Synthesis of phytochelatins in vetiver grass upon lead exposure in the presence of phosphorus

In a hydroponic setting, we investigated the possible role of phytochelatins (metal-binding peptides) in the lead (Pb) tolerance of vetiver grass (Vetiveria zizanioides L.). Pb was added to the nutrient medium at concentrations ranging from 0 to 1,200 mg L^?1. Furthermore, we simulated the effect of soil phosphorus (P) on potentially plant available Pb by culturing vetiver grass in P-rich nutrient media. After 7 days of exposure to Pb, we evaluated the Pb uptake by vetiver grass. Results indicate that vetiver can accumulate Pb up to 3,000 mg kg^?1 dry weight in roots with no toxicity. Formation of lead phosphate inhibited Pb uptake by vetiver, suggesting the need for an environmentally safe chelating agent in conjunction with phytoremediation to clean up soils contaminated with lead-based paint. Unambiguous characterization of phytochelatins (PC_n) was possible using high pressure liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESMS). Vetiver shows qualitative and quantitative differences in PC_n synthesis between root and shoot. In root tissue from vetiver exposed to 1,200 mg Pb L^-1, phytochelatins ranged from PC₁ to PC₃. Collision-induced dissociation of the parent ion allowed confirmation of each PC_n based on the amino acid sequence. Possible Pb-PC₁ and Pb₂-PC₁ complexes were reported in vetiver root at the highest Pb concentration. The data from these experiments show that the most probable mechanism for Pb detoxification in vetiver is by synthesizing PC_n and forming Pb–PC_n complexes.     

Surfactant protein A activation of atypical protein kinase C zeta in IkappaB-alpha-dependent anti-inflammatory immune regulation

The pulmonary collectin surfactant protein (SP)-A has a pivotal role in anti-inflammatory modulation of lung immunity. The mechanisms underlying SP-A-mediated inhibition of LPS-induced NF-kappaB activation in vivo and in vitro are only partially understood. We previously demonstrated that SP-A stabilizes IkappaB-alpha, the primary regulator of NF-kappaB, in alveolar macrophages (AM) both constitutively and in the presence of LPS. In this study, we show that in AM and PBMC from IkappaB-alpha knockout/IkappaB-beta knockin mice, SP-A fails to inhibit LPS-induced TNF-alpha production and p65 nuclear translocation, confirming a critical role for IkappaB-alpha in SP-A-mediated LPS inhibition. We identify atypical (a) protein kinase C (PKC) zeta as a pivotal upstream regulator of SP-A-mediated IkappaB-alpha/NF-kappaB pathway modulation deduced from blocking experiments and confirmed by using AM from PKCzeta-/- mice. SP-A transiently triggers aPKCThr(410/403) phosphorylation, aPKC kinase activity, and translocation in primary rat AM. Coimmunoprecipitation experiments reveal that SP-A induces aPKC/p65 binding under constitutive conditions. Together the data indicate that anti-inflammatory macrophage activation via IkappaB-alpha by SP-A critically depends on PKCzeta activity, and thus attribute a novel, stimulus-specific signaling function to PKCzeta in SP-A-modulated pulmonary immune response.     

Optimal molecular profiling of tissue and tissue components

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This article reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies, and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing, and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high quality, appropriately anatomically tagged scientific results. In optimized protocols is a source of inefficiency in current life science research. Improvement in this area will significantly increase life science quality and productivity. The article is divided into introduction, materials, protocols, and notes sections. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this article, readers are advised to read through the entire article first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.     

Phospholipids inhibit lipopolysaccharide (LPS)-induced cell activation: a role for LPS-binding protein

