Relationship between body composition, peripheral muscle strength and functional exercise capacity in patients with severe chronic obstructive pulmonary disease.

The influence of body composition and peripheral muscle strength on 6-minute walk distance was assessed by performing dual energy X-ray absorptiometry scanning, spirometry and dynamometry testing in 13 men and 13 women with severe chronic obstructive pulmonary disease. Multivariate modelling showed that 76% of the variance in 6-minute walk distance could be explained by an equation incorporating lung function, quadriceps strength and lean leg mass. These findings indicate an important role for lower limb strength measures in pulmonary rehabilitation training programmes.

Magnetic dichroism in K-shell photoemission from laser excited Li atoms.

Magnetic dichroism in the angular distribution has been demonstrated for single-electron photoemission from inner ns(2) subshells of gaseous atomic targets using the example of K-shell photoionization of polarized Li atoms laser prepared in the 1s(2)2p (2)P(3/2) excited state. The effect is pronounced for the conjugate shakeup and conjugate shakedown photoelectron lines, and less important, though observable, for the main and direct shakeup lines. The phenomenon is caused by configuration interaction in the final continuum state and is quantitatively described by the close-coupling R-matrix calculations.

Intensity inversion between main and satellite lines in atomic photoionization.

The 1s photoionization of atomic Li was studied by photoelectron spectroscopy in the photon energy region between 85 and 140 eV for the ground state and the three lowest excited configurations Li(*) 1s(2)nl, nl=2p, 3s, 3p. The importance of electron correlations was investigated by comparing the multielectron transitions, so-called shake-up and conjugate shake-up satellites, and the direct process, so-called main lines. The relative intensity of the satellites increases with the level of initial excitation of the Li atom. The shake-up process dominates for states with an n=3 valence electron and the satellites become stronger than the main lines. This spectacular effect can be explained by the spatial overlap of the initial and final state wave functions. Surprisingly, the spatial overlap affects shake-up and conjugate shake-up lines in the same way.

Outstanding spin-orbit-activated interchannel coupling in the Cs and Ba 3d photoemission.

Partial 3d5/2 photoionization cross sections of atomic Cs and Ba as well as the asymmetry parameter beta of the angular distribution of the Cs 3d5/2 photoelectrons were investigated near the threshold of the 3d3/2 channel at about 750 eV and 800 eV, respectively. Strong electron correlations, in particular, the spin-orbit activated interchannel coupling between the 3d5/2 and 3d3/2 channels, govern the observed spectra. The most striking effect was found for beta5/2 of Cs with a dramatic increase from beta=1.0 to beta=1.5 in the energy region where the mixing between both channels causes a pronounced minimum in the partial 3d5/2 cross section. This result indicates the decisive influence of the interference term on the asymmetry parameter beta with its dependence on the phase difference between the outgoing p and f waves.

Electrotransformation and expression of bacterial genes encoding hygromycin phosphotransferase and beta-galactosidase in the pathogenic fungus Histoplasma capsulatum.

We developed an efficient electrotransformation system for the pathogenic fungus Histoplasma capsulatum and used it to examine the effects of features of the transforming DNA on transformation efficiency and fate of the transforming DNA and to demonstrate fungal expression of two recombinant Escherichia coli genes, hph and lacZ. Linearized DNA and plasmids containing Histoplasma telomeric sequences showed the greatest transformation efficiencies, while the plasmid vector had no significant effect, nor did the derivation of the selectable URA5 marker (native Histoplasma gene or a heterologous Podospora anserina gene). Electrotransformation resulted in more frequent multimerization, other modification, or possibly chromosomal integration of transforming telomeric plasmids when saturating amounts of DNA were used, but this effect was not observed with smaller amounts of transforming DNA. We developed another selection system using a hygromycin B resistance marker from plasmid pAN7-1, consisting of the E. coli hph gene flanked by Aspergillus nidulans promoter and terminator sequences. Much of the heterologous fungal sequences could be removed without compromising function in H. capsulatum, allowing construction of a substantially smaller effective marker fragment. Transformation efficiency increased when nonselective conditions were maintained for a time after electrotransformation before selection with the protein synthesis inhibitor hygromycin B was imposed. Finally, we constructed a readily detectable and quantifiable reporter gene by fusing Histoplasma URA5 with E. coli lacZ, resulting in expression of functional beta-galactosidase in H. capsulatum. Demonstration of expression of bacterial genes as effective selectable markers and reporters, together with a highly efficient electrotransformation system, provide valuable approaches for molecular genetic analysis and manipulation of H. capsulatum, which have proven useful for examination of targeted gene disruption, regulated gene expression, and potential virulence determinants in this fungus.

Rare homologous gene targeting in Histoplasma capsulatum: disruption of the URA5Hc gene by allelic replacement.

