Enzymatic properties of an ecto-nucleoside triphosphate diphosphohydrolase from Legionella pneumophila: substrate specificity and requirement for virulence

Legionella pneumophila is the predominant cause of Legionnaires disease, a severe and potentially fatal form of pneumonia. Recently, we identified an ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila, termed Lpg1905, which enhances intracellular replication of L. pneumophila in eukaryotic cells. Lpg1905 is the first prokaryotic member of the CD39/NTPDase1 family of enzymes, which are characterized by the presence of five apyrase conserved regions and the ability to hydrolyze nucleoside tri- and diphosphates. Here we examined the substrate specificity of Lpg1905 and showed that apart from ATP and ADP, the enzyme catalyzed the hydrolysis of GTP and GDP but had limited activity against CTP, CDP, UTP, and UDP. Based on amino acid residues conserved in the apyrase conserved regions of eukaryotic NTPDases, we generated five site-directed mutants, Lpg1905E159A, R122A, N168A, Q193A, and W384A. Although the mutations E159A, R122A, Q193A, and W384A abrogated activity completely, N168A resulted in decreased activity caused by reduced affinity for nucleotides. When introduced into the lpg1905 mutant strain of L. pneumophila, only N168A partially restored the ability of L. pneumophila to replicate in THP-1 macrophages. Following intratracheal inoculation of A/J mice, none of the Lpg1905 mutants was able to restore virulence to an lpg1905 mutant during lung infection, thereby demonstrating the importance of NTPDase activity to L. pneumophila infection. Overall, the kinetic studies undertaken here demonstrated important differences to mammalian NTPDases and different sensitivities to NTPDase inhibitors that may reflect underlying structural variations.     

Immune suppression blocks sodium-sensitive hypertension following recovery from ischemic acute renal failure

The present study determined the effect of immune suppression with mycophenolate mofetil (MMF) on sodium-sensitive hypertension following recovery from ischemia reperfusion (I/R)-induced acute renal failure. Male Sprague-Dawley rats fed 0.4% NaCl chow were subjected to 40 min bilateral I/R or control sham surgery. After 35 days of recovery, when plasma creatinine levels had returned to normal, the rats were switched to 4.0% NaCl chow for 28 days and administered vehicle or MMF (20 mg.kg(-1).day(-1) ip). High-salt mean arterial pressure was significantly higher in I/R rats (144 +/- 16 mmHg) compared with vehicle-treated sham rats (122 +/- 2 mmHg). Treatment of I/R rats with MMF during the period of high salt intake prevented the salt-induced increase in arterial pressure (114 +/- 3 mmHg). Conscious creatinine clearance was lower in I/R rats (0.27 +/- 0.07 ml.min(-1).100 g body wt(-1)) compared with vehicle-treated sham rats (0.58 +/- 0.04 ml.min(-1).100 g body wt(-1)); MMF treatment prevented the decrease in creatinine clearance in I/R rats (0.64 +/- 0.07 ml.min(-1).100 g body wt(-1)). I/R injury also significantly increased glomerular tissue damage and increased the presence of ED-1 positive (macrophages) and S100A4 positive cells (fibroblasts) in the renal interstitium. The I/R rats treated with MMF exhibited a significant reduction in infiltrating macrophages and fibroblasts and decreased histological damage. The present data indicate that infiltrating immune cells mediate or participate in the development of sodium-sensitive hypertension and renal damage in rats apparently recovered from renal I/R injury.     

Localized decrease of beta-catenin contributes to the differentiation of human embryonic stem cells

Human embryonic stem cells (hESC) are pluripotent, and can be directed to differentiate into different cell types for therapeutic applications. To expand hESCs, it is desirable to maintain hESC growth without differentiation. As hESC colonies grow, differentiated cells are often found at the periphery of the colonies, but the underlying mechanism is not well understood. Here, we utilized micropatterning techniques to pattern circular islands or strips of matrix proteins, and examined the spatial pattern of hESC renewal and differentiation. We found that micropatterned matrix restricted hESC differentiation at colony periphery but allowed hESC growth into multiple layers in the central region, which decreased hESC proliferation and induced hESC differentiation. In undifferentiated hESCs, β-catenin primarily localized at cell-cell junctions but not in the nucleus. The amount of β-catenin in differentiating hESCs at the periphery of colonies or in multiple layers decreased significantly at cell-cell junctions. Consistently, knocking down β-catenin decreased Oct-4 expression in hESCs. These results indicate that localized decrease of β-catenin contributes to the spatial pattern of differentiation in hESC colonies.     