The inhibition of LPS-induced cell activation by specific antagonists is a long-known phenomenon; however, the underlying mechanisms are still poorly understood. It is commonly accepted that the membrane-bound receptors mCD14 and TLR4 are involved in the activation of mononuclear cells by LPS and that activation may be enhanced by soluble LPS-binding protein (LBP). Hexaacylated Escherichia coli lipid A has the highest cytokine-inducing capacity, whereas lipid A with four fatty acids (precursor IVa, synthetic compound 406) is endotoxically inactive, but expresses antagonistic activity against active LPS. Seeking to unravel basic molecular principles underlying antagonism, we investigated phospholipids with structural similarity to compound 406 with respect to their antagonistic activity. The tetraacylated diphosphatidylglycerol (cardiolipin, CL) exhibits high structural similarity to 406, and our experiments showed that CL strongly inhibited LPS-induced TNF-alpha release when added to the cells before stimulation or as a CL/LPS mixture. Also negatively charged and to a lesser degree zwitterionic diacyl phospholipids inhibited LPS-induced cytokine production. Using Abs against LBP, we could show that the activation of cells by LPS was dependent on the presence of cell-associated LBP, thus making LBP a possible target for the antagonistic action of phospholipids. In experiments investigating the LBP-mediated intercalation of LPS and phospholipids into phospholipid liposomes mimicking the macrophage membrane, we could show that preincubation of soluble LBP with phospholipids leads to a significant reduction of LPS intercalation. In summary, we show that LBP is a target for the inhibitory function of phospholipids.     

Aggregates are the biologically active units of endotoxin

For the elucidation of the very early steps of immune cell activation by endotoxins (lipopolysaccharide, LPS) leading to the production and release of proinflammatory cytokines the question concerning the biologically active unit of endotoxins has to be addressed: are monomeric endotoxin molecules able to activate cells or is the active unit represented by larger endotoxin aggregates? This question has been answered controversially in the past. Inspired by the observation that natural isolates of lipid A, the lipid moiety of LPS harboring its endotoxic principle, from Escherichia coli express a higher endotoxic activity than the same amounts of the synthetic E. coli-like hexaacylated lipid A (compound 506), we looked closer at the chemical composition of natural isolates. We found in these isolates that the largest fraction was hexaacylated, but also significant amounts of penta- and tetraacylated molecules were present that, when administered to human mononuclear cells, may antagonize the induction of cytokines by biologically active hexaacylated endotoxins. We prepared separate aggregates of either compound 506 or 406 (tetraacylated precursor IVa), mixed at different molar ratios, and mixed aggregates containing both compounds in the same ratios. Surprisingly, the latter mixtures showed higher endotoxic activity than that of the pure compound 506 up to an admixture of 20% of compound 406. Similar results were obtained when using various phospholipids instead of compound 406. These observations can only be understood by assuming that the active unit of endotoxins is the aggregate. We further confirmed this result by preparing monomeric lipid A and LPS by a dialysis procedure and found that, at the same concentrations, only the aggregates were biologically active, whereas the monomers showed no activity.     

Cell activation of human macrophages by lipoteichoic acid is strongly attenuated by lipopolysaccharide-binding protein

Lipoteichoic acid (LTA) represents immunostimulatory molecules expressed by Gram-positive bacteria. They activate the innate immune system via Toll-like receptors. We have investigated the role of serum proteins in activation of human macrophages by LTA from Staphylococcus aureus and found it to be strongly attenuated by serum. In contrast, the same cells showed a sensitive response to LTA and a significantly enhanced production of tumor necrosis factor alpha under serum-free conditions. We show that LTA interacts with the serum protein lipopolysaccharide-binding protein (LBP) and inhibits the integration of LBP into phospholipid membranes, indicating the formation of complexes of LTA and soluble LBP. The addition of recombinant human LBP to serum-free medium inhibited the production of tumor necrosis factor alpha and interleukins 6 and 8 after stimulation of human macrophages with LTA in a dose-dependent manner. Using anti-LBP antibodies, this inhibitory effect could be attributed to soluble LBP, whereas LBP in its recently described transmembrane configuration did not modulate cell activation. Also, using primary alveolar macrophages from rats, we show a sensitive cytokine response to LTA under serum-free culture conditions that was strongly attenuated in the presence of serum. In summary, our data suggest that innate immune recognition of LTA is organ-specific with negative regulation by LBP in serum-containing compartments and sensitive recognition in serum-free compartments like the lung.     

