Pneumocystis carinii in corticosteroid-treated voles: a comparison of three different staining methods.

Pneumocystis carinii (PC) is an opportunistic pathogen which causes clinical disease in immunocompromised hosts. Three different staining protocols were employed to detect this organism in lung samples of corticosteroid treated voles in order to discover a suitable method for large-scale screening. The procedures employed were: Grocotts methenamine silver (GMS)-stained paraffin sections, toluidine blue O-stained impression smears, and methenamine-silver-stained frozen sections. GMS-stained paraffin sections were relatively easy to interpret and gave more positive results than the other methods. It seemed to be the satisfactory method for large-scale population analyses. An unexpected result was that methylprednisolone treatment did not induce in voles a similarly fatal pneumocystosis infection as occurred in rats. All infections found in voles were mild. This might be due to species-dependent differences in metabolizing methylprednisolone.     

Sequence analysis of the cbb3 oxidases and an atomic model for the Rhodobacter sphaeroides enzyme

The cbb3-type oxidases are members of the heme-copper oxidase superfamily, distant by sequence comparisons, but sharing common functional characteristics. To understand the minimal common properties of the superfamily, and to learn about cbb3-type oxidases specifically, we have analyzed a wide set of heme-copper oxidase sequences and built a homology model of the catalytic subunit of the cbb3 oxidase from Rhodobacter sphaeroides. We conclude that with regard to the active site surroundings, the cbb3 oxidases greatly resemble the structurally known oxidases, while major differences are found in three segments: the additional N-terminal stretch of ca. 60 amino acids, the segment following helix 3 to the end of helix 5, and the C-terminus from helix 11 onward. The conserved core contains the active site tyrosine and also an analogue of the K-channel of proton transfer, but centered on a well-conserved histidine in the lower part of helix 7. Modeling the variant parts of the enzyme suggests that two periplasmic loops (between helices 3 and 4 and between helices 11 and 12) could interact with each other as a part of the active site structure and might have an important role in proton pumping. An analogue of the D-channel is not found, but an alternative channel might form around helix 9. A preliminary packing model of the trimeric enzyme is also presented.     

The calcium binding site in cytochrome aa3 from Paracoccus denitrificans.

A shift in the spectrum of heme a induced by calcium or proton binding, or by the proton electrochemical gradient, has been attributed to interaction of Ca2+ or H+ with the vicinity of the heme propionates in mitochondrial cytochrome c oxidase, and proposed to be associated with the exit path of proton translocation. However, this shift is absent in cytochrome c oxidases from yeast and bacteria [Kirichenko et al. (1998) FEBS Lett. 423, 329-333]. Here we report that mutations of Glu56 or Gln63 in a newly described Ca2+/Na+ binding site in subunit I of cytochrome c oxidase from Paracoccus denitrificans [Ostermeier et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 10547-10553] establish the Ca2+-dependent spectral shift in heme a. This shift is counteracted by low pH and by sodium ions, as was described for mammalian cytochrome c oxidase, but in the mutant Paracoccus enzymes Na+ is also able to shift the heme a spectrum, albeit to a smaller extent. We conclude that the Ca2+-induced shift in both Paracoccus and mitochondrial cytochrome aa3 is due to binding of the cation to the new metal binding site. Comparison of the structures of this site in the two types of enzyme allows rationalization of their different reactivity with cations. Structural analysis and data from site-directed mutagenesis experiments suggest mechanisms by which the cation binding may influence the heme spectrum.     

Alphavirus Replicase Protein NSP1 Induces Filopodia and Rearrangement of Actin Filaments

Expression of the NSP1 protein of Semliki Forest virus and Sindbis virus in cultured cells induced filopodia-like extensions containing NSP1 but not F actin. The actin stress fibers disappeared, whereas vimentin, keratin, and tubulin networks remained intact. The effects of NSP1 were dependent on its palmitoylation but not on its enzymatic activities and were also observed in virus-infected cells.     

