Mahtab Nourbakhsh

Differential Effects of Multiplicity of Infection on Helicobacter pylori-Induced Signaling Pathways and Interleukin-8 Gene Transcription

Interleukin-8 (IL-8) plays a central role in the pathogenesis of Helicobacter pylori infection. We used four different H. pylori strains isolated from patients with gastritis or duodenal ulcer disease to examine their differential effects on signaling pathways and IL-8 gene response in gastric epithelial cells. IL-8 mRNA level is elevated in response to high (100) multiplicity of infection (MOI) independent of cagA, vacA, and dupA gene characteristics. By lower MOIs (1 or 10), only cagA+ strains significantly induce IL-8 gene expression. This is based on differential regulation of IL-8 promoter activity. Analysis of intracellular signaling pathways indicates that H. pylori clinical isolates induce IL-8 gene transcription through NF-κB p65, but by a MOI-dependent differential activation of MAPK pathways. Thus, the major virulence factors of H. pylori CagA, VacA, and DupA might play a minor role in the level of IL-8 gene response to a high bacterial load.

Chondrogenic Differentiation of Human Adipose-Derived Stem Cells: A New Path in Articular Cartilage Defect Management?

According to data published by the Centers for Disease Control and Prevention, over 6 million people undergo a variety of medical procedures for the repair of articular cartilage defects in the U.S. each year. Trauma, tumor, and age-related degeneration can cause major defects in articular cartilage, which has a poor intrinsic capacity for healing. Therefore, there is substantial interest in the development of novel cartilage tissue engineering strategies to restore articular cartilage defects to a normal or prediseased state. Special attention has been paid to the expansion of chondrocytes, which produce and maintain the cartilaginous matrix in healthy cartilage. This review summarizes the current efforts to generate chondrocytes from adipose-derived stem cells (ASCs) and provides an outlook on promising future strategies.

Macrophage Migration Inhibitory Factor in Acute Adipose Tissue Inflammation

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine and has been implicated in inflammatory diseases. However, little is known about the regulation of MIF in adipose tissue and its impact on wound healing. The aim of this study was to investigate MIF expression in inflamed adipose and determine its role in inflammatory cell recruitment and wound healing. Adipose tissue was harvested from subcutaneous adipose tissue layers of 24 healthy subjects and from adipose tissue adjacent to acutely inflamed wounds of 21 patients undergoing wound debridement. MIF protein and mRNA expression were measured by ELISA and RT-PCR. Cell-specific MIF expression was visualized by immunohistochemistry. The functional role of MIF in cell recruitment was investigated by a chemotaxis assay and by flow cytometry of labeled macrophages that were injected into Mif –/–and wildtype mice. Wound healing was evaluated by an in vitro scratch assay on human fibroblast monolayers. MIF protein levels of native adipose tissue and supernatants from acutely inflamed wounds were significantly elevated when compared to healthy controls. MIF mRNA expression was increased in acutely inflamed adipose tissue indicating the activation of MIF gene transcription in response to adipose tissue inflammation. MIF is expressed in mature adipocytes and in infiltrated macrophages. Peripheral blood mononuclear cell migration was significantly increased towards supernatants derived from inflamed adipose tissue. This effect was partially abrogated by MIF-neutralizing antibodies. Moreover, when compared to wildtype mice, Mif –/–mice showed reduced infiltration of labeled macrophages into LPS-stimulated epididymal fat pads in vivo. Finally, MIF antibodies partially neutralized the detrimental effect of MIF on fibroblast wound healing. Our results indicate that increased MIF expression and rapid activation of the MIF gene in fat tissue adjacent to acute wound healing disorders may play a role in cell recruitment to the site of inflammation and wound healing.

Mapping of NRF binding motifs of NF-kappaB p65 subunit.

NF-kappaB repressing factor (NRF) is a nuclear transcription factor that binds to a specific DNA sequence in NF-kappaB target promoters. Previous reports suggested that NRF interferes with the transcriptional activity of NF-kappaB binding sites through a direct interaction with NF-kappaB subunits. The aim of this study was to map specific NRF binding domains in the NF-kappaB proteins, p65 and p50. Our data demonstrate that NRF is able to interact with the p65 subunit and inhibit its transcription enhancing activity in reporter gene experiments. Using tandem affinity purifications (TAP), we show that NRF protein significantly binds to the endogenous p65, subunit but not to the p50 subunit. The selective binding activity of the NRF protein is consistently mediated by the N-terminal domain of NRF (Amino acids 1-380). Moreover, the Rel homology domain (RHD) of p65 is sufficient for binding to the N-terminal domain of NRF. Using detailed peptide mapping studies, we finally identify three peptide motifs in p65 RHD showing distinctive binding specificities for the NRF protein. According to the predicted structure of p65, all three peptide motifs align within an exposed region of p65 and might hint at promising targets for inhibitors.

