A bovine dander allergen, comparative modeling, and similarities and differences in folding with related proteins

The site-specific recombinases FLP and Cre are useful for genomic engineering in many living systems. Manipulation of their enzymatic properties offers a means to improve their applicability in different host organisms. We chose to manipulate the thermolability of FLP recombinase. A lacZ-based recombination assay in Escherichia coli was used for selection in a protein evolution strategy that relied on error-prone PCR and DNA shuffling. Improved FLP recombinases were identified through cycles of increasing stringency imposed by both raising temperature and reducing protein expression, combined with repetitive cycles of screening at the same stringency to enrich for clones with improved fitness. An eighth generation clone (termed FLPe) showed improved properties in E. coli, in vitro, in human 293- and mouse ES-cells.     

Resolution of synthetic chi structures by the FLP site-specific recombinase

The FLP site-specific recombinase is encoded by the two micron circle, an endogenous plasmid of Saccharomyces cerevisiae. FLP-mediated recombination in vitro proceeds via a short-lived Holliday (chi) intermediate. We have made a synthetic chi structure containing two FLP recognition target (FRT) sequences in order to investigate resolution by purified FLP protein. We found that incubation of this model substrate with FLP generated two pairs of linear products in equal quantities. Thus, resolution was equally likely to occur in either direction. Alteration of FLP binding sites, so as to inhibit binding, affected the direction of resolution; cleavage was reduced adjacent to the altered binding site. The overall efficiency of resolution increased when one FLP binding site was mutated. In investigating the series of mutated chi structures we found that resolution requires only two intact FLP binding sites. However, the non-specific protein-DNA interaction of additional FLP molecules may also be required. Thus, resolution is more tolerant of the loss of FLP binding sites than is the complete recombination reaction.     

Natural killer cells determine development of allergen-induced eosinophilic airway inflammation in mice

The earliest contact between antigen and the innate immune system is thought to direct the subsequent antigen-specific T cell response. We hypothesized that cells of the innate immune system, such as natural killer (NK) cells, NK1.1(+) T cells (NKT cells), and gamma/delta T cells, may regulate the development of allergic airway disease. We demonstrate here that depletion of NK1.1(+) cells (NK cells and NKT cells) before immunization inhibits pulmonary eosinophil and CD3(+) T cell infiltration as well as increased levels of interleukin (IL)-4, IL-5, and IL-12 in bronchoalveolar lavage fluid in a murine model of allergic asthma. Moreover, systemic allergen-specific immunoglobulin (Ig)E and IgG2a levels and the number of IL-4 and interferon gamma-producing splenic cells were diminished in mice depleted of NK1.1(+) cells before the priming regime. Depletion of NK1.1(+) cells during the challenge period only did not influence pulmonary eosinophilic inflammation. CD1d1 mutant mice, deficient in NKT cells but with normal NK cells, developed lung tissue eosinophilia and allergen-specific IgE levels not different from those observed in wild-type mice. Mice deficient in gamma/delta T cells showed a mild attenuation of lung tissue eosinophilia in this model. Taken together, these findings suggest a critical role of NK cells, but not of NKT cells, for the development of allergen-induced airway inflammation, and that this effect of NK cells is exerted during the immunization. If translatable to humans, these data suggest that NK cells may be critically important for deciding whether allergic eosinophilic airway disease will develop. These observations are also compatible with a pathogenic role for the increased NK cell activity observed in human asthma.     

Cell-specific ecdysone-inducible expression of FLP recombinase in mammalian cells

The ability of site-specific recombinases, like FLP and Cre, to catalyze alterations in genomic DNA is well established, whereas their application to genetic engineering strategies has been restricted because of the inability to temporally regulate their expression and subsequent recombination events in specific populations of cells. We describe a regulatory system for ecdysone-controlled expression of FLP recombinase. Furthermore, we demonstrate that ecdysone-induced, FLP-mediated site-specific recombination events can be targeted to specific cells. This system can be applied to cell-lineage studies as well as to the design of gene-therapy strategies, particularly in stem cells.     