Estrogenic regulation of skeletal muscle proteome: a study of premenopausal women and postmenopausal MZ cotwins discordant for hormonal therapy

Female middle age is characterized by a decline in skeletal muscle mass and performance, predisposing women to sarcopenia, functional limitations, and metabolic dysfunction as they age. Menopausal loss of ovarian function leading to low circulating level of 17β‐estradiol has been suggested as a contributing factor to aging‐related muscle deterioration. However, the underlying molecular mechanisms remain largely unknown and thus far androgens have been considered as a major anabolic hormone for skeletal muscle. We utilized muscle samples from 24 pre‐ and postmenopausal women to establish proteome‐wide profiles, associated with the difference in age (30–34 years old vs. 54–62 years old), menopausal status (premenopausal vs. postmenopausal), and use of hormone replacement therapy (HRT; user vs. nonuser). None of the premenopausal women used hormonal medication while the postmenopausal women were monozygotic (MZ) cotwin pairs of whom the other sister was current HRT user or the other had never used HRT. Label‐free proteomic analyses resulted in the quantification of 797 muscle proteins of which 145 proteins were for the first time associated with female aging using proteomics. Furthermore, we identified 17β‐estradiol as a potential upstream regulator of the observed differences in muscle energy pathways. These findings pinpoint the underlying molecular mechanisms of the metabolic dysfunction accruing upon menopause, thus having implications for understanding the complex functional interactions between female reproductive hormones and health.     

Effects of palmitoylation of replicase protein nsP1 on alphavirus infection

The membrane-associated alphavirus RNA replication complex contains four virus-encoded subunits, the nonstructural proteins nsP1 to nsP4. Semliki Forest virus (SFV) nsP1 is hydrophobically modified by palmitoylation of cysteines 418 to 420. Here we show that Sindbis virus nsP1 is also palmitoylated on the same site (cysteine 420). When mutations preventing nsP1 palmitoylation were introduced into the genomes of these two alphaviruses, the mutant viruses remained viable and replicated to high titers, although their growth was slightly delayed. The subcellular distribution of palmitoylation-defective nsP1 was altered in the mutant: it no longer localized to filopodial extensions, and a fraction of it was soluble. The ultrastructure of the alphavirus replication sites appeared normal, and the localization of the other nonstructural proteins was unaltered in the mutants. In both wild-type- and mutant-virus-infected cells, SFV nsP3 and nsP4 could be extracted from membranes only by alkaline solutions whereas the nsP2-membrane association was looser. Thus, the membrane binding properties of the alphavirus RNA replication complex were not determined by the palmitoylation of nsP1. The nsP1 palmitoylation-defective alphaviruses produced normal plaques in several cell types, but failed to give rise to plaques in HeLa cells, although they induced normal apoptosis of these cells. The SFV mutant was apathogenic in mice: it caused blood viremia, but no infectious virus was detected in the brain.     

Occurrence of Pneumocystis carinii in Canine Distemper

Pneumocystis carinii is a eukaryotic opportunistic pathogen causing pneumonia (PCP) in immunosuppressed patients. It is best known in human medicine as a pathogen of AIDS pa-tients and in immunosuppressed transplant and cancer patients (Waltzer 1993).     

Adipocytes as a Link Between Gut Microbiota-Derived Flagellin and Hepatocyte Fat Accumulation

While the role of both elevated levels of circulating bacterial cell wall components and adipose tissue in hepatic fat accumulation has been recognized, it has not been considered that the bacterial components-recognizing adipose tissue receptors contribute to the hepatic fat content. In this study we found that the expression of adipose tissue bacterial flagellin (FLG)-recognizing Toll-like receptor (TLR) 5 associated with liver fat content (r = 0.699, p = 0.003) and insulin sensitivity (r = -0.529, p = 0.016) in humans (n = 23). No such associations were found for lipopolysaccharides (LPS)-recognizing TLR4. To study the underlying molecular mechanisms of these associations, human HepG2 hepatoma cells were exposed in vitro to the conditioned culture media derived from FLG or LPS-challenged human adipocytes. The adipocyte-mediated effects were also compared to the effects of direct HepG2 exposure to FLG and LPS. We found that the media derived from FLG-treated adipocytes stimulated fat accumulation in HepG2 cells, whereas either media derived from LPS-treated adipocytes or direct FLG or LPS exposure did not. This is likely due to that FLG-treatment of adipocytes increased lipolysis and secretion of glycerol, which is known to serve a substrate for triglyceride synthesis in hepatocytes. Similarly, only FLG-media significantly decreased insulin signaling-related Akt phosphorylation, IRS1 expression and mitochondrial respiratory chain ATP5A. In conclusion, our results suggest that the FLG-induced TLR5 activation in adipocytes increases glycerol secretion from adipocytes and decreases insulin signaling and mitochondrial functions, and increases fat accumulation in hepatocytes. These mechanisms could, at least partly, explain the adipose tissue TLR5 expression associated with liver fat content in humans.