URA5 genes encode orotidine-5'-monophosphate pyrophosphorylase (OMPpase), an enzyme involved in pyrimidine biosynthesis. We cloned the Histoplasma capsulatum URA5 gene (URA5Hc) by using a probe generated by PCR with inosine-rich primers based on relatively conserved sequences in OMPpases from other organisms. Transformation with this gene restored uracil prototrophy and OMPpase activity to UV-mutagenized ura5 strains of H. capsulatum. We attempted to target the genomic URA5 locus in this haploid organism to demonstrate homologous allelic replacement with transforming DNA, which has not been previously done in H. capsulatum and has been challenging in some other pathogenic fungi. Several strategies commonly used in Saccharomyces cerevisiae and other eukaryotes were unsuccessful, due to the frequent occurrence of ectopic integration, linear plasmid formation, and spontaneous resistance to 5-fluoroorotic acid, which is a selective agent for URA5 gene inactivation. Recent development of an efficient electrotransformation system and of a second selectable marker (hph, conferring hygromycin B resistance) for this fungus enabled us to achieve allelic replacement by using transformation with an insertionally inactivated Deltaura5Hc::hph plasmid, followed by dual selection with hygromycin B and 5-fluoroorotic acid, or by screening hygromycin B-resistant transformants for uracil auxotrophy. The relative frequency of homologous gene targeting was approximately one allelic replacement event per thousand transformants. This work demonstrates the feasibility but also the potential challenge of gene disruption in this organism. To our knowledge, it represents the first example of experimentally directed allelic replacement in H. capsulatum, or in any dimorphic systemic fungal pathogen of humans.

A system of rapid isolation of end-DNA from a small amount of fosmid DNA, with vector-based PCR for chromosome walking.

The pBAC 108L and pFos 1 vectors were developed as stable propagation vectors which, due to their extremely low copy number, facilitate the cloning of a large-sized insert containing repeated DNA. However, the low copy number requires laborious end-DNA preparation for end sequencing and chromosome walking. Here we describe efficient methods for end-DNA isolation. The entire process, including small-scale DNA preparation, restriction digestion, self-ligation, and PCR with vector-based primers, is carried out in 96-well formats. Using a Fosmid library of genomic DNA of Candida albicans, PCR products ranging in size from 0.1 to 8 kbp were generated from 118 end sequences in 140 reactions from 70 Fosmid clones. A single or a prominent band was found in 101 of these reactions. Twenty-six of these bands were tested for walking and all of them proved to be specific. Thus, the system overcomes the disadvantage caused by low copy number. This system allows rapid physical mapping of genomes, and is adaptable for several other vectors including BAC (bacterial artificial chromosome), PAC (P1-derived artificial chromosome), and YAC (yeast artificial chromosome).

The URA5 gene is necessary for histoplasma capsulatum growth during infection of mouse and human cells.

The Histoplasma capsulatum URA5 gene, which has recently been cloned and disrupted by allelic replacement, encodes orotidine-5'-monophosphate pyrophosphorylase. Inactivation of URA5 by either targeted or UV mutagenesis results in disruption of the pyrimidine biosynthetic pathway and uracil auxotrophy. We examined the effect of uracil auxotrophy due to a ura5 mutation on H. capsulatum virulence in both cell culture and whole-animal models. Uracil auxotrophs of two H. capsulatum restriction fragment length polymorphism classes were found to be avirulent in cultured murine and human cells, as well as in mice. Moreover, virulence could be restored either by supplying a functional URA5 gene in trans or by supplying exogenous uracil during infection in vitro. These experiments demonstrate that the pyrimidine biosynthetic pathway is essential for H. capsulatum growth and virulence.

Pathogenesis of Histoplasma capsulatum.

Histoplasma capsulatum is well adapted to be infectious and pathogenic for humans. As a soil fungus with no known requirement for interacting with a mammalian host as part of an obligate lifecycle, its plethora of strategies for successful pathogenesis is particularly remarkable. These features include the dimorphic mold-yeast transition, entry into host macrophages, subcellular localization, intracellular survival and proliferation during active infection, and persistence during clinically inapparent infection with the capacity for reactivation. To thrive within the harsh environment of a professionally phagocytic and antimicrobial host cell, H. capsulatum displays mechanisms for modulating its microenvironmental pH level, resisting host reactive oxygen and nitrogen intermediates and degradative enzymes, and withstanding nutrient starvation conditions, including acquisition of iron and calcium and biosynthesis of nucleic acid precursors. Attention has been focused on identifying virulence-associated phenotypic traits and genes that are differentially expressed under relevant conditions, such as yeast morphotype-specific genes and genes that are up-regulated during infection. These studies, together with the increasing ability to perform molecular genetic manipulations in this fungus, may yield novel antifungal drug or vaccine targets as well as elucidating pathogenic mechanisms.