Function of 90-kDa heat shock protein in cellular differentiation of human embryonal carcinoma cells

Summary Heat shock proteins (HSPs) have been recognized as molecules that maintain cellular homeostasis during changes in the environment. Here we report that HSP90 functions not only in stress responses but also in certain aspects of cellular differentiation. We found that HSP90 slowed remarkably high expression in undifferentiated human embryonal carcinoma (EC) cells, which were subsequently dramatically down-regulated during in vitro cellular differentiation, following retinoic acid (RA) treatment, at the protein level. Surprisingly, heat shock treatment also triggered the down-regulation of HSP90 within 48 h at the protein level. Furthermore, the heat treatment induced cellular differentiation into neural cells. This down-regulation of HSP90 by heat treatment was shifted to an up-regulation attern after cellular differentiation in response to RA treatment. In order to clarify the functions of HSP90 in cellular differentiation, we conducted various experiments, including overexpression of HSP90 via gene transfer. We showed that the RA-induced differentiation of EC cells into a neural cell lineage was inhibited by overexpression of the HSP90α or-β isoform via the gene transfer method. On the other hand, the overexpression of HSP90β alone impaired cellular differentiation into trophoectoderm. These results show that down-regulation of HSP90 is a physiological critical event in the differentiation of human EC cells and that specific HSP90 isoforms may be involved in differentiation into specific cell lineages.     

Dietary troglitazone decreases oxidative stress in early stage type II diabetic rats

Oxidative stress is involved in the initiation and development of atherosclerosis in diabetes. We tested the hypothesis that oxidative stress is already increased in early stage type II diabetes, and that troglitazone may prevent the increase. Three groups of 20 week old rats were studied: untreated Otsuka Long-Evans Tokushima Fatty (OLETF) rats, as an animal model of type II diabetes, OLETF rats treated with troglitazone, and control Long-Evans Tokushima Otsuka (LETO) rats. Plasma lipid hydroperoxides (LOOH) concentration, as an indication of lipid peroxidation, and superoxide dismutase (SOD) activity in the thoracic aorta were measured. Plasma LOOH concentration was significantly higher in non-treated OLETF rats compared to LETO rats and treatment with troglitazone completely prevented this increase. SOD activity was significantly decreased in non-treated OLETF rats compared to LETO rats and troglitazone attenuated the diminution of it. These observations demonstrate oxidative stress is already increased in the early stage of type II diabetes and we confirmed troglitazone has the effect of an antioxidant in vivo.     

Molecular Mechanism of Peptide-Specific Pheromone Signaling in Enterococcus faecalis: Functions of Pheromone Receptor TraA and Pheromone-Binding Protein TraC Encoded by Plasmid pPD1

Conjugative transfer of the Enterococcus faecalis plasmid pPD1 is activated by cPD1, one of several peptide sex pheromones secreted by plasmid-free recipient cells, and is blocked by a donor-produced peptide inhibitor, iPD1. Using a tritiated pheromone, [³H]cPD1, we investigated how pPD1-harboring donor cells receive these peptide signals. Donor cells rapidly incorporated [³H]cPD1. The cell extract but not the membrane fraction of the donor strain exhibited significant [³H]cPD1-binding activity. On the basis of these data and those of tracer studies, it was demonstrated that cPD1 was internalized, where it bound to a high-molecular-weight compound. The cell extract of a strain carrying the traA-bearing multicopy plasmid (pDLHH21) also exhibited high [³H]cPD1-binding activity. A recombinant TraA exhibited a dissociation constant of 0.49 ± 0.08 nM against [³H]cPD1. iPD1 competitively inhibited [³H]cPD1 binding to TraA, whereas pheromones and inhibitors relating to other plasmid systems did not. These results show that TraA is a specific intracellular receptor for cPD1 and that iPD1 acts as an antagonist for TraA. A strain carrying the traC-bearing multicopy plasmid (pDLES23) exhibited significant [³H]cPD1-binding activity. A strain carrying traC-disrupted pPD1 (pAM351CM) exhibited lower [³H]cPD1-binding activity as well as lower sensitivity to cPD1 than a wild-type donor strain. Some of the other pheromones and inhibitors inhibited [³H]cPD1 binding to the traC transformant like cPD1 and iPD1 did. These results show that TraC, as an extracellular less-specific pheromone-binding protein, supports donor cells to receive cPD1.     

