Preparation of a new benzylureido-ß-cyclodextrin-based column and its application for the determination of phenylmercapturic acid and benzylmercapturic acid enantiomers in human urine by LC/MS/MS.

A benzylureido-ß-cyclodextrin was synthesized by the reaction of 6-amino-ß-cyclodextrin with an active benzyl isocyanate. Then, it was bonded to silica gel by a thiol-ene addition reaction, obtaining a new benzylureido-ß-cyclodextrin-based chiral stationary phase (BzCDP). Its chemical structure was characterized by infrared spectroscopy, elemental analysis, and solid-state nuclear magnetic resonance spectroscopy. The BzCDP was successfully used to separate phenylmercapturic acid (PMA) and benzylmercapturic acid (BMA) enantiomers, which were confirmed as biomarkers of exposure to benzene and toluene in human urine. The enantiomeric separations were also optimized through the investigation of related factors. The resolutions of PMA and BMA enantiomers could be up to 2.25 and 2.14, respectively, within 30 min under reversed-phase chromatography. Based on the optimal chromatographic and mass spectrometry conditions, a new LC-MS/MS quantitative method for the PMA and BMA enantiomers was established by negative ion multiple reaction monitoring (MRM) and an isotope-labeled PMA (d2-PMA) as an internal standard. The limits of detection (LODs) of enantiomers were less than 0.17 µg/L for PMA and 0.14 µg/L for BMA, and the averaged recoveries of enantiomers were in the range of 86~100% for PMA and 86~113% for BMA. The method had good reproducibility levels with the RSDs (3.5~11.3% for intra-day and 3.9~13.1% for inter-day). The method was successfully applied to urine testing of 60 painting and printing workers. The results showed that only L-PMA was detected in the urine of the Printers, while a high content of L-PMA (27.5~106 µg/L) and D-PMA (19.9~82.8 µg/L) can be detected simultaneously in the urine of the Painters, indicating that benzene pollution was more serious in this group. The positive rate of BMA was rather higher, indicating that toluene pollution was more common than benzene. BMA also existed in the form of two enantiomers (L-BMA and D-BMA), but the difference between the two types of occupational groups was small. It is a meaningful work to deeply study the existence and content of chiral markers in human urine, which will help to better understand and evaluate the harmful effects of benzene series on human beings. Graphical abstract.

Two sorting motifs, a ubiquitination motif and a tyrosine motif, are involved in HIV-1 and simian immunodeficiency virus Nef-mediated receptor endocytosis.

HIV-1 and SIV Nef proteins downregulate cell surface CD4 and MHC class I (MHC-I) molecules of infected cells, which are necessary for efficient viral replication and pathogenicity. We previously reported that K144 in HIV-1 Nef is di-ubiquitinated, and K144R substitution impairs Nef-mediated CD4 downregulation. In this report, we extend the role of ubiquitination at this lysine residue from Nef-mediated CD4 downregulation to Nef-mediated MHC-I downregulation and from HIV Nef to SIV Nef. All HIV-1 Nef mutants that contain K144R substitution are inactive in MHC-I downregulation. Tested MHC-I alleles include HLA-ABC endogenously expressed and HLA-A2 exogenously expressed in Jurkat T cells. CD4 downregulation by SIV Nef involves K176 that aligns with K144 in HIV-1 Nef, as well as an N-terminal tyrosine motif Y28Y39 not present in HIV-1 Nef. Dual mutation at K176 and Y28Y39 completely impaired SIV Nef-mediated CD4 and MHC-I downregulation, whereas a single mutation at K176 or Y28Y39 did not. The involvement of tyrosine motif in SIV Nef-mediated CD4 and MHC-I downregulation prompted us to investigate a putative tyrosine motif (Y202Y/F203) in HIV-1 Nef that is conserved among HIV-1 species. Single mutation at the tyrosine motif Y202F203 in HIV-1 Nef (NA7) greatly impaired Nef-mediated CD4 downregulation, which is similar to what we observed previously with the single mutation at lysine K144. Thus, our study demonstrated that Nef-mediated receptor endocytosis involves the ubiquitination motif and tyrosine motif.

A New Benzyl Phenethanolamine-ß-Cyclodextrin Bonded Phase: Chiral Chromatography Performance and Application for LC-MS/MS Analysis of Pesticide Enantiomers in Fruits and Vegetables.