Transforming growth factor-beta signaling helps specify tumor type in DMBA and hormone-induced mammary cancers

To determine the role of transforming growth factor-beta (TGF-beta) signaling in mammary development and tumor formation, we previously generated transgenic mice that expressed a dominant-negative form of the TGF-beta type II receptor (DNIIR) under the control of DNA regulatory elements from the metallothionein promoter (MT-DNIIR-28). In this report, we tested the hypothesis that loss of TGF-beta signaling in the mammary gland alters the development of chemically or hormonally induced tumors in mice. Four groups of mice were used in the study: wild-type and MT-DNIIR-28 mice on zinc with pituitary isograft, and wild-type and MT-DNIIR-28 mice on zinc with pituitary isograft treated with the carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA). Tumor-free survival over time, tumor growth rate, and tumor pathology were measured. Statistically significant differences in tumor free survival over time or tumor growth rate were not detected in wild-type versus transgenic mice treated with DMBA. In contrast, tumor-free survival was significantly altered in transgenic mice that were treated with the pituitary isograft alone with MT-DNIIR mice developing tumors more quickly. Alterations in the types of tumors that formed in wild-type versus MT-DNIIR DMBA-treated mice were detected. In wild-type mice, tumors with squamous differentiation or bicellular adenomyoepitheliomas were most common. Adenomyoepitheliomas were not detected in transgenic mice. Furthermore, there was reduced staining for alpha smooth muscle actin and keratin 14, markers for myoepithelial cells, in the glandular portion of tumors in transgenic mice. The pathology of tumors induced by pituitary isograft alone was also markedly different in wild-type and transgenic mice. All the tumors classified from wild-type mice demonstrated some form of squamous differentiation, whereas squamous differentiation was not detected in the pituitary-induced transgenic tumors. The results suggest that TGF-beta acts as a tumor suppressor for hormone-induced cancers and that TGF-beta has a role in determining tumor pathology by regulating myoepithelial or squamous differentiation, maintenance, or transformation.     

Extraction of Tissue Antigens for Functional Assays

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined ¹. This is particularly true in autoimmune diseases and cancer². Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes ^1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays ⁶. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.     

Interference of low‐molecular substances with the thioflavin‐T fluorescence assay of amyloid fibrils

Abnormal fibrillization of amyloidogenic peptides/proteins has been linked to various neurodegenerative diseases such as Alzheimer's and Parkinson's disease as well as with type‐II diabetes mellitus. The kinetics of protein fibrillization is commonly studied by using a fluorescent dye Thioflavin T (ThT) that binds to protein fibrils and exerts increased fluorescence intensity in bound state. Recently, it has been demonstrated that several low‐molecular weight compounds like Basic Blue 41, Basic Blue 12, Azure C, and Tannic acid interfere with the fluorescence of ThT bound to Alzheimers' amyloid‐β fibrils and cause false positive results during the screening of fibrillization inhibitors. In the current study, we demonstrated that the same selected substances also decrease the fluorescence signal of ThT bound to insulin fibrils already at submicromolar or micromolar concentrations. Kinetic experiments show that unlike to true inhibitors, these compounds did neither decrease the fibrillization rate nor increase the lag‐period. Absence of soluble insulin in the end of the experiment confirmed that these compounds do not disaggregate the insulin fibrils and, thus, are not fibrillization inhibitors at concentrations studied. Our results show that interference with ThT test is a general phenomenon and more attention has to be paid to interpretation of kinetic results of protein fibrillization obtained by using fluorescent dyes. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Synaptic underpinnings of altered hippocampal function in glutaminase-deficient mice during maturation

Glutaminase-deficient mice (GLS1 hets), with reduced glutamate recycling, have a focal reduction in hippocampal activity, mainly in CA1, and manifest behavioral and neurochemical phenotypes suggestive of schizophrenia resilience. To address the basis for the hippocampal hypoactivity, we examined synaptic plastic mechanisms and glutamate receptor expression. Although baseline synaptic strength was unaffected in Schaffer collateral inputs to CA1, we found that long-term potentiation was attenuated. In wild-type (WT) mice, GLS1 gene expression was highest in the hippocampus and cortex, where it was reduced by about 50% in GLS1 hets. In other brain regions with lower WT GLS1 gene expression, there were no genotypic reductions. In adult GLS1 hets, NMDA receptor NR1 subunit gene expression was reduced, but not AMPA receptor GluR1 subunit gene expression. In contrast, juvenile GLS1 hets showed no reductions in NR1 gene expression. In concert with this, adult GLS1 hets showed a deficit in hippocampal-dependent contextual fear conditioning, whereas juvenile GLS1 hets did not. These alterations in glutamatergic synaptic function may partly explain the hippocampal hypoactivity seen in the GLS1 hets. The maturity-onset reduction in NR1 gene expression and in contextual learning supports the premise that glutaminase inhibition in adulthood should prove therapeutic in schizophrenia.