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The passivation effect of Fe 3 O 4 /mulberry pole biochar (Fe-MBC) prepared at different carbonization temperatures on soil available arsenic content was studied through soil culture experiments, and Fe-MBC-800 (prepared by carbonization at 800℃) with good passivation effect was selected and characterized. The effects of 1%-7% (mass fraction of biochar to soil) Fe-MBC-800, MBC-800, and Fe 3 O 4 on soil pH value, soil electrical conductivity, soil arsenic form, rice biomass, and total arsenic (As) content in rice were studied using a pot experiment. The results showed that:①Fe-MBC-800 successfully loaded Fe 3 O 4 , and its main functional groups were C=O double bond, O-H bond, C-O bond, and Fe-O bond. The specific surface areas of Fe-MBC-800, MBC-800, and Fe 3 O 4 were 209.659 m 2 ·g -1 , 517.714 m 2 ·g -1 , and 68.025 m 2 ·g -1 , respectively. ②The addition of Fe-MBC-800 could increase the soil pH value, decrease the soil EC value, increase the content of residual arsenic in soil, and reduce the content of water-soluble arsenic and available arsenic in the soil. Under the treatment using 7% Fe-MBC-800 ( ω ) amendments, the content of water-soluble arsenic and available arsenic in the soil decreased by 81.6% and 56.33%, respectively. ③When the addition ratio of Fe-MBC-800 in the soil was 5%-7%, it could promote the growth of rice plants, increase rice biomass, and reduce the bioaccumulation of arsenic by between 62.5% and 68.75%.

In this study, original mulberry-biochar (M-BC) and magnetic iron oxide/mulberry stem biochar (Fe-BC) materials were prepared and characterized using mulberry stems as the raw material. The effects of carbonized temperature of Fe-BC and M-BC on dissolved organic carbon (DOC) and arsenic(As) speciation in soil leaching solutions were studied using soil incubation experiments. The results showed that:① Fe-BC was mainly composed of Fe 3 O 4 and was magnetic, and the main functional groups were a C＝O double bond, O-H bond, C-O bond, and Fe-O bond. The point of zero charge values (pH zpc ) of Fe-BC-400, Fe-BC-500, and Fe-BC-600 were 8.92, 8.74, and 9.19, respectively, and the specific surface areas of Fe-BC-400, Fe-BC-500, and Fe-BC-600 were 447.412, 482.697, and 525.708 m 2 ·g -1 , respectively. ② With the increase in the carbonization temperature of M-BC and Fe-BC, the ρ (DOC) of soil leaching solution decreased 11.6-315.6 mg·L -1 and 78-365.6 mg·L -1 , respectively. The DOC concentration of soil leaching solution was negatively correlated with soil EC. On day 35 of the incubation experiments, compared with that in soil after incubation without biochar (control), the As concentration of the soil leaching solution with Fe-BC-600 decreased by 55.96%, and there was no significant correlation between the As concentration of the soil leaching solution and the DOC concentration of the soil. ③ The available As concentration on day 35 in soil after incubation with Fe-BC was lower than that of the control group; the available As concentration on day 35 in soil incubated with Fe-BC-600 was reduced by 39.21%. ④ The residue As concentration on day 35 in soil incubated with M-BC decreased by 17.76%-49.11%. The residue As content on day 35 in soil incubated with Fe-BC-600 increased by 80%. Fe-BC-600 was most beneficial to reduce the DOC concentration and the available As content in soil leaching solution and increased the residue As content, thus reducing the bioavailability of soil arsenic. Therefore, this study can provide a theoretical basis for magnetic iron oxide/biochar remediation in arsenic-contaminated soil.

This paper describes a simple and convenient method for the rapid screening of potential inhibitors of bacterial adhesion and for the quantitative evaluation of the efficacy of the inhibitors using arrays of self-assembled monolayers (SAMs) of alkanethiolates on gold that are presented on a 96-well microtiter plate. The SAMs present mixtures of alpha-D-mannopyranoside (a ligand that promotes the adhesion of uropathogenic Escherichia coli by binding to the FimH proteins on the tip of type 1 pili), and tri(ethylene glycol) moieties (organic groups that resist nonspecific adsorption of proteins and cells). The SAMs provide surfaces for studies of adhesion of uropathogenic E. coli to specific ligands; they also provide excellent resistance to nonspecific adhesion. Using arrays of mannoside-presenting SAMs, inhibitors of bacterial adhesion were easily screened by observing the number of bacteria that adhered to the surface of the SAMs in the presence of inhibitor. The potency of the inhibitor was quantified by measuring the percentage of inhibition as a function of the concentration of the inhibitor. The properties of SAMs, when combined with the convenience and standardization of a microtiter plate, make arrays of SAMs a versatile tool that can be applied to high-throughput screening of inhibitors of bacterial, viral, and mammalian cell adhesion and of strongly binding ligands for proteins.

