Growth and Cadmium Phytoextraction by Swiss Chard,Maize, Rice,Noccaea caerulescens,and Alyssum murale in pH Adjusted Biosolids Amended Soils

Past applications of biosolids to soils at some locations added higher Cd levels than presently permitted. Cadmium phytoextraction would alleviate current land use constraints. Unamended farm soil, and biosolids amended farm and mine soils were obtained from a Fulton Co., IL biosolids management facility. Soils contained 0.16, 22.8, 45.3 mg Cd kg^–1 and 43.1, 482, 812 mg Zn kg^–1 respectively with initial pH 6.0, 6.1, 6.4. In greenhouse studies, Swiss chard (Beta vulgaris var. cicla), a Cd-accumulator maize (inbred B37 Zea mays) and a southern France Cd-hyperaccumulator genotype of Noccaea caerulescens were tested for Cd accumulation and phytoextraction. Soil pH was adjusted from ～5.5–7.0. Additionally 100 rice (Oryza sativa) genotypes and the Ni-hyperaccumulator Alyssum murale were screened for potential phytoextraction use.

Chard suffered phytotoxicity at low pH and accumulated up to 90 mg Cd kg^–1 on the biosolids amended mine soil. The maize inbred accumulated up to 45 mg Cd kg^–1 with only mild phytotoxicity symptoms during early growth at pH > 6.0. N. caerulescens did not exhibit phytotoxicity symptoms at any pH, and accumulated up to 235 mg Cd kg^–1 in 3 months. Reharvested N. caerulescens accumulated up to 900 mg Cd kg^–1 after 10 months. Neither Alyssum nor 90% of rice genotypes survived acceptably.

Both N. caerulescens and B37 maize show promise for Cd phytoextraction in IL and require field evaluation; both plants could be utilized for nearly continuous Cd removal. Other maize inbreds may offer higher Cd phytoextraction at lower pH, and mono-cross hybrids higher shoot biomass yields. Further, maize grown only for biomass Cd maximum removal could be double-cropped.     

Phytochemicals from the stem wood of Sorbus lanata (D. Don.) Schauer

Three new phenolic compounds, sorlanin (4-(3-(hydroxymethyl)-5-methoxy-7-phenyl-2,3-dihydrobenzo[b][1,4]dioxin-2-yl)-2-methoxyphenol, 1), sorbanin (2-((3,5-dimethoxy-[1,1′-biphenyl]-4-yl)oxy)-1-(4-hydroxy-3-methoxyphenyl)propane-1,3-diol, 2) and sorbalanin (4-(3-(hydroxymethyl)-5,6-dimethoxy-2,3-dihydrobenzo[b][1,4]dioxino[2,3-g]benzofuran-2-yl)-2-methoxyphenol, 3), together with eight known compounds, polystachyol (4), isolariciresinol (5), dihydrodehydrodiconiferyl alcohol (6), tuberculatin (7), ovafolinin E (8), aucuparin (9), 2′-methoxyaucuparin (10), and tetracosyl-3-(3,4-dihydroxyphenyl)acrylate (11), were isolated from Sorbus lanata. The structures of these phytoconstituents were elucidated through extensive spectroscopic techniques, including UV, IR, 1D and 2D NMR, ESI-MS and HRESI-MS experiments. All the compounds except 9 and 10 were isolated for the first time from the genus Sorbus. The isolated compounds were also tested in DPPH radical scavenging reaction where compounds 6, 7, 10 and 11 showed significant activities with IC₅₀ values of 9.2, 11.7, 23.0 and 33.7 μM, respectively.     

Application of Multiplexed Kinase Inhibitor Beads to Study Kinome Adaptations in Drug-Resistant Leukemia

Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in an imatinib-resistant (MYL-R) and -sensitive (MYL) cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKKα, PKCβ, NEK9) or decreased (Abl, Kit, JNK, ATM, Yes) abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942). MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244). Furthermore, the combined inhibition of MEK and IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify dysregulated kinases and to interrogate kinome dynamics as cells respond to targeted kinase inhibition.     

Specificity and Effector Functions of Human RSV-Specific IgG from Bovine Milk

Background

Respiratory syncytial virus (RSV) infection is the second most important cause of death in the first year of life, and early RSV infections are associated with the development of asthma. Breastfeeding and serum IgG have been shown to protect against RSV infection. Yet, many infants depend on bovine milk-based nutrition, which at present lacks intact immunoglobulins.

Objective

To investigate whether IgG purified from bovine milk (bIgG) can modulate immune responses against human RSV.

Methods

ELISAs were performed to analyse binding of bIgG to human respiratory pathogens. bIgG or hRSV was coated to plates to assess dose-dependent binding of bIgG to human Fcγ receptors (FcγR) or bIgG-mediated binding of myeloid cells to hRSV respectively. S. Epidermidis and RSV were used to test bIgG-mediated binding and internalisation of pathogens by myeloid cells. Finally, the ability of bIgG to neutralise infection of HEp2 cells by hRSV was evaluated.

Results

bIgG recognised human RSV, influenza haemagglutinin and Haemophilus influenza. bIgG bound to FcγRII on neutrophils, monocytes and macrophages, but not to FcγRI and FcγRIII, and could bind simultaneously to hRSV and human FcγRII on neutrophils. In addition, human neutrophils and dendritic cells internalised pathogens that were opsonised with bIgG. Finally, bIgG could prevent infection of HEp2 cells by hRSV.

