielding a recombinant, pWS3. Lac+ colonies of the Escherichia coli M182 (lacIOPZYA), isolated after transformation with pWS3, produced a pre-S2 peptide-ß-galactosidase fusion protein. This fusion protein, which comprised as much as 3% of the total bacterial protein, was purified to >90% homogeneity by affinity chromatography on p-aminophenyl-ß-D-thiogalactoside-Sepharose. It is immunoprecipitable with rabbit antibodies to a synthetic peptide corresponding to amino acids 120-145 of the pre-S2 region of serotype adw [pre-S(120-145)] or with antibodies to hepatitis B virus. pre-S(120-145) completely blocked the binding of either antibody to the pre-S2 peptide-ß-galactosidase fusion protein. These results indicate that there are antigenic determinants on the fusion protein that are closely related to, if not identical to, determinants on synthetic pre-S(120-145) and on pre-S2 sequences of native hepatitis B virus particles. Thus, bacteria transformed with pWS3 can provide an abundant source of pre-S2-ß-galactosidase fusion protein, which may prove useful either as a diagnostic reagent possessing marker enzyme activity suitable for ELISA tests or as an immunogen with potential to contribute to active prophylaxis of hepatitis B

Synthesis of Reovirus Oligo Adenylic Acid In Vivo and In Vitro

The formation of reovirus double-stranded (ds) RNA and of oligo adenylic acid (oligo A) is inhibited by 5 μg of actinomycin D per ml added at the time of viral infection. Viral proteins are synthesized and assembled into dsRNA-deficient particles under these conditions. The addition of cycloheximide to infected cells during the mid-logarithmic phase of viral replication terminates protein and dsRNA synthesis, but allows continued oligo A synthesis for about 1 h. The 3H-labeled oligo A formed in the presence of cycloheximide is incorporated into particles whose density in CsCl is identical to that of reovirions. Using the large particulate or virus factory-containing cytoplasmic fraction of infected L-cells, we have established an in vitro system for the synthesis of oligo A. The in vitro product migrates slightly faster in sodium dodecyl sulfate acrylamide gels than marker oligo A. Oligo A synthesis in vitro continues for about 1 h, requires, the presence of only one ribonucleoside triphosphate (ATP), is not inhibited by DNase or RNase, but is abruptly terminated by the addition of chymotrypsin to the reaction mixture. Oligo A formed both in vivo and in vitro is released from the factory fraction by chymotrypsin digestion. The enzymes which catalyze the synthesis of oligo A, dsRNA, and single-stranded RNA all exhibit a similar temperature dependence with an optimum of ∼45 C. These results indicate that oligo A is formed within the core of the nascent virion after the completion of dsRNA synthesis; they suggest that the oligo A polymerase is an alternative activity of the virion-bound transcriptase and that it is regulated by outer capsomere proteins

Mechanism of Reovirus Double-Stranded Ribonucleic Acid Synthesis In Vivo and In Vitro

The complementary strands of reovirus double-stranded ribonucleic acid (ds RNA) are synthesized sequentially in vivo and in vitro. In both cases, preformed plus strands serve as templates for the synthesis of the complementary minus strands. The in vitro synthesis of dsRNA is catalyzed by a large particulate fraction from reovirus-infected cells. Treatment of this fraction with chymotrypsin or with detergents which solubilize cellular membranes does not alter its capacity to synthesize dsRNA. The enzyme or enzymes responsible for dsRNA synthesis remain sedimentable at 10,000 × g after these enzyme or detergent treatments, indicating their particulate nature. Pretreatment of this fraction with ribonuclease, however, abolishes its ability to catalyze dsRNA synthesis, emphasizing the single-stranded nature of the template and its location in a structure permeable to ribonuclease. In contrast, the newly formed dsRNA is resistant to ribonuclease digestion at low salt concentrations and hence is thought to reside within a ribonuclease-impermeable structure

DNA (Cytosine-C5) methyltransferase inhibition by oligodeoxyribonucleotides containing 2-(1H)-pyrimidinone (zebularine aglycon) at the enzymatic target site

20 pages, 7 figures, 1 table.-- PMID: 19467223 [PubMed].-- PMCID: PMC2756644.-- NIHMSID: NIHMS130041.-- Printed version published Sep 15, 2009.Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors.Partial support for this work was provided by a grant from the NIH/NCI (R21CA91315) to J.K.C. and a fellowship from the Graduate College at UNMC to D.V.B. We are grateful to S. Kumar of New England Biolabs for providing us with Eschericia coli strain ER1727 containing the pUHE25HhaI plasmid. This research was also supported in part with funds from the Intramural Research Program of the NIH, Center for Cancer Research, NCI Frederick.Peer reviewe