Mechanisms of bacterial pathogenesis have become an increasingly important subject as pathogens have become increasingly resistant to current antibiotics. The adhesion of microorganisms to the surface of host tissue is often a first step in pathogenesis and is a plausible target for new antiinfective agents. Examination of bacterial adhesion has been difficult both because it is polyvalent and because bacterial adhesins often recognize more than one type of cell-surface molecule. This paper describes an experimental procedure that measures the forces of adhesion resulting from the interaction of uropathogenic Escherichia coli to molecularly well defined models of cellular surfaces. This procedure uses self-assembled monolayers (SAMs) to model the surface of epithelial cells and optical tweezers to manipulate the bacteria. Optical tweezers orient the bacteria relative to the surface and, thus, limit the number of points of attachment (that is, the valency of attachment). Using this combination, it was possible to quantify the force required to break a single interaction between pilus and mannose groups linked to the SAM. These results demonstrate the deconvolution and characterization of complicated events in microbial adhesion in terms of specific molecular interactions. They also suggest that the combination of optical tweezers and appropriately functionalized SAMs is a uniquely synergistic system with which to study polyvalent adhesion of bacteria to biologically relevant surfaces and with which to screen for inhibitors of this adhesion.

This paper describes the use of laminar flow of liquids in capillary systems to pattern the cell culture substrate, to perform patterned cell deposition, and to pattern the cell culture media. We demonstrate the patterning of the cell culture substrate with different proteins, the patterning of different types of cells adjacent to each other, the patterned delivery of chemicals to adhered cells, and performing enzymatic reactions over select cells or over a portion of a cell. This method offers a way to simultaneously control the characteristics of the surface to which cells are attached, the type of cells that are in their vicinity, and the kind of media that cells or part of a cell are exposed to. The method is experimentally simple, highly adaptable, and requires no special equipment except for an elastomeric relief that can be readily prepared by rapid prototyping.

Peptide binding to class II MHC proteins occurs in acidic endosomal compartments following dissociation of class II-associated invariant chain peptide (CLIP). Based on peptide binding both to empty class II MHC and to molecules preloaded with peptides including CLIP, we find evidence for two isomeric forms of empty MHC. One (inactive) does not bind peptide. The other (active) binds peptide rapidly, with k(on) 1000-fold faster than previous estimates. The active isomer can be formed either by slow isomerization of the inactive molecule or by dissociation of a preformed peptide/MHC complex. In the absence of peptide, the active isomer is unstable, rapidly converting to the inactive isomer. These results demonstrate that fast peptide binding is an inherent property of one isomer of empty class II MHC. Dissociation of peptides such as CLIP yields this transient, peptide-receptive isomer.

The NH2-terminal peptide of myelin basic protein (MBP) bound to the class II major histocompatibility complex (MHC) protein I-Au is an immunodominant epitope in experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis. However, the MBP-I-Au complex is very unstable. To investigate this, we performed site-directed mutagenesis of the I-Au MHC protein and the MBP peptide. Biochemical, T cell activation, and molecular modeling studies of mutant complexes demonstrate that the MBP peptide's key residue for MHC binding, lysine 4, is buried in the P6 pocket of I-Au, which is predominantly hydrophobic. This implies that the MBP-I-Au complex differs from more stable complexes in two respects: (a) the peptide leaves the NH2-terminal region of the MHC peptide-binding cleft unoccupied; (b) the peptide is not anchored by typical favorable interactions between peptide side chains and MHC pockets. To test these hypotheses, a modified MBP peptide was designed based on molecular modeling, with the aim of producing strong I-Au binding. Extension of the NH2 terminus of MBP with six amino acids from the ova peptide, and replacement of the lysine side chain in the P6 pocket with an aromatic anchor, results in >1,000-fold increased binding stability. These results provide an explanation for the unusual peptide-MHC-binding kinetics of MBP, and should facilitate an understanding of why mice are not tolerant to this self-peptide- MHC complex.

