Open microwave cavity for use in a Purcell enhancement cooling scheme.

A microwave cavity is described which can be used to cool lepton plasmas for potential use in synthesis of antihydrogen. The cooling scheme is an incarnation of the Purcell effect: when plasmas are coupled to a microwave cavity, the plasma cooling rate is resonantly enhanced through increased spontaneous emission of cyclotron radiation. The cavity forms a three electrode section of a Penning-Malmberg trap and has a bulged cylindrical geometry with open ends aligned with the magnetic trapping axis. This allows plasmas to be injected and removed from the cavity without the need for moving parts while maintaining high quality factors for resonant modes. The cavity includes unique surface preparations for adjusting the cavity quality factor and achieving anti-static shielding using thin layers of nichrome and colloidal graphite, respectively. Geometric design considerations for a cavity with strong cooling power and low equilibrium plasma temperatures are discussed. Cavities of this weak-bulge design will be applicable to many situations where an open geometry is required.

Electron Plasmas Cooled by Cyclotron-Cavity Resonance.

We observe that high-Q electromagnetic cavity resonances increase the cyclotron cooling rate of pure electron plasmas held in a Penning-Malmberg trap when the electron cyclotron frequency, controlled by tuning the magnetic field, matches the frequency of standing wave modes in the cavity. For certain modes and trapping configurations, this can increase the cooling rate by factors of 10 or more. In this Letter, we investigate the variation of the cooling rate and equilibrium plasma temperatures over a wide range of parameters, including the plasma density, plasma position, electron number, and magnetic field.

Synthesis and characterization of diverse Pt nanostructures in Nafion.

With the aid of TEM characterization, we describe two distinct Pt nanostructures generated via the electroless reduction of Pt(NH3)4(NO2)2 within Nafion. Under one set of conditions, we produce bundles of Pt nanorods that are 2 nm in diameter and 10-20 nm long. These bundled Pt nanorods, uniformly distributed within 5 µm of the Nafion surface, are strikingly similar to the proposed hydrated nanomorphology of Nafion, and therefore strongly suggestive of Nafion templating. By altering the reaction environment (pH, reductant strength, and Nafion hydration), we can also generate nonregular polyhedron Pt nanoparticles that range in size from a few nanometers in diameter up to 20 nm. These Pt nanoparticles form a dense Pt layer within 100-200 nm from the Nafion surface and show a power-law dependence of particle size and distribution on the distance from the Nafion membrane surface. Control over the distribution and the type of Pt nanostructures in the surface region may provide a cost-effective, simple, and scaleable pathway for enhancing manufacturability, activity, stability, and utilization efficiency of Pt catalysts for electrochemical devices.

Toll-like receptor expression and function in subsets of human gammadelta T lymphocytes.

Two subsets of human gammadelta T cells can be identified by T cell receptor (TCR) V gene usage. Vdelta2Vgamma9 T cells dominate in peripheral blood and recognize microbe- or tumour-derived phosphoantigens. Vdelta1 T cells are abundant in mucosal tissue and recognize stress-induced MHC-related molecules. Toll-like receptors (TLRs) are known to co-stimulate interferon-gamma (IFN-gamma) production in peripheral blood gammadelta T cells and in Vdelta2Vgamma9 T cell lines. By microarray analysis, we have identified a range of genes differentially regulated in freshly isolated gammadelta T cells by TCR versus TCR plus TLR3 stimulation. Furthermore, we have investigated TLR expression in freshly isolated Vdelta1 and Vdelta2 subsets and cytokine/chemokine production in response to TLR1/2/6, 3 and 5 ligands. TLR1,2,6,7 RNA was abundantly expressed in both subsets, whereas TLR3 RNA was present at low levels, and TLR5 and 8 RNA only marginally in both subsets. Despite abundant RNA expression, TLR1 was rarely detectable by flow cytometry. In contrast, TLR2 and TLR6 proteins were detected in purified Vdelta1 and Vdelta2 T cells, and TLR3 protein was detected intracellularly in both subsets. TLR1/2/6, 3 and 5 ligands co-stimulated the IFN-gamma and chemokine secretion in TCR-activated Vdelta1 and Vdelta2 subsets, although the levels of IFN-gamma secreted by Vdelta1 T cells were much lower than those produced by Vdelta2 T cells. Our results reveal comparable expression of TLRs and functional responses to TLR ligands in freshly isolated Vdelta1 and Vdelta2 T cells and underscore the intrinsically different capacity for IFN-gamma secretion of Vdelta1 versus Vdelta2 T cells.

Lack of c-jun expression in a transformed cell line isolated by glucocorticoid promotion of ras-transfected rat embryo fibroblasts.

Rat embryo fibroblasts (REFs) are inefficiently transformed by the T24-ras oncogene. A contributing factor to cellular resistance to transformation is the limited tolerance to p21-ras oncoprotein expression. Here we present data suggesting that long-term glucocorticoid treatment of ras oncogene-transfected REFs results in increased tolerance to p21-ras oncoproteins, leading to expression of the transformed phenotype. Stably transformed cell lines that expressed high levels of H-ras and could be maintained in the absence of hormone were isolated. In three out of four lines studied, the AP-1-dependent collagenase gene was expressed at a low level. In one of these lines, low collagenase expression was paralleled by lack of c-jun mRNA. Immunochemical analysis revealed that progression to hormone independence was not paralleled by mutations in the p53 gene. We propose that a decreased expression of AP-1-driven genes may result in increased tolerance to p21-ras oncoprotein.