The contribution of impaired working memory monitoring to performance of the self-ordered pointing task in normal aging and Parkinson''s disease

IFI 16 is an interferon-inducible nucleoprotein expressed by human monocytes. IFI 16 and a related mouse protein, p202, control cellular proliferation by binding and modulating the functions of cell cycle regulatory factors including p53 and the retinoblastoma gene product, pRb. In this study, we examined IFI 16 expression in myeloid precursor cells cultured in vitro in colony-forming assays using granulocyte (G-) and granulocyte-macrophage (GM-) colony-stimulating factor (CSF). IFI 16 was expressed in 100% of CD34+ cells isolated from human bone marrow. When the CD34+ cells were induced to differentiate, two sub-populations of cells were identified by two-color cytofluorography: the CD14+ (monocytoid) cells all expressed IFI 16, whereas the CD14- (polymorphonuclear precursor) cells did not. The strongest expression of IFI 16 was in the cells staining brightest for CD14, whereas depletion of CD14+ monocytoid cells from mixed monocytic/granulocytic cultures largely abolished IFI 16-stained cells. Furthermore, in eight independent colony-forming assays, the number of IFI 16+ cells correlated closely with the numbers of monocyte precursors identified morphologically (R2 = 0.99), but was unrelated to the numbers of myelocytes, promyelocytes, and metamyelocytes; nor was IFI 16 expressed by erythroid or eosinophil precursors. We conclude that IFI 16 is expressed in CD34+ and monocytoid daughter cells, but is rapidly and markedly down-regulated at the corresponding stages of polymorphonuclear and erythroid development. This differential expression of IFI 16 in myeloid precursor subpopulations and its perceived molecular properties are consistent with a possible role in regulating myelopoiesis.     

Evaluation of estrogen treatment in female patients with dementia of the Alzheimer type

Increased numbers of eosinophilic granulocytes and eosinophil degranulation have been observed in various inflammatory conditions. Biopsies from the fallopian tubes of 28 patients were examined for the content of eosinophilic granulocytes. Peritoneal fluids (PF) from 89 patients were analyzed for the concentrations of eosinophilic granulocytes and the granule protein eosinophil cationic protein (ECP). The ECP levels in the PF were substantially elevated during genital inflammation (P < 0.001). Furthermore, one week after laparotomy for adhesiolysis there were both increased PF-ECP levels (P < 0.01) and raised concentrations of eosinophilic granulocytes (P < 0.001) when compared to the reference group. Staining of the biopsies with monoclonal antibodies revealed an increased total number as well as a number of activated eosinophilic granulocytes in specimens from adhesions and sactosalpinx compared to specimens from normal fimbriae. The findings indicate that eosinophilic granulocytes may have a possible role in the fibrotic process of pelvic adhesion disease.     

The formation of intramolecular disulfide bridges is required for induction of the Sindbis virus mutant ts23 phenotype

The Sindbis virus envelope protein spike is a hetero-oligomeric complex composed of a trimer of glycoprotein E1-E2 heterodimers. Spike assembly is a multistep process which occurs in the endoplasmic reticulum (ER) and is required for the export of E1 from the ER. PE2 (precursor to E2), however, can transit through the secretory pathway and be expressed at the cell surface in the absence of E1. Although oligomer formation does not appear to be required for the export of PE2, there is evidence that defects in E1 folding can affect PE2 transit from the ER. Temperature-sensitive mutant ts23 of Sindbis virus contains two amino acid substitutions in E1, while PE2 and capsid protein have the wild-type sequence; however, at the nonpermissive temperature, both E1 and PE2 are retained within the ER and can be isolated in protein aggregates with the molecular chaperone GRP78-BiP. We previously demonstrated that the temperature sensitivity for ts23 was lost as oligomer formation took place at the permissive temperature, suggesting that temperature sensitivity is initiated early in the process of viral spike assembly (M. Carleton and D. T. Brown, J. Virol. 70:952-959, 1996). Experiments described herein investigated the defects in envelope protein maturation that occur in ts23-infected cells and which result in retention of both envelope proteins in the ER. The data demonstrate that in ts23-infected cells incubated at the nonpermissive temperature, E1 folding is disrupted early after synthesis, resulting in the rapid incorporation of both E1 and PE2 into disulfide-stabilized aggregates. Furthermore, the aberrant E1 conformation which is responsible for induction of the ts phenotype requires the formation of intramolecular disulfide bridges formed prior to E1 association with PE2 and the completion of E1 folding.     

