Glutamate 59 is critical for transport function of the amino acid cotransporter KAAT1

KAAT1 is a neutral amino acid transporter activated by K⁺ or by Na⁺ (9). The protein shows significant homology with members of the Na⁺/Cl^–-dependent neurotransmitter transporter super family. E59G KAAT1, expressed in Xenopus oocytes, exhibited a reduced leucine uptake [20–30% of wild-type (WT)], and kinetic analysis indicated that the loss of activity was due to reduction of V_max and apparent affinity for substrates. Electrophysiological analysis revealed that E59G KAAT1 has presteady-state and uncoupled currents larger than WT but no leucine-induced currents. Site-directed mutagenesis analysis showed the requirement of a negative charge in position 59 of KAAT1. The analysis of permeant and impermeant methanethiosulfonate reagent effects confirmed the intracellular localization of glutamate 59. Because the 2-aminoethyl methanethiosulfonate hydrobromid inhibition was not prevented by the presence of Na⁺ or leucine, we concluded that E59 is not directly involved in the binding of substrates. N-ethylmaleimide inhibition was qualitatively and quantitatively different in the two transporters, WT and E59G KAAT1, having the same cysteine residues. This indicates an altered accessibility of native cysteine residues due to a modified spatial organization of E59G KAAT1. The arginine modifier phenylglyoxal effect supports this hypothesis: not only cysteine but also arginine residues become more accessible to the modifying reagents in the mutant E59G. In conclusion, the results presented indicate that glutamate 59 plays a critical role in the three-dimensional organization of KAAT1. amino acid transport; structure/function; amino acid modifiers; Manduca sexta     

Campylobacter jejuni binds intestinal H(O) antigen (Fuc alpha 1, 2Gal beta 1, 4GlcNAc), and fucosyloligosaccharides of human milk inhibit its binding and infection

The most common cause of infant mortality is diarrhea; the most common cause of bacterial diarrhea is Campylobacter jejuni, which is also the primary cause of motor neuron paralysis. The first step in campylobacter pathogenesis is adherence to intestinal mucosa. We found that such binding was inhibited in vitro by human milk and, with high avidity, by alpha1,2-fucosylated carbohydrate moieties containing the H(O) blood group epitope (Fuc alpha 1,2Gal beta 1,4GlcNAc em leader ). In studies on the mechanism of adherence, campylobacter, which normally does not bind to Chinese hamster ovary cells, bound avidly when the cells were transfected with a human alpha1,2-fucosyltransferase gene that caused overexpression of H-2 antigen; binding was specifically inhibited by H-2 ligands (lectins Ulex europaeus and Lotus tetragonolobus and H-2 monoclonal antibody), H-2 mimetics, and human milk oligosaccharides. Human milk oligosaccharides inhibited campylobacter colonization of mice in vivo and human intestinal mucosa ex vivo. Campylobacter colonization of nursing mouse pups was inhibited if their dams had been transfected with a human alpha1,2-fucosyltransferase gene that caused expression of H(O) antigen in milk. We conclude that campylobacter binding to intestinal H-2 antigen is essential for infection. Milk fucosyloligosaccharides and specific fucosyl alpha1,2-linked molecules inhibit this binding and may represent a novel class of antimicrobial agents.     

Invasion of mammalian host cells by Plasmodium sporozoites

Malaria is transmitted through the bite of an infected mosquito, which introduces Plasmodium sporozoites into the mammalian host. Sporozoites rapidly reach the liver of the host where they are sequestered, a process probably mediated by circumsporozoite (CS) protein. Once in the liver, sporozoites migrate through several hepatocytes by breaching their plasma membranes before infecting a final hepatocyte with formation of a vacuole around the sporozoite, where development occurs into blood stage parasites. We propose that migration through several host cells activates sporozoites for ultimate productive invasion. This migration triggers sporozoite exocytosis, which is necessary for hepatocyte invasion, probably because it provides molecules, such as thrombospondin-related anonymous protein (TRAP), likely required for sporozoite invasion with the formation of a vacuole. How sporozoites migrate from the skin to the liver and invade hepatocytes remains unclear. Understanding this initial stage of malaria is crucial for the development of new approaches against the disease.     