DNA (Cytosine-C5) methyltransferase inhibition by oligodeoxyribonucleotides containing 2-(1H)-pyrimidinone (zebularine aglycon) at the enzymatic target site

20 pages, 7 figures, 1 table.-- PMID: 19467223 [PubMed].-- PMCID: PMC2756644.-- NIHMSID: NIHMS130041.-- Printed version published Sep 15, 2009.Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors.Partial support for this work was provided by a grant from the NIH/NCI (R21CA91315) to J.K.C. and a fellowship from the Graduate College at UNMC to D.V.B. We are grateful to S. Kumar of New England Biolabs for providing us with Eschericia coli strain ER1727 containing the pUHE25HhaI plasmid. This research was also supported in part with funds from the Intramural Research Program of the NIH, Center for Cancer Research, NCI Frederick.Peer reviewe

Loss of Dnmt3b function upregulates the tumor modifier Ment and accelerates mouse lymphomagenesis

DNA methyltransferase 3B (Dnmt3b) belongs to a family of enzymes responsible for methylation of cytosine residues in mammals. DNA methylation contributes to the epigenetic control of gene transcription and is deregulated in virtually all human tumors. To better understand the generation of cancer-specific methylation patterns, we genetically inactivated Dnmt3b in a mouse model of MYC-induced lymphomagenesis. Ablation of Dnmt3b function using a conditional knockout in T cells accelerated lymphomagenesis by increasing cellular proliferation, which suggests that Dnmt3b functions as a tumor suppressor. Global methylation profiling revealed numerous gene promoters as potential targets of Dnmt3b activity, the majority of which were demethylated in Dnmt3b–/– lymphomas, but not in Dnmt3b–/– pretumor thymocytes, implicating Dnmt3b in maintenance of cytosine methylation in cancer. Functional analysis identified the gene Gm128 (which we termed herein methylated in normal thymocytes [Ment]) as a target of Dnmt3b activity. We found that Ment was gradually demethylated and overexpressed during tumor progression in Dnmt3b–/– lymphomas. Similarly, MENT was overexpressed in 67% of human lymphomas, and its transcription inversely correlated with methylation and levels of DNMT3B. Importantly, knockdown of Ment inhibited growth of mouse and human cells, whereas overexpression of Ment provided Dnmt3b+/+ cells with a proliferative advantage. Our findings identify Ment as an enhancer of lymphomagenesis that contributes to the tumor suppressor function of Dnmt3b and suggest it could be a potential target for anticancer therapies

Gender injustice in compensating injury to autonomy in English and Singaporean negligence law

The extent to which English law remedies injury to autonomy (ITA) as a stand-alone actionable damage in negligence is disputed. In this article I argue that the remedy available is not only partial and inconsistent (Keren-Paz in Med Law Rev, 2018) but also gendered and discriminatory against women. I first situate the argument within the broader feminist critique of tort law as failing to appropriately remedy gendered harms, and of law more broadly as undervaluing women’s interest in reproductive autonomy. I then show by reference to English remedies law’s first principles how imposed motherhood cases—Rees v Darlington and its predecessor McFarlane v Tayside Health Board—result in gender injustice when compared with other autonomy cases such as Chester v Afshar and Yearworth v North Bristol NHS Trust: A minor gender-neutral ITA is better remedied than the significant gendered harm of imposing motherhood on the claimant; men’s reproductive autonomy is protected to a greater extent than women’s; women’s reproductive autonomy is protected by an exceptional, derisory award. Worst of all, courts refuse to recognise imposed motherhood as detriment; and the deemed, mansplained, nonpecuniary joys of motherhood are used to offset pecuniary upkeep costs, forcing the claimant into a position she sought to avoid and thus further undermining her autonomy. The recent Singaporean case ACB v Thomson Medical Pte Ltd, awarding compensation for undermining the claimant’s genetic affinity in an IVF wrong-sperm-mix-up demonstrates some improvement in comparison to English law, and some shared gender injustices in the context of reproductive autonomy. ACB’s analysis is oblivious to the nature of reproductive autonomy harm as gendered; and prioritises the father’s interest in having genetic affinity with the baby over a woman’s interest in not having motherhood imposed upon her

Potent Inhibition of HhaI DNA Methylase by the Aglycon of 2-(1H)-Pyrimidinone Riboside (Zebularine) at the GCGC Recognition Domain

A short oligodeoxynucleotide (ODN) with 2-(1H)-pyrimidinone at the HhaI DNA methyltransferase target site (GCGC) is shown to induce a level of inhibition of methyl transfer and thermal stability of the complex with the enzyme identical to that achieved with a similar ODN substituted with 5-azacytosine. The drugs responsible for these effects - zebularine and 5-azacytidine/2′-deoxy-5-azacytidine - are contrasted in terms of chemical stability and possible metabolic activation by a brief structure-activity analysis.Peer reviewe