Glucocorticoid hormones may partially substitute for adenovirus E1A in cooperation with ras.

The effects of hormonal promotion of T24-ras oncogene-transfected rat embryo fibroblasts (REF) were compared to cotransformation of these cells with adenovirus E1A and ras. Cotransfection of E1A + ras resulted in the appearance of morphologically transformed cells which were very efficiently established into cell lines. Addition of glucocorticoid hormones to T24-ras-transfected REF cells resulted in cells with a transformed morphology and a capacity to form foci. These foci were, however, inefficiently established into stable cell lines. Removal of hormone from growing cells resulted in retarded growth, suggesting that the hormone acted as a growth factor on these cells. Both E1A-transformed cells and hormone-treated ras-transformed cells showed a reduction in synthesis of high molecular weight tropomyosin isoforms and a decreased expression of surface fibronectin. Control experiments demonstrated that the effects of hormone were mediated through the glucocorticoid receptor. Our findings suggest that glucocorticoid hormones may promote the in vitro growth of ras-initiated REF cells into stably transformed cell lines, but that this ability is limited compared to that of adenovirus E1A.

Fetal Doppler flow measurements in the early second trimester: a correlation with fetal outcome.

In 31 women with a singleton pregnancy, abdominal duplex doppler examinations were performed at W16-W17, W26 (+/- 2 weeks) and W34 (+/- 2 weeks) in order to study the flow velocity wave (FVW) indices in the early second trimester and their predictive value for fetal outcome. 24 women with a normal pregnancy outcome were considered as the reference group. In the early second trimester, end-diastolic block occurs frequently at the 150 Hz thump filter setting (15/24 in the fetal descending aorta, 19/24 in the umbilical artery). At the 50 Hz filter setting, end diastolic block appeared in 1/24 cases in the aorta and in 2/24 cases in the umbilical artery. The finding did not persist throughout pregnancy. The flow-velocity indices in the early second trimester in the small-for-dates were comparable to the normal group. We conclude that high peripheral resistance is a common finding in the fetal circulation in early pregnancy. It is not predictive of subsequent growth retardation.

A viable mouse polyomavirus mutant without immortalizing or transforming activities.

The polyomavirus mutant, dl1041, has a 375-base pair deletion. It removes most of the sequences that are unique to rodent polyomaviruses and encodes part of the large and middle T-antigens. The mutant was conditionally viable, although both the immortalizing and transforming functions of the T-antigens produced by this mutant were found to be defective. However, the dl1041 mutant was found to be capable of DNA replication in rapidly growing mouse 3T6 cells. In contrast, dl1041 DNA synthesis could not be detected in serum-deprived mouse 3T3 cells. In these cells, the low efficiency of dl1041 DNA replication could be attributed to deficiencies in both large and middle T-antigen, suggesting a link between the mitogenic and oncogenic activities of these proteins. Transfection of growing mouse 3T6 cells with dl1041 DNA resulted in the formation of infectious virus, demonstrating that the dl1041 mutant is able to complete an infection cycle. The ability to activate the viral late promoter in trans was retained by the dl1041 mutant large T-antigen, suggesting that immortalization and trans-activation of the late promoter represent two distinct activities of the protein. An essential element of the immortalizing activity in the large T-antigen polypeptide chain appeared to be in a segment consisting of amino acid residues 136-184, since the dl1041 deletion abolished the activity and the 184 amino acid residue N-terminal dl1354 fragment of large T-antigen retained the activity.

Complete transformation of embryonal rat fibroblasts by polyomavirus occurs during passage in vitro.

The tumorigenicity of secondary rat embryo fibroblasts transfected with a plasmid harboring a replication origin-defective polyomavirus was found to increase during in vitro propagation. Thus, polyomavirus-transfected cells were found to be more than 10,000-fold more tumorigenic when injected into syngenic rats at 3 months after transfection compared to those injected at an earlier time point. Furthermore, most clones of polyomavirus-transfected cells did not grow in semisolid medium at 52 days after transfection but did grow at 95 days. Addition of glucocorticoid hormones, but not of 25% fetal calf serum, to the growth medium of the early passage cells resulted in limited anchorage-independent growth. An altered level of expression of a number of proteins was found in cells analyzed at different times after transfection. Notably, the expression of a component of the actin filament system, tropomyosin 2, was shown to decrease during growth in vitro. The development of a more fully transformed phenotype at late passages correlated with loss of the requirement for large T-antigen for growth. Thus, cells transfected with a polyomavirus mutant encoding a thermolabile large T-antigen did not grow at the restrictive temperature at 6 weeks after transfection, but grew well at 5 months after transfection. We suggest that these phenomena may be explained by assuming that establishment of rodent fibroblasts, and thereby sensitivity to transformation by middle T-antigen, is not an immediate consequence of expression of large T-antigen but occurs after a period of growth in vitro.