Established human papillomavirus type 16-expressing tumors are effectively eradicated following vaccination with long peptides

Peptide-based vaccines aimed at the induction of effective T cell responses against established cancers have so far only met with limited clinical success and clearly need to be improved. In a preclinical model of human papillomavirus (HPV)16-induced cervical cancer we show that prime-boost vaccinations with the HPV16-derived 35 amino-acid long peptide E7(43-77), containing both a CTL epitope and a Th epitope, resulted in the induction of far more robust E7-specific CD8(+) T cell responses than vaccinations with the minimal CTL epitope only. We demonstrate that two distinct mechanisms are responsible for this effect. First, vaccinations with the long peptide lead to the generation of E7-specific CD4(+) Th cells. The level of the induced E7-specific CD8(+) T cell response proved to be dependent on the interactions of these Th cells with professional APC. Second, we demonstrate that vaccination with the long peptide and dendritic cell-activating agents resulted in a superior induction of E7-specific CD8(+) T cells, even when T cell help was excluded. This suggests that, due to its size, the long peptide was preferably endocytosed, processed, and presented by professional APCs. Moreover, the efficacy of this superior HPV-specific T cell induction was demonstrated in therapeutic prime-boost vaccinations in which the long peptide admixed with the dendritic cell-activating adjuvant oligodeoxynucleotide-CpG resulted in the eradication of large, established HPV16-expressing tumors. Because the vaccine types used in this study are easy to prepare under good manufacturing practice conditions and are safe to administer to humans, these data provide important information for future clinical trials.     

Immobilized particles in gel matrix-type porous media. Nonhomogeneous cell distribution

The conventional random pore model assumes a homogeneous cell distribution in the gel matrix used to immobilize cells. However, the validity of this model is restricted to values of the exponent alpha, between 1.8 and 2.25, of a model power function relating the diffusivity coefficient in the matrix with the overall cell volume fraction in the system. Based on the analysis of published data for diffusion in gels with immobilized cells and on the homogeneous approach for the random pore model developed in a previous work, a new, nonhomogeneous approach is proposed for alpha values outside the range 1.8-2.25. To explain these data, two main types of nonhomogeneous cell distribution were considered: (1) nonhomogeneous cell distribution in the gel for alpha > 2.25 (type 1) and (2) nonhomogeneity related with anisotropy of cell space orientation when alpha < 1.8 (type 2). In the case of nonhomogeneity of type 1, the cell volume fraction in the layers occupied by cells must be considered in place of the concept previously used for homogeneous distribution, viz., the average cell volume fraction. This model underlines that accumulation of cells in a thin layer close to the surface improves their nutrient intake. For nonhomogeneity of type 2, the tortuosity of such a system is smaller than should be expected if spherical cells were considered, thereby changing the effective diffusion. The model proposed in this work proved to fit into several real cases reported in the literature.     

Relation between fitness tests and match performance in elite Italian soccer referees

This study examined the relation between field-test results and match performance in elite Italian soccer referees. Subjects (n = 22) were all experienced elite-level referees enrolled in the Commissione Arbitri Nazionali (CAN) and thus officiating in the Serie A and B Italian championships. Referees were, on separate occasions, tested for fitness (50-m, 200-m, and 12-minute run tests) and observed a minimum of 1 and a maximum of 3 times (n = 39) during Serie A matches. Match analyses were performed considering 11 match activity categories. Analyses of correlations were performed considering 50-m, 200-m, and 12-minute run test performances as independent variables and total distance, maximal speed distance (runs performed at speeds faster than 24 km.h-1), and high-intensity activity distance (runs performed at speeds faster than 18 km.h-1, high intensity activity [HIA]) as dependent variables. Statistical significance was set at p 相似文献   

Carbamylation of proteins in 2-D electrophoresis--myth or reality?

