Syndecan-1 expression is regulated in an isoform-specific manner by the farnesoid-X receptor

Syndecan-1 (SDC1), a transmembrane heparan sulfate proteoglycan that participates in the binding and internalization of extracellular ligands, was identified in a screen designed to isolate genes that are regulated by the farnesoid X-receptor (FXR, NR1H4). Treatment of human hepatocytes with either naturally occurring (chenodeoxycholic acid) or synthetic (GW4064) FXR ligands resulted in both induction of SDC1 mRNA and enhanced binding, internalization, and degradation of low density lipoprotein. Transient transfection assays, using wild-type and mutant SDC1 promoter-luciferase genes, led to the identification of a nuclear hormone receptor-binding hexad arranged as a direct repeat separated by one nucleotide (DR-1) in the proximal promoter that was necessary and sufficient for activation by FXR. The wild-type, but not a mutated DR-1 element, conferred FXR responsiveness to a heterologous thymidine kinase promoter-reporter gene. Four murine FXR isoforms have been identified recently that differ either at their amino terminus and/or by the presence or absence of four amino acids in the hinge region. Interestingly, the activities of the human SDC1 promoter-reporter constructs were highly induced by the two FXR isoforms that do not contain the four-amino acid insert and were unresponsive to the isoforms containing the four amino acids. Thus, current studies demonstrate that hepatic SDC1 is induced in an FXR isoform-specific manner. Increased expression of SDC1 may account in part for the hypotriglyceridemic effect that can result from the administration of chenodeoxycholic acid to humans.     

Feedback regulation of the Bradyrhizobium japonicum nodulation genes

Lipochitin Nod signals are produced by rhizobia and are required for the establishment of a nitrogen-fixing symbiosis with a legume host. The nodulation genes encode products required for the synthesis of this signal and are induced in response to plant-produced flavonoid compounds. The addition of chitin and lipo-chitin oligomers to Bradyrhizobium japonicum cultures resulted in a significant reduction in the expression of a nod–lacZ fusion. Intracellular expression of NodC, encoding a chitin synthase, also reduced nod gene expression. In contrast, expression of the ChiB chitinase increased nod gene expression. The chain length of the oligosaccharide was important in feedback regulation, with chitotetraose molecules the best modulators of nod gene expression. Feedback regulation is mediated by the induction of nolA by chitin, resulting in elevated levels of the repressor protein, NodD2.     

Differential expression of cell-wall-related genes during the formation of tracheary elements in the Zinnia mesophyll cell system

Plants, animals and some fungi undergo processes of cell specialization such that specific groups of cells are adapted to carry out particular functions. One of the more remarkable examples of cellular development in higher plants is the formation of water-conducting cells that are capable of supporting a column of water from the roots to tens of metres in the air for some trees. The Zinnia mesophyll cell system is a remarkable tool with which to study this entire developmental pathway in vitro. We have recently applied an RNA fingerprinting technology, to allow the detection of DNA fragments derived from RNA using cDNA synthesis and subsequent PCR-amplified fragment length polymorphisms (cDNA-AFLP), to systematically characterize hundreds of the genes involved in the process of tracheary element formation. Building hoops of secondary wall material is the key structural event in forming functional tracheary elements and we have identified over 50 partial sequences related to cell walls out of 600 differentially expressed cDNA fragments. The Zinnia system is an engine of gene discovery which is allowing us to identify and characterize candidate genes involved in cell wall biosynthesis and assembly.     

Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population

Mammary epithelium can functionally regenerate upon transplantation. This renewal capacity has been classically ascribed to the function of a multipotent mammary gland stem cell population, which has been hypothesized to be a primary target in the etiology of breast cancer. Several complementary approaches were employed in this study to identify and enrich mammary epithelial cells that retain stem cell characteristics. Using long-term BrdU labeling, a population of label retaining cells (LRCs) that lack expression of differentiation markers has been identified. LRCs isolated from mammary primary cultures were enriched for stem cell antigen-1 (Sca-1) and Hoechst dye-effluxing "side population" properties. Sca-1(pos) cells in the mammary gland were localized to the luminal epithelia by using Sca-1(+/GFP) mice, were progesterone receptor-negative, and did not bind peanut lectin. Finally, the Sca-1(pos) population is enriched for functional stem/progenitor cells, as demonstrated by its increased regenerative potential compared with Sca-1(neg) cells when transplanted into the cleared mammary fat pads of host mice.     

