Mapping novel traits by array-assisted bulk segregant analysis in Saccharomyces cerevisiae

We demonstrate a new method, microarray-assisted bulk segregant analysis, for mapping traits in yeast by genotyping pooled segregants. We apply a probabilistic model to the progeny of a single cross and as little as two microarray hybridizations to reliably map an auxotrophic marker, a Mendelian trait, and a major-effect quantitative trait locus.     

Interaction of KAI1 on tumor cells with DARC on vascular endothelium leads to metastasis suppression

CD82, also known as KAI1, was recently identified as a prostate cancer metastasis suppressor gene on human chromosome 11p1.2 (ref. 1). The product of CD82 is KAI1, a 40- to 75-kDa tetraspanin cell-surface protein also known as the leukocyte cell-surface marker CD82 (refs. 1,2). Downregulation of KAI1 has been found to be clinically associated with metastatic progression in a variety of cancers, whereas overexpression of CD82 specifically suppresses tumor metastasis in various animal models. To define the mechanism of action of KAI1, we used a yeast two-hybrid screen and identified an endothelial cell-surface protein, DARC (also known as gp-Fy), as an interacting partner of KAI1. Our results indicate that the cancer cells expressing KAI1 attach to vascular endothelial cells through direct interaction between KAI1 and DARC, and that this interaction leads to inhibition of tumor cell proliferation and induction of senescence by modulating the expression of TBX2 and p21. Furthermore, the metastasis-suppression activity of KAI1 was significantly compromised in DARC knockout mice, whereas KAI1 completely abrogated pulmonary metastasis in wild-type and heterozygous littermates. These results provide direct evidence that DARC is essential for the function of CD82 as a suppressor of metastasis.     

Helicobacter pylori vacuolating cytotoxin (VacA) alters cytoskeleton-associated proteins and interferes with re-epithelialization of wounded gastric epithelial monolayers

In previous studies, we demonstrated that Helicobacter pylori vacuolating cytotoxin (VacA) inhibits gastric epithelial cell proliferation and inhibits epidermal growth factor (EGF)-activated signal transduction. Cell proliferation and migration, both essential for mucosal healing are dependent on the cell cytoskeleton. Other investigators demonstrated that VacA induces vacuolation of eukaryotic cells. Since in some cells, control of actin cytoskeleton involves GTP-binding proteins of Rho family, in this study we examined whether VacA affects wound re-epithelialization, cell cytoskeleton-associated proteins Rho, Rac1 in a gastric epithelial (RGM1) cell monolayer wound model, and whether these changes correlate with vacuolation. VacA treatment significantly inhibited wound re-epithelialization, cell proliferation vs control. VacA-induced cell vacuolation strongly correlated with inhibition of wound re-epithelialization. Furthermore, VacA reduced Rac-1 protein expression and distribution, and C3-mediated ADP-ribosylation of Rho. These findings suggest that VacA may interfere with repair of gastric mucosal injury and ulcer re-epithelialization by altering cytoskeleton-dependent cell functions and signaling.     

Glutamine administration ameliorates sepsis-induced kidney injury by downregulating the high-mobility group box protein-1-mediated pathway in mice

Acute kidney injury (AKI) is a severe complication of sepsis. High-mobility group box (HMGB)-1 was implicated as a late mediator of lethal systemic inflammation in sepsis. Since glutamine (GLN) was shown to have anti-inflammatory and antioxidant properties, we hypothesized that GLN administration may downregulate an HMGB-1-mediated pathway and thus ameliorate sepsis-induced AKI. Mice were randomly assigned to a normal group (NC), a septic saline group (SS), or a septic GLN group (SG). Sepsis was induced by cecal ligation and puncture (CLP). The SS group was injected with saline, and the SG group was given 0.75 g GLN/kg body wt once via a tail vein 1 h after CLP. Mice were killed 2, 6, and 24 h after CLP, and blood and kidneys of the animals were harvested for further analysis. The results showed that sepsis resulted in higher mRNA and/or protein expressions of kidney HMGB-1, toll-like receptor (TLR) 4, myeloid differentiation primary-response protein (MyD) 88, and receptor of advanced glycation end products (RAGE) compared with normal mice. Septic mice with GLN administration exhibited decreased HMGB-1, TLR4, RAGE, and phosphorylated NF-κB p65 protein expressions and reduced nitrotyrosine levels in kidney tissues. The histological findings showed that damage to the kidneys was less severe, and survival improved in the SG group. These results indicated that a single dose of GLN administered after the initiation of sepsis plays a prophylactic role in downregulating the expressions of HMGB-1-related mediators and decreasing oxidative stress in the kidneys, which may consequently have ameliorated AKI induced by sepsis.     

Ultrafiltration improves ELISA and Endopep MS analysis of botulinum neurotoxin type A in drinking water

The objective of this study was to adapt and evaluate two in vitro botulinum neurotoxin (BoNT) detection methods, including the Botulinum Toxin ELISA and the Endopep MS (a mass spectrometric-based endopeptidase method), for use with drinking water samples. The method detection limits (MDL) of the ELISA and Endopep MS were 260 pg/mL and 21 pg/mL of BoNT/A complex toxin, respectively. Since toxin could be present in water samples at highly dilute concentrations, large volume (100-L) samples of municipal tap water from five US municipalities having distinct water compositions were dechlorinated, spiked with 5 μg BoNT/A, and subjected to tangential-flow ultrafiltration (UF) using hollow fiber dialyzers. The recovery efficiency of BoNT/A using UF and quantified by ELISA ranged from 11% to 36% while efficiencies quantified by MS ranged from 26% to 55%. BoNT/A was shown to be stable in dechlorinated municipal tap water stored at 4°C for up to four weeks. In addition, toxin present in UF-concentrated water samples was also shown to be stable at 4°C for up to four weeks, allowing holding of samples prior to analysis. Finally, UF was used to concentrate a level of toxin (7 pg/mL) which is below the MDL for direct analysis by both ELISA and Endopep MS. Following UF, toxin was detectable in these samples using both in vitro analysis methods. These data demonstrate that UF-concentration of toxin from large volume water samples followed by use of existing analytical methods for detection of BoNT/A can be used in support of a monitoring program for contaminants in drinking water.     