Stimulation of Macrophages with the β-Glucan Produced by Aureobasidium pullulans Promotes the Secretion of Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL)

A β-glucan produced by Aureobasidium pullulans (AP-PG) is consisting of a β-(1,3)-linked main chain with β-(1,6)-linked glucose side residues. Various β-glucans consisting of β-(1,3)-linked main chain including AP-PG are believed to exhibit anti-tumor activities, and actually, anti-tumor activities of AP-PG in mice have been demonstrated. In this study, we demonstrate that stimulation with AP-PG induces TRAIL expression in mouse and human macrophage-like cell lines. TRAIL is known to be a cytokine which specifically induces apoptosis in transformed cells, but not in untransformed cells. The expression of TRAIL mRNA after stimulation with AP-PG was increased in RAW264.7 cells, Mono Mac 6 cells, and macrophage-differentiated THP-1 cells. The mRNA expression of TNF-α and FasL is only weakly increased after stimulation with AP-PG. The induction activity of TRAIL by curdlan, a bacterial β-glucan, was very similar to that by AP-PG in RAW264.7 cells, but weaker in macrophage-differentiated THP-1 cells. Activation of caspases was found in HeLa cells after treatment with the supernatant of cultured medium from AP-PG-stimulated Mono Mac 6 cells, and was inhibited by the anti-TRAIL neutralizing antibody. These findings suggest that the stimulation with AP-PG effectively induces TRAIL in macrophages, and that it may be related to apoptosis induction of tumor cells.     

Angiographic Lesion Complexity Score and In-Hospital Outcomes after Percutaneous Coronary Intervention

Objective

We devised a percutaneous coronary intervention (PCI) scoring system based on angiographic lesion complexity and assessed its association with in-hospital complications.

Background

Although PCI is finding increasing application in patients with coronary artery disease, lesion complexity can lead to in-hospital complications.

Methods

Data from 3692 PCI patients were scored based on lesion complexity, defined by bifurcation, chronic total occlusion, type C, and left main lesion, along with acute thrombus in the presence of ST-segment elevation myocardial infarction (1 point assigned for each variable).

Results

The patients’ mean age was 67.5 +/- 10.8 years; 79.8% were male. About half of the patients (50.3%) presented with an acute coronary syndrome, and 2218 (60.1%) underwent PCI for at least one complex lesion. The patients in the higher-risk score groups were older (p < 0.001) and had present or previous heart failure (p = 0.02 and p = 0.01, respectively). Higher-risk score groups had significantly higher in-hospital event rates for death, heart failure, and cardiogenic shock (from 0 to 4 risk score; 1.7%, 4.5%, 6.3%, 7.1%, 40%, p < 0.001); bleeding with a hemoglobin decrease of >3.0 g/dL (3.1%, 11.0%, 13.1%, 10.3%, 28.6%, p < 0.001); and postoperative myocardial infarction (1.5%, 3.1%, 3.8%, 3.8%, 10%, p = 0.004), respectively. The association with adverse outcomes persisted after adjustment for known clinical predictors (odds ratio 1.72, p < 0.001).

Conclusion

The complexity score was cumulatively associated with in-hospital mortality and complication rate and could be used for event prediction in PCI patients.     

Prevalence and Trends of Staphylococcus aureus Bacteraemia in Hospitalized Patients in South Africa, 2010 to 2012: Laboratory-Based Surveillance Mapping of Antimicrobial Resistance and Molecular Epidemiology

Introduction

We aimed to obtain an in-depth understanding on recent antimicrobial resistance trends and molecular epidemiology trends of S. aureus bacteraemia (SAB).

Methods

Thirteen academic centres in South Africa were included from June 2010 until July 2012. S. aureus susceptibility testing was performed on the MicroScan Walkaway. Real-time PCR using the LightCycler 480 II was done for mecA and nuc. SCCmec and spa-typing were finalized with conventional PCR. We selected one isolate per common spa type per province for multilocus sequence typing (MLST).

