Kalpana Rengarajan

Convergence of redox-sensitive and mitogen-activated protein kinase signaling pathways in tumor necrosis factor-α-mediated monocyte chemoattractant protein-1 induction in vascular smooth muscle cells

Monocyte chemoattractant protein-1 (MCP-1) is an important component of the inflammatory response of the vessel wall and has been shown to be regulated by cytokines, such as tumor necrosis factor-alpha (TNF-alpha). However, the precise signaling pathways leading to MCP-1 induction have not been fully elucidated in vascular smooth muscle cells (VSMCs). Cytokine signal transduction involves protein kinases as well as reactive oxygen species (ROS). The relation between these 2 factors is not clear. In this study, we show that TNF-alpha induces a parallel phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38MAPK) and increases MCP-1 mRNA expression in cultured VSMCs. Inhibition of ERK1/2 but not p38MAPK caused a partial attenuation of MCP-1 induction (43+/-10% inhibition). Incubation of VSMCs with multiple antioxidants (diphenylene iodonium, liposomal superoxide dismutase, catalase, N-acetylcysteine, dimethylthiourea, and pyrrolidine dithiocarbamate) had no effect on TNF-alpha-mediated MCP-1 upregulation. However, simultaneous blockade of the ERK1/2 and ROS pathways by using PD098059 combined with diphenylene iodonium or N-acetylcysteine potently enhanced the ability of MAPK kinase inhibitors to abrogate MCP-1 mRNA expression (100+/-2% inhibition). Thus, parallel ROS-dependent and ERK1/2-dependent pathways converge to regulate TNF-alpha-induced MCP-1 gene expression in VSMCs. These data unmask a complex but organized integration of ROS and protein kinases that mediates cytokine-induced vascular inflammatory gene expression.

Environmental infection control considerations for Ebola

Environmental infection control considerations for Ebola John J. Lowe PhD *, Patricia L. Olinger BS, RBP , Shawn G. Gibbs PhD, CIH , Kalpana Rengarajan PhD, RBP , Elizabeth L. Beam RN, Kathleen C. Boulter BAN , Michelle M. Schwedhelm MSN, A. Kim Hayes RN , Christopher J. Kratochvil MD , Sharon Vanairsdale MS, APRN, Brian Frislie CEH , Jerry Lewis , Angela L. Hewlett MD, Philip W. Smith MD, Bryce Gartland MD , Bruce S. Ribner MD

Extra-hepatic expression of serum albumin mRNA in mouse retina

Purpose. In some mammals, serum albumin protein exists in the interphotoreceptor space (IPS), the space between photoreceptor cells and the retinal pigment epithelium. Serum albumin is synthesized largely in the liver, though low levels of extra-hepatic expression have been documented in several other tissues, including fetal rat kidney, pancreas, lung, and heart. The purpose of this study was to investigate whether serum albumin protein and mRNA are found in mouse retina. Methods. Using albumin rabbit antibodies and HRP goat anti-(rabbit IgG), we performed immunoassays on mouse IPS washes to detect the presence of serum albumin protein. Protein extracts from IPS washes were subjected to Affigel Blue chromatography. This resin has an affinity for serum albumin. Reverse transcription-polymerase chain reaction (RT-PCR) of retina total RNA was performed to search for albumin mRNA. Also, real-time reverse transcription polymerase chain reaction (RT-RT-PCR) was employed to look at the levels of expression in different age groups. Results. A constituent of the IPS washes specifically bound and eluted from Affigel Blue column, suggesting that the washes contained serum albumin. SDS PAGE revealed that the size of the constituent was 67 kDa, the size of serum albumin. This 67 kDa band reacted with mouse serum antibody. An RT-PCR amplified fragment of serum albumin mRNA from retina displayed the expected size. The sequence of this fragment is identical to authentic serum albumin cDNA sequence. RPE and choroid were negative for serum albumin mRNA. However, rd1 - /rd1 - retina was positive, suggesting that at least some serum albumin is synthesized in the inner layers of the retina. RT-RT-PCR showed that serum albumin mRNA levels in whole retina reached a maximum at about postnatal day 15 and gradually decreased to about one-sixth of maximum at 12 months age. Conclusions. Serum albumin protein and mRNA were found in mouse IPS and retina, suggesting that the protein is synthesized in the retina. The previously demonstrated ability of serum albumin to bind fatty acids and retinoids and its presence in the mouse IPS suggest a role for serum albumin in transporting retinoids in the retina or IPS, especially at young ages when concentrations appear greatest.