Biosynthesis of d-arabinose in Mycobacterium smegmatis: specific labeling from d-glucose

d-Arabinose is a major sugar in the cell wall polysaccharides of Mycobacterium tuberculosis and other mycobacterial species. The reactions involved in the biosynthesis and activation of d-arabinose represent excellent potential sites for drug intervention since d-arabinose is not found in mammalian cells, and the cell wall arabinomannan and/or arabinogalactan appear to be essential for cell survival. Since the pathway involved in conversion of d-glucose to d-arabinose is unknown, we incubated cells of Mycobacterium smegmatis individually with [1-(14)C]glucose, [3,4-(14)C]glucose, and [6-(14)C]glucose and compared the specific activities of the cell wall-bound arabinose. Although the specific activity of the arabinose was about 25% lower with [6-(14)C]glucose than with other labels, there did not appear to be selective loss of either carbon 1 or carbon 6, suggesting that arabinose was not formed by loss of carbon 1 of glucose via the oxidative step of the pentose phosphate pathway, or by loss of carbon 6 in the uronic acid pathway. Similar labeling patterns were observed with ribose isolated from the nucleic acid fraction. Since these results suggested an unusual pathway of pentose formation, labeling studies were also done with [1-(13)C]glucose, [2-(13)C]glucose, and [6-(13)C]glucose and the cell wall arabinose was examined by NMR analysis. This method allows one to determine the relative (13)C content in each carbon of the arabinose. The labeling patterns suggested that the most likely pathway was condensation of carbons 1 and 2 of fructose 6-phosphate produced by the transaldolase reaction with carbons 4, 5, and 6 (i.e., glyceraldehyde 3-phosphate) formed by fructose-1,6 bisphosphate aldolase. Cell-free enzyme extracts of M. smegmatis were incubated with ribose 5-phosphate, xylulose 5-phosphate, and d-arabinose 5-phosphate under a variety of experimental conditions. Although the ribose 5-phosphate and xylulose 5-phosphate were converted to other pentoses and hexoses, no arabinose 5-phosphate (or free arabinose) was detected in any of these reactions. In addition, these enzyme extracts did not convert arabinose 5-phosphate to any other pentose or hexose. In addition, incubation of [(14)C]glucose 6-phosphate and various nucleoside triphosphates (ATP, CTP, GTP, TTP, and UTP) with cytosolic or membrane fractions from the mycobacterial cells did not result in formation of a nucleotide form of arabinose, although other radioactive sugars including rhamnose and galactose were found in the nucleotide fraction. Furthermore, no radioactive arabinose was found in the nucleotide fraction isolated from M. smegmatis cells grown in [(3)H]glucose, nor was arabinose detected in a large-scale extraction of the sugar nucleotide fraction from 300 g of cells. The logical conclusion from these studies is that d-arabinose is probably produced from d-ribose by epimerization of carbon 2 of the ribose moiety of polyprenylphosphate-ribose to form polyprenylphosphate-arabinose, which is then used as the precursor for formation of arabinosyl polymers.     

The role of apolipoprotein A-I helix 10 in apolipoprotein-mediated cholesterol efflux via the ATP-binding cassette transporter ABCA1

Recent studies of Tangier disease have shown that the ATP-binding cassette transporter A1 (ABCA1)/apolipoprotein A-I (apoA-I) interaction is critical for high density lipoprotein particle formation, apoA-I integrity, and proper reverse cholesterol transport. However, the specifics of this interaction are unknown. It has been suggested that amphipathic helices of apoA-I bind to a lipid domain created by the ABCA1 transporter. Alternatively, apoA-I may bind directly to ABCA1 itself. To better understand this interaction, we created several truncation mutants of apoA-I and then followed up with more specific point mutants and helix translocation mutants to identify and characterize the locations of apoA-I required for ABCA1-mediated cholesterol efflux. We found that deletion of residues 221-243 (helix 10) abolished ABCA1-mediated cholesterol efflux from cultured RAW mouse macrophages treated with 8-bromo-cAMP. Point mutations in helix 10 that affected the helical charge distribution reduced ABCA1-mediated cholesterol efflux versus the wild type. We noted a strong positive correlation between cholesterol efflux and the lipid binding characteristics of apoA-I when mutations were made in helix 10. However, there was no such correlation for helix translocations in other areas of the protein as long as helix 10 remained intact at the C terminus. From these observations, we propose an alternative model for apolipoprotein-mediated efflux.     

