The influence of lead and arsenite on the inhibition of human breast cancer MCF-7 cell proliferation by American ginseng root (Panax quinquefolius L.)

American ginseng root (Panax quinquefolius) has a number of purported therapeutic effects, including inhibition of cancer cell proliferation. The ability of environmentally relevant heavy metals to alter ginseng effects on cancer cell growth was the subject of this study. A water extract of American ginseng root was applied alone or in combination with physiologically relevant doses of either lead (Pb) or arsenite to MCF-7 breast cancer cells in vitro and effects on cell proliferation were determined. Ginseng alone produced a significant dose-dependent inhibition of MCF-7 cell proliferation starting at 0.5 mg ml(-1). Treatment of MCF-7 cells with 2.5 microM arsenite significantly decreased MCF-7 cell proliferation (p < 0.01). When cells were treated with arsenite (1.25 or 2.5 microM) in combination with ginseng extract (0.5 mg ml(-1)), there was an apparent synergistic inhibition of cell proliferation. Treatment of MCF-7 breast cancer cells with 50 microM Pb significantly decreased cell proliferation relative to control (p < 0.01), and concomitant ginseng and Pb treatment did not lead to a further decrease. These results suggest that contaminant heavy metals, some of which have been detected in ginseng root extracts or commercial ginseng preparations, may alter the biological activity of ginseng.     

Effect of sinomenine on gene expression of the IL-1 beta-activated human synovial sarcoma

Sinomenine is an alkaloid with pharmacological effects of anti-inflammation, anti-angiogenesis, anti-arthritis and immunosuppression. This study aimed to investigate the effect of sinomenine on gene expression of human synovial sarcoma cells (Hs701.T) activated by IL-1 beta. The proliferative effect of sinomenine was examined in the presence or absence of IL-1 beta by the [3H]-thymidine incorporation and MTT assay, respectively. Using DNA microarray technology and RT-PCR, the activating action of IL-1 beta and modulatory effect of sinomenine on Hs701.T were simultaneously determined. Results showed that IL-1 beta could stimulate the proliferation and gene expression of Hs701.T cells. Sinomenine could significantly inhibit proliferation of IL-1 beta-activated Hs701.T cells and suppress expression of 17 genes including IL-6, PlGF, Daxx, and HSP27. These genes were found to be important in tumor progression through the mediation of inflammation, cell adhesion, proliferation, apoptosis and angiogenesis. In conclusion, our study provides supplementary information for the further studies on the pharmacological effects of sinomenine acting on synovial sarcoma.     

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).Download video file.^{(51M, mov)}     

The Genome Sequence of Trypanosoma brucei gambiense,Causative Agent of Chronic Human African Trypanosomiasis

Background

Trypanosoma brucei gambiense is the causative agent of chronic Human African Trypanosomiasis or sleeping sickness, a disease endemic across often poor and rural areas of Western and Central Africa. We have previously published the genome sequence of a T. b. brucei isolate, and have now employed a comparative genomics approach to understand the scale of genomic variation between T. b. gambiense and the reference genome. We sought to identify features that were uniquely associated with T. b. gambiense and its ability to infect humans.

Methods and Findings

An improved high-quality draft genome sequence for the group 1 T. b. gambiense DAL 972 isolate was produced using a whole-genome shotgun strategy. Comparison with T. b. brucei showed that sequence identity averages 99.2% in coding regions, and gene order is largely collinear. However, variation associated with segmental duplications and tandem gene arrays suggests some reduction of functional repertoire in T. b. gambiense DAL 972. A comparison of the variant surface glycoproteins (VSG) in T. b. brucei with all T. b. gambiense sequence reads showed that the essential structural repertoire of VSG domains is conserved across T. brucei.

Conclusions

This study provides the first estimate of intraspecific genomic variation within T. brucei, and so has important consequences for future population genomics studies. We have shown that the T. b. gambiense genome corresponds closely with the reference, which should therefore be an effective scaffold for any T. brucei genome sequence data. As VSG repertoire is also well conserved, it may be feasible to describe the total diversity of variant antigens. While we describe several as yet uncharacterized gene families with predicted cell surface roles that were expanded in number in T. b. brucei, no T. b. gambiense-specific gene was identified outside of the subtelomeres that could explain the ability to infect humans.     

Impact of defective interfering particles on virus replication and antiviral host response in cell culture-based influenza vaccine production

During the replication of influenza viruses, defective interfering particles (DIPs) can be generated. These are noninfectious deletion mutants that require coinfection with a wild-type virus but interfere with its helper virus replication. Consequently, coinfected cells mainly produce DIPs. Little is known about how such noninfectious virus particles affect the virus yield of cell culture-based influenza vaccine production. We compared infections of Madin-Darby canine kidney cells with two seed virus preparations of the influenza virus strain A/Puerto Rico/8/34 that contain different amounts of DIPs. A combination of conventional RT-PCR, RT-qPCR, and flow cytometry revealed that DI genomes indeed strongly accumulate in coinfected cells and impede the viral RNA synthesis. Additionally, cells infected at the higher DIP concentration showed a stronger antiviral response characterized by increased interferon-β expression and apoptosis induction. Furthermore, in the presence of DIPs, a significant fraction of cells did not show any productive accumulation of viral proteins at all. Together, these effects of DIPs significantly reduce the virus yield. Therefore, the accumulation of DIPs should be avoided during influenza vaccine production which can be achieved by quality controls of working seed viruses based on conventional RT-PCR. The strategy for the depletion of DIPs presented here can help to make cell culture-based vaccine production more reliable and robust.     

ATPase Subdomain IA Is a Mediator of Interdomain Allostery in Hsp70 Molecular Chaperones

The versatile functions of the heat shock protein 70 (Hsp70) family of molecular chaperones rely on allosteric interactions between their nucleotide-binding and substrate-binding domains, NBD and SBD. Understanding the mechanism of interdomain allostery is essential to rational design of Hsp70 modulators. Yet, despite significant progress in recent years, how the two Hsp70 domains regulate each other''s activity remains elusive. Covariance data from experiments and computations emerged in recent years as valuable sources of information towards gaining insights into the molecular events that mediate allostery. In the present study, conservation and covariance properties derived from both sequence and structural dynamics data are integrated with results from Perturbation Response Scanning and in vivo functional assays, so as to establish the dynamical basis of interdomain signal transduction in Hsp70s. Our study highlights the critical roles of SBD residues D481 and T417 in mediating the coupled motions of the two domains, as well as that of G506 in enabling the movements of the α-helical lid with respect to the β-sandwich. It also draws attention to the distinctive role of the NBD subdomains: Subdomain IA acts as a key mediator of signal transduction between the ATP- and substrate-binding sites, this function being achieved by a cascade of interactions predominantly involving conserved residues such as V139, D148, R167 and K155. Subdomain IIA, on the other hand, is distinguished by strong coevolutionary signals (with the SBD) exhibited by a series of residues (D211, E217, L219, T383) implicated in DnaJ recognition. The occurrence of coevolving residues at the DnaJ recognition region parallels the behavior recently observed at the nucleotide-exchange-factor recognition region of subdomain IIB. These findings suggest that Hsp70 tends to adapt to co-chaperone recognition and activity via coevolving residues, whereas interdomain allostery, critical to chaperoning, is robustly enabled by conserved interactions.