Results

S. aureus from 2709 patients were included, and 1231 (46%) were resistant to methicillin, with a significant decline over the three-year period (p-value = 0.003). Geographical distribution of MRSA was significantly higher in Gauteng compared to the other provinces (P<0.001). Children <5 years were significantly associated with MRSA with higher rates compared to all other age groups (P = 0.01). The most prevalent SCCmec type was SCCmec type III (531 [41%]) followed by type IV (402 [31%]). Spa-typing discovered 47 different spa-types. The five (87%) most common spa-types were t037, t1257, t045, t064 and t012. Based on MLST, the commonest was ST612 clonal complex (CC8) (n = 7) followed by ST5 (CC5) (n = 4), ST36 (CC30) (n = 4) and ST239 (CC8) (n = 3).

Conclusions

MRSA rate is high in South Africa. Majority of the isolates were classified as SCCmec type III (41%) and type IV (31%), which are typically associated with hospital and community- acquired infections, respectively. Overall, this study reveals the presence of a variety of hospital-acquired MRSA clones in South Africa dominance of few clones, spa 037 and 1257. Monitoring trends in resistance and molecular typing is recommended to detect changing epidemiological trends in AMR patterns of SAB.     

Quantitative Mass Density Image Reconstructed from the Complex X-Ray Refractive Index

We demonstrate a new analytical X-ray computed tomography technique for visualizing and quantifying the mass density of materials comprised of low atomic number elements with unknown atomic ratios. The mass density was obtained from the experimentally observed ratio of the imaginary and real parts of the complex X-ray refractive index. An empirical linear relationship between the X-ray mass attenuation coefficient of the materials and X-ray energy was found for X-ray energies between 8 keV and 30 keV. The mass density image of two polymer fibers was quantified using the proposed technique using a scanning-type X-ray microbeam computed tomography system equipped with a wedge absorber. The reconstructed mass density agrees well with the calculated one.     

Directed evolution of squalene synthase for dehydrosqualene biosynthesis

Squalene synthase (SQS) catalyzes the first step of sterol/hopanoid biosynthesis in various organisms. It has been long recognized that SQSs share a common ancestor with carotenoid synthases, but it is not known how these enzymes selectively produce their own product. In this study, SQSs from yeast, human, and bacteria were independently subjected to directed evolution for the production of the C₃₀ carotenoid backbone, dehydrosqualene. This was accomplished via high-throughput screening with Pantoea ananatis phytoene desaturase, which can selectively convert dehydrosqualene into yellow carotenoid pigments. Genetic analysis of the resultant mutants revealed various mutations that could effectively convert SQS into a “dehydrosqualene synthase.” All of these mutations are clustered around the residues that have been proposed to be important for NADPH binding.     

Cryptic diversity of free-living parabasalids, Pseudotrichomonas keilini and Lacusteria cypriaca n. g., n. sp., as inferred from small subunit rDNA sequences

Ultrastructural and molecular phylogenetic evidence indicate that the Parabasalia consists of seven main subgroups: the Trichomonadida, Honigbergiellida, Hypotrichomonadida, Tritrichomonadida, Cristamonadida, Spirotrichonymphida, and Trichonymphida. Only five species of free-living parabasalids are known: Monotrichomonas carabina, Ditrichomonas honigbergii, Honigbergiella sp., Tetratrichomonas undula, and Pseudotrichomonas keilini. Phylogenetic analyses show that free-living species do not form a clade and instead branch in several different positions within the context of their parasitic relatives. Because the diversity of free-living parabasalids is poorly understood, the systematics of these lineages is in a significant state of disarray. In order to better understand the phylogenetic distribution of free-living parabasalids, we sequenced the small subunit rDNA from three different strains reminiscent of P. keilini; the strains were isolated from different geographical locations: (1) mangrove sediments in Japan and (2) sediments in Cyprus. These data demonstrated that the free-living parabasalids P. keilini and Lacusteria cypriaca n. g., n. sp., form a paraphyletic assemblage near the origin of a clade consisting mostly of parasitic trichomonadids (e.g. Trichomonas vaginalis). This paraphyletic distribution of similar morphotypes indicates that free-living trichomonadids represent a compelling example of morphostasis that provides insight into the suite of features present in the most recent free-living ancestor of their parasitic relatives.