Elevated chemokine responses are maintained in lungs after clearance of viral infection

We observed two patterns of chemokine expression in the lungs of mice infected with murine gammaherpesvirus 68: peaks of chemokine expression correlated with or occurred after the peak of viral gene expression. Chemokine expression remained elevated through 29 days postinfection.     

Characterization of early,chitin-induced gene expression in Arabidopsis

Three genes (i.e., a zinc finger protein, a lectin-like protein, and AtMPK3), previously shown to respond to chitin elicitation in microarray experiments, were used to examine the response of Arabidopsis spp. to chitin addition. Maximum induction for all three genes was found upon addition of crab-shell chitin at 100 mg per liter. Threefold induction was found with a chitin concentration as low as 10(-4) mg per liter. The specificity of this response was examined using purified chitin oligomers (degree of polymerization = 2 to 8). The larger chitin oligomers (hexamer to octamer), were most effective in inducing expression of the three genes assayed. Gene induction was observed after the addition of 1 nM chitin octamer. The protein kinase inhibitors staurosporine and K252a effectively suppressed chitin-induced gene expression, while the protein phosphatase inhibitors calyculin A and okadaic acid induced the accumulation of mRNA in the absence of chitin. The phosphorylation event necessary for transmission of the chitin signal was completed within the first 20 min of chitin addition. The level of chitin-induced gene expression of the lectin-like protein and AtMPK3 was not significantly changed in mutants blocked in the jasmonic acid (JA, jar1)-, ethylene (ein2)-, or salicylic acid (SA, pad4, npr1, and eds5)-dependent pathway. In contrast, expression of mRNA for the zinc finger protein was reduced in the mutants affected in the JA- or SA-dependent pathway.     

Bacterial expression and characterization of mature apolipoprotein A-I

Plasma levels of apolipoprotein A-I (apoA-I) are correlated with reduced incidence of heart disease due to the critical role of this protein in reverse cholesterol transport. Because of its diversity of function and poorly understood structure, much research has sought to understand how the structure of apoA-I facilitates its function. A popular approach has been the use of site-directed mutagenesis followed by structural and functional studies. There are a wide variety of expression systems available to produce these mutant proteins including eukaryotic cell lines and prokaryotic cells such as Escherichia coli. Expression in a bacterial system is generally favorable because it can produce large amounts of pure protein quickly and economically through the use of affinity tags on the expressed protein. Unfortunately, many of these systems are not ideal for the production of apolipoproteins because, in many cases, the proteolytic digestion required to remove the affinity tag also cleaves the target protein. Here we describe a method that produces large amounts of recombinant protein that is easily purified using a histidine (His) affinity tag that is cleaved with IgA protease from Neisseria gonorrhoeae. This enzyme does not cleave the wild type apoA-I sequence, leaving intact, mature apoA-I (containing a Thr-Pro- on the N-terminus). We show that this recombinant protein is similar to wild type protein in structure and function using circular dichroism analysis, lipid clearance assays, recombinant particle formation and cholesterol efflux assays. This system is particularly useful for the bacterial production of apolipoproteins because of the extreme specificity of IgA protease for its target cleavage site.     

A modified nick translation method used with FISH that produces reliable results with archival tissue sections

Nick translation is used to label DNA and RNA to produce probes for in situ hybridization and Northern and Southern blotting. Fluorescence in situ hybridization (FISH) is a widely applied technique used to determine chromosomal and genetic anomalies in many biological samples. Initially the technique was applied to metaphase preparations, but the usefulness of detecting genetic anomalies in solid tumors in situ has resulted in the development of modified protocols. Formalin fixed paraffin processed tissue sections present novel challenges when applying FISH; the probes must be small (between 200 and 600 base pairs) and pretreatment is necessary before the probes can be applied to tissue sections, to promote probe access to target DNA. Here we report on a modification of a nick translation method to produce a probe that can reliably be used with FISH in paraffin processed tissue sections.