Eosinophilic cationic protein in chronic eosinophilic pneumonia and eosinophilic granuloma

We measured eosinophilic cationic protein (ECP) concentrations in the circulation and bronchoalveolar lavage (BAL) fluids from patients with chronic eosinophilic pneumonia, patients with eosinophilic granuloma, and normal control subjects. Significantly increased ECP concentrations were found in the circulation of patients with chronic eosinophilic pneumonia and with eosinophilic granuloma compared with those found in control subjects. The ECP concentrations were well correlated to eosinophil counts in the circulation of patients with chronic eosinophilic pneumonia, while they were not in patients with eosinophilic granuloma. Chronic eosinophilic pneumonia patients had prominently increased ECP concentrations in BAL fluids compared with those found in control subjects, while eosinophilic granuloma patients did not. Those concentrations in chronic eosinophilic pneumonia patients were well correlated to eosinophil counts in the BAL fluid. Corticosteroid therapy remarkably decreased circulating ECP concentrations in three patients with chronic eosinophilic pneumonia, but it had no significant effects in two patients with eosinophilic granuloma. Measurement of ECP concentrations seems to be useful to evaluate the disease activity of chronic eosinophilic pneumonia.     

Differential allelic expression of PrP mRNA in carriers of the E200K mutation

Creutzfeldt-Jakob disease (CJD) linked to the E200K mutation of the prion protein (PrP) gene presents with a wide range of age at disease onset. Since most patients are heterozygous for the mutation, we tested whether differential expression of mutant versus wild-type (wt) PrP may affect the age at disease onset in carriers of the mutation. We measured wt and mutant PrP protein and mRNA in Epstein-Barr virus (EBV)-transformed B cells of either E200K CJD patients or healthy E200K carriers. Our results suggests that while in most healthy carriers the expression of wt PrP was higher than that of E200K PrP, most of the E200K CJD patients express equal levels of both PrP proteins. Similar results were obtained for either PrP protein or PrP mRNA. These results suggest that preferential expression of PrP from the wt allele may modulate the outbreak of the disease in carriers of prion mutations. This notion is consistent with the results obtained in transgenic mice carrying a human PrP gene, which suggest that endogenous PrP protects mice from contracting scrapie after inoculation with human CJD brain. Similar mechanisms may prevail in other inherited diseases with variable phenotypes.     

A comparison of DNA polymerase alpha from untransformed and SV40-transformed human fibroblasts

1. DNA polymerase alpha (pol alpha) isolated from Simian virus 40 (SV40)-transformed cells showed more than 3-fold higher specific activity than pol alpha from normal cells. The enzymes from untransformed and transformed cells also differed in molecular size, thermolability, sensitivity to inhibitors and specificity of template-primer utilization. 2. Western analysis using anti-Tag to probe both a crude cell homogenate and partially purified pol alpha from SV40 transformed cells showed multiple immunoreactive bands with different molecular sizes. 3. While alpha polymerases from both normal and transformed cells exhibited tightly associated primase activity, they showed different DNA binding affinities. 4. These data suggest that T antigen binding to pol alpha alters the initiation of DNA replication and/or the function of pol alpha in SV40-transformed cells, and that pol alpha from SV40-transformed human fibroblasts have different catalytic subunit characteristics than pol alpha from untransformed cells.