Diffusion coefficients of retinoids

Purpose. Diffusion coefficients of various retinoids have not been measured previously. It is important to know the diffusion coefficients of the retinoids because this property might be rate-limiting in dark adaptation. Also, retinoid diffusion is important to explore given that rhodopsin regeneration is not impaired in IRBP knockout mice. Methods. Measurements of lateral diffusion coefficients (D) of 9-cis-retinal, all-trans-retinal, and all-trans-retinol were made by Fourier transform pulsed-gradient spin-echo NMR measurements (FT-PGSE NMR) in several solvents. Also, 3 H-all-trans-retinoic acid was used to measure diffusion from an aqueous agarose matrix and absorption into a toluene based scintillation fluid in a biphase assay. Results. In a 1:1 mixture of CD 3 OD:D 2 O the D’s of the retinoids were, 2.4 to 3.0 × 10 -6 cm 2 /s. In the biphase assay, 3 H-all trans-retinoic acid exhibited a diffusion coefficient of 2.3 × 10 -6 cm 2 /s. Conclusions. The lower than expected D for retinoids and our calculations suggest that mechanisms in addition to pure aqueous diffusion may be needed to account for normal rhodopsin regeneration rates in the mammalian retina.

Implementing Poliovirus Containment Requirements in a Global Academic Research Collaboration: A Case Study

The Global Polio Eradication Initiative has established the goal of eliminating wild poliovirus circulation to form a polio-free world. To help achieve this goal, the WHO developed GAPIII, a global guideline implemented at the national level to minimize the risk of facility-associated poliovirus infections. The guidelines require research facilities to either destroy or contain wild type, vaccine-derived poliovirus; oral poliovirus vaccine viruses; and potentially infectious poliovirus materials for an area where polio was found or oral polio vaccine was in use. These requirements are applicable to facilities that conduct research unrelated to poliovirus but intend to use samples that may contain potentially infectious materials, such as infant stool specimens. In this case study, we demonstrate our approach to comply with the GAPIII guidelines, identify opportunities to improve the guidelines for international public health research, and provide recommendations for biosafety officers to help research facilities comply with changing global guidelines such as poliovirus containment.

Is Your Institution Disposing of Culture Media Containing Antibiotics

The disposal of culture media containing antibiotics is a daily practice in research laboratories at any level (ie, academia, industry, federal/state). Generally, good microbiological practices recommend that the biohazard be treated before disposal of the culture media. This brief summary aims to provide a different angle in the waste management of culture media containing antibiotics from the perspective of handling the biohazard (microbiological agent) and the chemical hazard (antibiotic) before disposal. Available practices and suggested discussion points are presented to initiate conversation among biosafety professionals and within institutions.

Biosafety Lessons Learned When Zika Virus Met Academic Research

Early 2016, investigators at Emory University challenged the Biosafety Office with the task of reviewing and guiding how research with Zika virus, an emerging infectious disease, could be conducted at the institution. The challenge was not new, as the Biosafety Office has experience with reviewing other flaviviruses, such as West Nile, dengue, and yellow fever. Many of the practices and procedures were already in place. However, Zika virus posed a critical new challenge—the potential for reproductive hazards not seen in other flaviviruses. The reproductive hazard changed the risk assessment. This article aims to present the rationale and strategies used by Emory University to guide and implement the recommendations set forth by the Institutional Biosafety Committee for in vivo and in vitro research.