Responses of vertebrates to pastoralism,military land use and landscape position in an Australian tropical savanna

Despite the hegemony of pastoralism over most of Australia’s tropical savannas, its impacts upon biodiversity are poorly known. There is even less knowledge about the impacts of military training, a recent, but rapidly expanding, alternative land use. We compare impacts of these land uses upon mammals, birds, reptiles and frogs at a site in north‐eastern Australia, with sampling from 24 quadrats stratified by four landscape positions (upper slope to riparian) and three current land‐use types (pastoralism, military training and undisturbed). Prior to exclusion in 1967, the whole study area had been subjected to grazing over the course of approximately 100 years, so differences observed strictly reflect responses to changed land use (largely cessation from grazing) over the period of 32 years subsequent to the imposition of the present regime. The four classes of vertebrates showed contrasting responses. Frog distribution was unrelated to land use, but strongly associated with landscape position. Reptiles showed a very strong response to land‐use type but not to landscape position. The total abundance and richness of reptiles was greater in ungrazed (i.e. military and undisturbed) than in grazed quadrats. The total abundance and species richness of birds varied strongly with landscape position but was unrelated to land use. However, many individual bird species showed significant responses to land‐use type, and bird species composition was significantly related to both land‐use type and landscape position. The richness of the mammal fauna was weakly related to landscape position and not related to land‐use type. A few individual mammal species showed significant responses to either or both factors, but mammal species composition was significantly (albeit weakly) related only to land‐use type. With due regard to some interpretative constraints in the study design, and the history of the site prior to this study, these results suggest that pastoralism leads to a substantial rearrangement of the vertebrate fauna, and particularly so for reptiles and those mammals and birds associated with the ground and understorey layers. Given the extent of pastoralism across the tropical savannas, these results suggest that this industry has contributed to major and widespread change in the savanna fauna. In contrast to pastoralism, military land use (at least at the relatively low intensity examined here) produced little change in vertebrate assemblages.     

The ultrastructure of mouse testicular interstitial tissue containing plutonium-239 and its significance in explaining the observed distribution of plutonium in the testis.

The technique of autoradiography with Araldite-embedded sections was used to study the distribution of 239Pu in mouse testis at various times post-injection. Adjacent sections were examined with both the light microscope and electron microscope. The autoradiographs showed that from 1 week to 3 months postinjection, most 239Pu in located in interstitial tissue. The major change in distribution observed was that the early diffuse deposit in interstitial tissue is concentrated in macrophages with increasing time post-injection. This is a real change of distribution as the amount of 239Pu in mouse testis remains constant from 1 week to 3 months post-injection. Study of the ultrastructure of interstitial tissue indicated that the accumulation of 239Pu in macrophages may be brought about in two ways. First, there may be phagocytosis of dead cells containing 239Pu. Second, 239Pu may follow the transfer of waste products of hormone synthesis from Leydig cells into macrophages. The significance of these observations is discussed with regard to the deposition of 239Pu in human testis.     

Heavy meromyosin binding to microfilaments involved in cell and morphogenetic movements

Certain microfilaments found in migratory single cells and in morphogenetically-active epithelial cells are shown to bind heavy meromyosin prepared from skeletal muscle. This ‘actin-like’ feature of the filaments is compatible with the hypothesis that such filaments are components of contractile systems in these cells.     

CHEMOSENSING: SELECTIVITY, SENSITIVITY AND ADDITIVE EFFECTS ON A STIMULANT-INDUCED ACTIVITY OF OLFACTORY PREPARATIONS

Stimulant-induced changes in soluble preparations from rabbit olfactory epithelium were monitored by ultraviolet difference spectroscopy. A time-dependent decrease of absorbance, maximum at 267 nm, was generated when specific stimulants were mixed with the olfactory preparations. Only seven of the 37 organic chemicals tested generated the absorbance change at 267 nm. The olfactory preparations exhibited a high degree of selectivity and sensitivity; measurable absorbance changes were observed when the active stimulant concentrations were 1 × 10^?5m . The experimental results suggest a complex mechanism of conformational change for the initial stimulant-induced molecular interactions in biological chemosensing.     

EPR studies of the Mn(II) complex with elongation factor Tu and GDP Identification of oxygen ligands to Mn(II) by observation of 17O superhyperfine coupling

The coordination sphere of Mn(II) in the complex with GDP and elongation factor Tu from Escherichia coli has been probed by EPR spectroscopy with 17O-labeled ligands. Inhomogeneous broadening in the EPR signals for Mn(II) due to unresolved superhyperfine coupling to the 17O nucleus was used to identify directly bound oxygen ligands. Results with GDP selectively enriched with 17O either in the alpha-phosphate or in the beta-phosphate revealed that GDP was a beta-monodentate ligand for Mn(II) in the complex with the protein. Results with 17O-enriched water showed that two water molecules are coordinated to the Mn(II). The EPR spectrum for the complex is characteristic of octahedral coordination for Mn(II). Hence, three ligands from the protein are required to complete the sextet of ligands.