Mutational analysis of polyomavirus small-T-antigen functions in productive infection and in transformation.

The function of polyomavirus small T antigen in productive infection and in transformation was studied. Transfection of permissive mouse cells with mixtures of mutants that express only one type of T antigen showed that small T antigen increased large-T-antigen-dependent viral DNA synthesis approximately 10-fold. Under the same conditions, small T antigen was also essential for the formation of infectious virus particles. To analyze these activities of small T antigen, mutants producing protein with single amino acid replacements were constructed. Two mutants, bc1073 and bc1075, were characterized. Although both mutations led to the substitution of amino acid residues of more than one T antigen, the phenotype of both mutants was associated with alterations of the small T antigen. Both mutant proteins had lost their activity in the maturation of infectious virus particles. The bc1075 but not the bc1073 small T antigen had also lost its ability to stimulate viral DNA synthesis in mouse 3T6 cells. Finally, both mutants retained a third activity of small T antigen: to confer on rat cells also expressing middle T antigen the ability to grow efficiently in semisolid medium. The phenotypes of the mutants in these three assays suggest that small T antigen has at least three separate functions.

Adenovirus proteins and MHC expression.

Adenoviruses are able to specifically down-regulate the cell surface expression of MHC class I antigens. Most viral serotypes achieve these ends by synthesizing a protein that binds to class I antigens in the endoplasmic reticulum (ER) and impedes the transport of these molecules to the cell surface. However, viruses belonging to the highly oncogenic subgenus A do not affect the class I antigen expression during acute infection. Instead, they are distinct from other adenoviruses in that they specifically down-regulate the level of mRNAs, encoding MHC class I antigens, in virally transformed cells. The virus-induced reduction of class I antigen expression drastically diminishes the ability of CTLs to recognize cells infected or transformed by adenovirus. A number of issues concerning these viral mechanisms for class I antigen modulation need to be addressed. The molecular mechanism by which the E1A gene product of subgenus A viruses diminishes class I mRNA levels has not been elucidated. Also, the details of the interaction between the E19 protein and class I molecules should be studied, preferably by X-ray crystallography of the complexes. This would clarify the role of the antigen-binding site as well as other portions of the class I molecule in the binding to the E19 protein. Of general importance for our understanding of the sorting and intracellular transport of proteins is the exact delimitation of the signal for ER localization, which is present in the COOH-terminus of the E19 protein. The putative interaction of this peptide sequence with components of the ER membrane should also be studied. Finally, the study of the pathophysiological role of the MHC class I down-regulation will undoubtedly yield new insights into how the immune system combats virally infected and transformed cells.

Glucocorticoids facilitate the stable transformation of embryonal rat fibroblasts by a polyomavirus large tumor antigen-deficient mutant.

The addition of glucocorticoids to the growth medium could substitute for the expression of the polyomavirus large tumor antigen in the transformation of rat fibroblasts in vitro. After transfection with a large tumor antigen-deficient mutant of polyomavirus, pbc1051, high-frequency permanent transformation was observed, if the cells were grown in medium containing dexamethasone. Growth of pbc1051-transfected rat fibroblasts was strictly dependent on the presence of glucocorticoids during the initial phase of transformation. In the second phase, the growth of pbc1051-transfected cells was stimulated by dexamethasone, but the hormone was not essential for growth. After approximately 10 weeks in culture, pbc1051-transfected cells had progressed to hormone independent growth. Rat embryo cells transfected with wild-type polyomavirus DNA had the second phase in which growth was stimulated by glucocorticoid, and after this phase growth was steroid independent. Addition of glucocorticoids to rat fibroblasts transfected with a plasmid encoding only the middle-sized tumor antigen resulted in only a weak stimulation of growth. In contrast, embryo cells transfected with a plasmid containing the human homologue of the cellular T24 Ha-ras gene linked to murine sarcoma virus and simian virus 40 enhancers could be efficiently established as cell lines in medium supplemented with glucocorticoids. The data suggest that, in the transformation of primary rodent cells by polyomavirus, the activity of large tumor antigen can be substituted for by stimulating normal cellular functions with dexamethasone.

Differential association between two human MHC class I antigens and an adenoviral glycoprotein.

The glycoprotein E19, encoded in early region 3 of adenovirus-2, forms complexes with major histocompatibility complex class I antigens. As a result of the complex formation, the intracellular transport of the class I antigens is abrogated, and adenovirus-infected cells display gradually diminishing quantities of cell surface-expressed class I molecules. To assess whether the E19 protein interacts equally well with different class I antigens, the associations between the viral protein and HLA-A2 and HLA-B7 antigens have been estimated. By infecting transfected HeLa cells expressing various amounts of HLA-A2 and HLA-B7 molecules, respectively, with various infectious doses of adenovirus-2, experimental conditions could be established that allowed quantitative estimates of the interactions to be determined. It was found that HLA-A2 molecules and the E19 protein interacts with a binding constant that is more than twice as high as that for HLA-B7 antigens and the viral protein. It is suggested that the pathogenicity of the virus may be dependent on the HLA-type of the infected individual.