Conclusions

The data presented here show that bIgG binds to hRSV and other human respiratory pathogens and induces effector functions through binding to human FcγRII on phagocytes. Thus bovine IgG may contribute to immune protection against RSV.     

Hot topic or hot air? Climate change and malaria resurgence in East African highlands

Climate has a significant impact on malaria incidence and we have predicted that forecast climate changes might cause some modifications to the present global distribution of malaria close to its present boundaries. However, it is quite another matter to attribute recent resurgences of malaria in the highlands of East Africa to climate change. Analyses of malaria time-series at such sites have shown that malaria incidence has increased in the absence of co-varying changes in climate. We find the widespread increase in resistance of the malaria parasite to drugs and the decrease in vector control activities to be more likely driving forces behind the malaria resurgence.     

Direct determination of homovanillic acid release from the human brain, an indicator of central dopaminergic activity

The plasma concentration of the dopamine (DA) metabolite, homovanillic acid (HVA), is used as an indicator of central nervous system dopaminergic activity. Using percutaneously inserted catheters we were able to obtain blood samples simultaneously from the right and left internal jugular veins. Veno-arterial HVA plasma concentration differences combined with adjusted organ plasma flows were used, according to the Fick Principle, to determine the HVA overflow from the brain. The HVA overflow from the liver was also measured. HVA overflow from the brain represented 12% of the total body HVA production. A similar amount was released from the liver, illustrating the limited validity of peripheral plasma HVA measurements as an indicator of central dopaminergic activity. HVA release from the human brain displayed a degree of asymmetry, the overflow into the left internal jugular vein being 36% greater than that into the right. Cerebral venous blood flow scans indicated that cortical cerebral regions drained preferentially into the right internal jugular; by inference the higher HVA overflow on the left originated from dopamine-rich subcortical brain areas. Since HVA in plasma may arise from the metabolism of DA existing either as a neurotransmitter or a norepinephrine (NE) precursor we measured the internal jugular vein plasma concentrations of NE, and its metabolite dihydroxyphenylglycol (DHPG), to determine whether they displayed a similar pattern of release to HVA. The overflow of both NE and DHPG into the right internal jugular vein was approximately double that on the left. Since the overflow of HVA did not parallel that of NE and DHPG it may be inferred that the origin of much of the subcortically produced HVA is from dopaminergic neurons and not from the metabolism of precursor DA in noradrenergic neurones or cerebrovascular sympathetic nerves.     

Site-specific PEGylation of engineered cysteine analogues of recombinant human granulocyte-macrophage colony-stimulating factor

Granulocyte macrophage colony-stimulating factor (GM-CSF) stimulates proliferation of hematopoietic cells of the macrophage and granulocyte lineages and is used clinically to treat neutropenia and other myeloid disorders. Because of its short circulating half-life, GM-CSF is administered to patients by daily injection. We describe here the engineering of highly potent, long-acting human GM-CSF proteins through site-specific modification of GM-CSF cysteine analogues with a cysteine-reactive poly(ethylene glycol) (PEG) reagent. Thirteen cysteine analogues of GM-CSF were constructed, primarily in nonhelical regions of the protein believed to lie away from the major receptor binding sites. The GM-CSF cysteine analogues were properly processed but insoluble following secretion into the Escherichia coli periplasm. The proteins were refolded and purified by column chromatography. Ten of the cysteine analogues could be modified with a 5-kDa maleimide PEG, and seven of the mono-PEGylated proteins were purified by ion-exchange column chromatography. Biological activities of the 13 cysteine analogues and 7 PEGylated cysteine analogues were comparable to that of wild-type GM-CSF in an in vitro cell proliferation assay using human TF-1 cells. One cysteine analogue was modified with larger 10-, 20-, and 40-kDa PEGs, with only minimal loss of in vitro bioactivity. Pharmacokinetic experiments in rats demonstrated that the PEGylated proteins had up to 47-fold longer circulating half-lives than wild-type GM-CSF. These data demonstrate the utility of site-specific PEGylation for creating highly potent, long-acting GM-CSF analogues and provide further evidence that the nonhelical regions of human GM-CSF examined are largely nonessential for biological activity of the protein.     

Genetic polymorphism of alpha 2HS-glycoprotein.

A genetic polymorphism of the human serum glycoprotein, alpha 2HS-glycoprotein, can be recognized using isoelectric focusing in polyacrylamide, followed by silver-stain immunofixation. In a North American Caucasian population, two common alleles and one rare allele have been recognized, with frequencies as follows: AHSG*1: .6419, AHSG*2: .3535, and AHSG*3: .0046; polymorphism information content (PIC): .36. A black population from various islands of the Caribbean has the two most common alleles, plus a variant (B) not found in the white population. Allele frequencies in the blacks were: AHSG*1: .6901, AHSG*2: .2606, AHSG*B: .0493; PIC: .396. Family studies confirmed the allele designations. Alleles in both populations were in Hardy-Weinberg equilibrium. This polymorphism will be useful as a marker on chromosome 3q and for forensic studies. The serum concentration associated with AHSG*1 may be somewhat greater than that associated with AHSG*2. Differences between the allele products remained after removal of sialic acid from the glycoprotein with neuraminidase. The silver-stain immunofixation technique used for this polymorphism has wide application for the study of polymorphisms where the protein is present in low concentration or where only low titer antiserum is available.