A facile method to synthesize histones with posttranslational modification mimics

Using an evolved pyrrolysyl-tRNA synthetase-tRNA(Pyl) pair, a Se-alkylselenocysteine was genetically incorporated into histone H3 with a high protein expression yield. Quantitative oxidative elimination of Se-alkylselenocysteine followed by Michael addition reactions with various thiol nucleophiles generated biologically active mimics of H3 with posttranslational modifications including lysine methylation, lysine acetylation, and serine phosphorylation.     

Restriction enzyme-free construction of random gene mutagenesis libraries in Escherichia coli

Directed evolution relies on both random and site-directed mutagenesis of individual genes and regulatory elements to create variants with altered activity profiles for engineering applications. Central to these experiments is the construction of large libraries of related variants. However, a number of technical hurdles continue to limit routine construction of random mutagenesis libraries in Escherichia coli, in particular, inefficiencies during digestion and ligation steps. Here, we report a restriction enzyme-free approach to library generation using megaprimers termed MegAnneal. Target DNA is first exponentially amplified using error-prone polymerase chain reaction (PCR) and then linearly amplified with a single 3′ primer to generate long, randomly mutated, single-stranded megaprimers. These are annealed to single-stranded dUTP-containing template plasmid and extended with T7 polymerase to create a complementary strand, and the resulting termini are ligated with T4 DNA ligase. Using this approach, we are able to reliably generate libraries of approximately 10⁷ colony-forming units (cfu)/μg DNA/transformation in a single day. We have created MegAnneal libraries based on three different single-chain antibodies and identified variants with enhanced expression and ligand-binding affinity. The key advantages of this approach include facile amplification, restriction enzyme-free library generation, and a significantly reduced risk of mutations outside the targeted region and wild-type contamination as compared with current methods.     

Zinc fluxes into developing barley grains: use of stable Zn isotopes to separate root uptake from remobilization in plants with contrasting Zn status

Background and Aims

Zn imported into developing cereal grains originates from either de novo Zn uptake by the roots or remobilization of Zn from vegetative tissues. The present study was focused on revealing the quantitative importance of the two pathways for grain Zn loading and how their relative contribution varies with the overall plant Zn status.

Methods

The stable isotope ⁶⁷Zn was used to trace Zn uptake and remobilization fluxes in barley (Hordeum vulgare L.) plants growing in hydroponics at 0.1?μM (low Zn), 1.5?μM (medium Zn) or 5?μM Zn (high Zn). When grain development reached 15?days after pollination the Zn source was changed to an enriched ⁶⁷Zn isotope and plants were harvested after 6 to 48?h. Zn concentrations and isotope ratios were determined using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS).

Results

Plants with low Zn status absorbed 3-fold more Zn than plants with medium or high Zn status when roots were exposed to an external concentration of 1.5?μM ⁶⁷Zn. Stems and ears were the primary recipients of the de novo incorporated Zn with preferential allocation to the developing grains over time. The leaves received in all cases a very small proportion (<5?%) of the newly absorbed Zn and the proportion did not increase over time. Zn fluxes derived from uptake and remobilization were almost equal in plants with low Zn status, while at high Zn status remobilization delivered 4 times more Zn to the developing grains than did root Zn uptake.

Conclusions

Stable isotopes in combination with ICP-MS provided a strong tool for quantification of Zn fluxes in intact plants. The importance of Zn remobilization compared to de novo root absorption of Zn increased with increasing plant Zn status. Very little de novo absorbed Zn was translocated to the leaves during generative growth stages.     

Abnormal NF-kappa B function characterizes human type 1 diabetes dendritic cells and monocytes

Dendritic cell (DC) differentiation is abnormal in type 1 diabetes mellitus (T1DM). However, the nature of the relationship between this abnormality and disease pathogenesis is unknown. We studied the LPS response in monocytes and monocyte-derived DCs isolated from T1DM patients and from non-T1DM controls. In T1DM patients, late LPS-mediated nuclear DNA binding by RelA, p50, c-Rel, and RelB was impaired as compared with type 2 DM, rheumatoid arthritis, and healthy subjects, associated with impaired DC CD40 and MHC class I induction but normal cytokine production. In TIDM monocytes, RelA and RelB were constitutively activated, and the src homology 2 domain-containing protein tyrosine phosphatase (SHP-1), a negative regulator of NF-kappaB, was overexpressed. Addition of sodium stibogluconate, a SHP-1 inhibitor, to DCs differentiating from monocyte precursors restored their capacity to respond to LPS in approximately 60% of patients. The monocyte and DC NF-kappaB response to LPS is thus a novel phenotypic and likely pathogenetic marker for human T1DM. SHP-1 is at least one NF-kappaB regulatory mechanism which might be induced as a result of abnormal inflammatory signaling responses in T1DM monocytes.