We have used molecular modeling to design substitutions in an invariant chain-derived peptide (CLIP), so as to alter the stability of its complex with class II major histocompatibility complex (MHC) proteins. We sought first to test whether CLIP binds in the same way to different class II MHC proteins. We designed destabilizing substitutions of two residues (Met 91 and Met 99) previously predicted to act as the major anchor residues for binding to all class II MHC and measured their effect on CLIP's dissociation rate from a series of three murine I-A MHC proteins. Even a conservative substitution preserving size and hydrophobicity but reducing flexibility (leucine, a branched residue) caused large accelerations in dissociation rates (up to 25-fold) at either position in all three MHC alleles, supporting the consistent role of these positions as the major anchors for MHC binding. These data also support the view that the special flexibility of the methionine side chains at these positions is essential for binding to diverse MHC molecules. We also used molecular modeling to design allele-specific enhancements of peptide binding. Designed substitutions of CLIP Pro 96 by Ala (for Ad), Glu (Ak), and Tyr (Au) each yielded strong enhancement of binding (up to 128-fold) for their targeted allele and only moderate or destabilizing effects to the other alleles. These results demonstrate the accuracy of the molecular models and the predictive value of this modeling. Moreover, they provide strong evidence for the proposed general model of invariant chain association, indicating that it binds to all class II MHC in the same conformation.

The region comprising residues 83-107 of the extracytoplasmic domain of the class II MHC-associated invariant chain protein is essential for its functional interaction with MHC proteins. A nested set of peptides that encompass this region, designated the class II invariant chain-derived peptides (CLIP), bind to many MHC proteins and inhibit the binding of antigenic peptides. The kinetics of the reactions between CLIP and five different murine class II MHC proteins have been determined. Specificity of CLIP binding was confirmed by competition with antigenic peptides. Large differences in the reaction rates were observed. For example, half-times of dissociation ranged from 4.4 min to 17.5 h, a > 200-fold difference. These results demonstrate that CLIP bind to MHC heterodimers at a site that involves the polymorphic residues. These data support the hypothesis that the CLIP binding site is within the peptide binding groove. It is further suggested that these differences in kinetic stabilities of CLIP-MHC protein complexes might affect the diversity of endogenous peptides bound to class II MHC proteins.

Studies on the kinetics of antigenic peptide binding to major histocompatibility complex class II molecules have been used extensively to probe major histocompatibility complex (MHC) structure as well as to investigate the molecular mechanism of peptide recognition. Previous experiments have frequently been carried out in the presence of a cocktail of protease inhibitors to inhibit the proteolysis of MHC heterodimers. By using high performance size exclusion chromatography to measure fluorescent peptide binding to MHC protein, we have found that the addition of a commonly used mixture of protease inhibitors leads to a significant reduction in peptide binding to the class II heterodimer.

Activation of epidermal growth factor (EGF) receptors stimulates inositol phosphate production in rat hepatocytes via a pertussis toxin-sensitive mechanism, suggesting the involvement of a G protein in the process. Since the first event after receptor-G protein interaction is exchange of GTP for GDP on the G protein, the effect of EGF was measured on the initial rates of guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) association and [alpha-32P]GDP dissociation in rat hepatocyte membranes. The initial rate of [35S]GTP gamma S binding was stimulated by EGF, with a maximal effect observed at 8 nM EGF. EGF also increased the initial rate of [alpha-32P]GDP dissociation. The effect of EGF on [35S]GTP gamma S association was blocked by boiling the peptide for 5 min in 5 mM dithiothreitol or by incubation of the membranes with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). EGF-stimulated [35S]GTP gamma S binding was completely abolished in hepatocyte membranes prepared from pertussis toxin-treated rats and was inhibited in hepatocyte membranes that were treated directly with the resolved A-subunit of pertussis toxin. The amount of guanine nucleotide binding affected by occupation of the EGF receptor was approximately 6 pmol/mg of membrane protein. Occupation of angiotensin II receptors, which are known to couple to G proteins in hepatic membranes, also stimulated [35S]GTP gamma S association with and [alpha-32P]GDP dissociation from the membranes. The effect of angiotensin II on [alpha-32P]GDP dissociation was blocked by the angiotensin II receptor antagonist [Sar1,Ile8]angiotensin II, demonstrating that the guanine nucleotide binding was receptor-mediated. In A431 human epidermoid carcinoma cells, EGF stimulates inositol lipid breakdown, but the effect is not blocked by treatment of the cells with pertussis toxin. In these cells, EGF had no effect on [35S]GTP gamma S binding. Occupation of the beta-adrenergic receptor in A431 cell membranes with isoproterenol did stimulate [35S] GTP gamma S binding, and the effect could be completely blocked by l-propranolol. These results support the concept that in hepatocyte membranes, EGF receptors interact with a pertussis toxin-sensitive G protein via a mechanism similar to other hormone receptor-G protein interactions, but that in A431 human epidermoid carcinoma cells, EGF may activate phospholipase C via different mechanisms.