Carbamylation is widely quoted as being a problem in 2-D gel analysis and the associated sample preparation steps. This modification occurs when iso-cyanate, a urea break-down product, covalently modifies lysine residues, thus inducing a change in isoelectric point. Urea is used at up to 9 M concentrations in sample preparation and 2-D gels because of its ability to disrupt protein structure and effect denaturation without the need for ionic surfactants such as SDS. We have studied carbamylation using 7 M urea and 2 M thiourea, under a range of experimental temperatures to establish when, and if, it occurs and what can be done to minimize the modification. The actual time required for protein extraction from a tissue is usually short compared to the time required for procedures such as reduction and alkylation and IPG rehydration and focusing. Therefore, it is the temperature during these post-extraction procedures that is the most critical factor. Our experiments have shown that carbamylation does not occur during electrophoresis in the presence of urea, even with prolonged run-times. However, under poorly controlled sample preparation and storage conditions, it can become a major event.     

Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography-tandem mass spectrometry as methyl ester tri(N-pentafluoropropionyl) derivative

Asymmetric dimethylarginine (ADMA; N(G),N(G)-dimethyl-L-arginine) is the most important endogenous inhibitor of nitric oxide synthase and a potential risk factor for cardiovascular diseases. This article describes a gas chromatographic-tandem mass spectrometric (GC-tandem MS) method for the accurate quantification of ADMA in human plasma or serum and urine using de novo synthesized [2H(3)]-methyl ester ADMA (d(3)Me-ADMA) as the internal standard. Aliquots (100 microl) of plasma/serum ultrafiltrate or native urine and of aqueous solutions of synthetic ADMA (1 microM for plasma and serum; 20 microM for urine) are evaporated to dryness. The residue from plasma/serum ultrafiltrate or urine is treated with a 100 microl aliquot of 2M HCl in methanol, whereas the residue of the ADMA solution is treated with a 100 microl aliquot of 2M HCl in tetradeuterated methanol. Methyl esters are prepared by heating for 60 min at 80 degrees C. After cooling to room temperature, the plasma or urine sample is combined with the d(3)Me-ADMA sample, the mixture is evaporated to dryness, the residue treated with a solution of pentafluoropropionic (PFP) anhydride in ethyl acetate (1:4, v/v) and the sample is incubated for 30 min at 65 degrees C. Solvent and reagents are evaporated under a stream of nitrogen gas, the residue is treated with a 200 microl aliquot of 0.4M borate buffer, pH 8.5, and toluene (0.2 ml for plasma, 1 ml for urine). Reaction products are extracted by vortexing for 1 min, the toluene phase is decanted, and a 1 microl aliquot is injected into the GC-tandem MS instrument. Quantitation is performed by selected reaction monitoring (SRM) of the common product ion at m/z 378 which is produced by collision-induced dissociation of the ions at m/z 634 for endogenous ADMA and m/z 637 for d(3)Me-ADMA. In plasma and urine of healthy humans ADMA was measured at concentrations of 0.39+/-0.06 microM (n=12) and 3.4+/-1.1 micromol/mmol creatinine (n=9), respectively. The limits of detection and quantitation of the method are approximately 10 amol and 320 pM of d(3)Me-ADMA, respectively.     

Study of the serum albumin-polyethyleneglycol interaction to predict the protein partitioning in aqueous two-phase systems

The theoretical framework based only on the excluded volume forces is not enough to explain the bovine serum albumin partitioning behaviour in aqueous biphasic systems. The goal of this work is to look at the phase separation via the polymer effect on the water structure. Our findings suggest that polyethyleneglycol 600-protein interaction is conducted by van der Waals forces between the hydrophobic surfaces from PEG and protein molecules, which implies the rupture of hydrogen bonds from the structured water in their neighbours. Therefore, the protein will concentrate in the most water-structured phase (polyethyleneglycol) in order to reach the minimal free energy condition. When polyethyleneglycol molecular weight increases, its exclusion from protein surface prevails, thus pushing the bovine serum albumin to the bottom phase.