Identification of Actively Translated mRNAs

Ribosomes are large protein-RNA complexes involved in translation of mRNA nucleotide sequences into proteins. Multiple ribosomes, polyribosomes (polysomes), bind to a single mRNA in order to initiate translation and protein synthesis. In order to distinguish actively translated RNAs, total polysomes can be isolated from cell lysates and purified by centrifugation through sucrose density gradients. The polysome fraction represents all actively translated cellular RNAs that can be specifically detected using common RNA detection assays, e.g., RT-PCR. The quantity of the target RNA in polysomes fraction indicates its translation state. This chapter provides a protocol for the isolation and fractionation of polysomes from mammalian cell lines.

The Effect of Lipoaspirates on Human Keratinocytes

BACKGROUND One increasingly important trend in plastic, reconstructive, and aesthetic surgery is the use of fat grafts to improve cutaneous wound healing. In clinical practice, lipoaspirates (adipose tissue harvested by liposuction) are re-injected in a procedure called lipofilling. Previous studies, however, mainly evaluated the regenerative effect of isolated adipocytes, adipose-derived stem cells, and excised en bloc adipose tissue on keratinocytes, whereas no study to date has examined the effect of lipoaspirates. OBJECTIVES The authors aimed to investigate differences in the regenerative property of en bloc adipose tissue and lipoaspirates on keratinocytes. METHODS Human keratinocytes, lipoaspirates, and en bloc adipose tissue from 36 healthy donors were isolated. In vitro proliferation, differentiation, migration, stratification, and wound healing of keratinocyte monolayers were measured. Furthermore, secreted levels of VEGF, bFGF, IGF-1, MMP-9, and MIF were detected by ELISA. RESULTS Migration, proliferation, and wound healing of keratinocytes were increased by lipoaspirates. Interestingly, the effect of lipoaspirates on keratinocyte proliferation was significantly higher than by en bloc adipose tissue after 5 days. The differentiation of keratinocytes was equally attenuated by lipoaspirates and en bloc adipose tissue. Stratification of keratinocyte layers was enhanced by lipoaspirates and en bloc fat when compared to controls. Lipoaspirates secrete higher levels of bFGF, whereas higher levels of VEGF and IGF-1 are released by en bloc adipose tissue. CONCLUSION We show that lipoaspirates and en bloc adipose tissue have a regenerative effect on keratinocytes. One reason for the higher effect of lipoaspirates on keratinocyte proliferation may be the secretion of different cytokines.

Single Nucleotide Mapping of RNA 5′ and 3′ Ends

Nuclease protection assay is a sensitive method for detection, quantitation, and mapping of a specific RNA in an extremely heterogeneous mixture of RNAs, such as total cellular RNA. The assay is based on a small volume solution hybridization of a single-stranded synthetic antisense and labeled RNA probe to a RNA sample. Thus, it is much more efficient than the common immobilized hybridization on a membrane, such as in northern-blot analysis. After solution hybridization, different nucleases are used to remove any remaining single-stranded nucleotides within the probe and sample RNA by digestion. Then, the remaining probe-target hybrids are purified and separated on a denaturing polyacrylamide gel. Using a radioactive labeled probe, the protected probe can be visualized by direct autoradiography and the copy number can be calculated based on the specific radioactivity of the RNA probe and the length of protected fragment. Because of its high sensitivity and resolution, nuclease protection assay is the most effective procedure for mapping internal and external boundaries in mRNA compared to other RNA detection methods such as RT-PCR.

Full-Length Characterization of Transcribed Genomic Regions

In the last years, an enormous progress has been made in the identification of genomic sequences. Given that genomic sequences can have various functions (e.g., structural organization, gene regulation, transcriptional start, and protein coding), molecular characterization is essential for progressing from the initial identification of genomic sequences to the delineation of a specific biological mechanism. Mapping of transcribed sequences is the initial step in functional characterization of genomic sequences. Northern blot analysis allows for a direct and detailed characterization of transcribed sequences, like size and splicing variants, and provides a relative comparison of transcript abundance between different cellular conditions. This method includes separation of total cellular RNA by size via gel electrophoresis, RNA transfer to a membrane, and RNA hybridization with a complementary labeled genomic probe.

Rapid mapping of RNA 3' and 5' ends.

In recent years, an enormous progress has been made in applied genomics leading to identification and isolation of novel cDNAs. However, most attempts result in the acquisition of transcribed sequences that represent only a part of the mRNAs complete sequence. Rapid Amplification of cDNA Ends (RACE) is a technique used in molecular biology to obtain the full length sequence of an RNA transcript found within a cell. Since the first report of this technique, many significant improvements have been made on the basic approach. This chapter describes the most recent update of the relatively simple and versatile classic RACE protocol.