BACKGROUND: At present, the research on achiral drug and pesticide residue detection methods is still the mainstay at home and abroad, and there is still a lack of systematic research on the enantiomeric analysis of chiral drugs and pesticides. OBJECTIVE: In order to prepare a novel chiral stationary phase, whose "multi-mode" chiral separation chromatographic performance and its utility was verified. METHOD: An S-(-)-2-benzylamino-1-phenylethanol mono-derivative ß-cyclodextrin bonded stationary phase (BzCSP) was prepared based on the "thiol-ene" addition reaction. The chiral compounds including four types of chiral compounds were used as "probes," and their chiral chromatographic properties were evaluated. Furthermore, a new LC-MS/MS method for the determination of the enantiomeric residues of three chiral pesticides in five kinds of fruits and vegetables was established. RESULTS: The study found that the novel stationary phase was suitable for a variety of chromatographic modes (normal phase mode, reversed-phase mode, polar organic mode). The resolutions of hexaconazole (Hex), tebuconazole (Teb), and triticonazole (Trit) enantiomers could be up to 2.31, 1.68, and 1.48, respectively, within 30 min under reversed-phase chromatography. Based on the optimal chromatographic and mass spectrum conditions, a new LC-MS/MS quantitative method for the Hex, Teb, and Trit enantiomers was established by multi-reaction positive ion monitoring (MRM). The detection limits (LODs) of enantiomers were less than 0.89 µg/kg for Hex, 0.93 µg/kg for Teb, and 0.93 µg/kg for Trit, and the averaged recoveries of enantiomers were in the range of 75.8-106.3% for Hex, 77.4-116.3% for Teb, and 78.7-113.4% for Trit. The method had good reproducibility with the RSDs (<5%) for intraday and (<7%) for interday. CONCLUSIONS: The established method had the characteristics of good selectivity, high sensitivity, strong resistance to matrix interference, and good reproducibility. It is indicated that the stationary phase prepared by the "thiol-ene" addition reaction is a new type of multi-mode stationary phase, which has a good development value. HIGHLIGHTS: The study reported a new method for the rapid preparation of a rare "multi-mode" chiral stationary phase BzCSP based on the "thiol-ene" addition reaction and verified the practicability of BzCSP including good selectivity, high sensitivity, strong resistance to matrix interference, and good reproducibility.

Alkylating HIV-1 Nef - a potential way of HIV intervention.

BACKGROUND: Nef is a 27 KDa HIV-1 accessory protein. It downregulates CD4 from infected cell surface, a mechanism critical for efficient viral replication and pathogenicity. Agents that antagonize the Nef-mediated CD4 downregulation may offer a new class of drug to combat HIV infection and disease. TPCK (N-alpha-p-tosyl-L-phenylalanine chloromethyl ketone) and TLCK (N-alpha-p-tosyl-L-lysine chloromethyl ketone) are alkylation reagents that chemically modify the side chain of His or Cys residues in a protein. In search of chemicals that inhibit Nef function, we discovered that TPCK and TLCK alkylated HIV Nef. METHODS: Nef modification by TPCK was demonstrated on reducing SDS-PAGE. The specific cysteine residues modified were determined by site-directed mutagenesis and mass spectrometry (MS). The effect of TPCK modification on Nef-CD4 interaction was studied using fluorescence titration of a synthetic CD4 tail peptide with recombinant Nef-His protein. The conformational change of Nef-His protein upon TPCK-modification was monitored using CD spectrometry RESULTS: Incubation of Nef-transfected T cells, or recombinant Nef-His protein, with TPCK resulted in mobility shift of Nef on SDS-PAGE. Mutagenesis analysis indicated that the modification occurred at Cys55 and Cys206 in Nef. Mass spectrometry demonstrated that the modification was a covalent attachment (alkylation) of TPCK at Cys55 and Cys206. Cys55 is next to the CD4 binding motif (A56W57L58) in Nef required for Nef-mediated CD4 downregulation and for AIDS development. This implies that the addition of a bulky TPCK molecule to Nef at Cys55 would impair Nef function and reduce HIV pathogenicity. As expected, Cys55 modification reduced the strength of the interaction between Nef-His and CD4 tail peptide by 50%. CONCLUSIONS: Our data suggest that this Cys55-specific alkylation mechanism may be exploited to develop a new class of anti HIV drugs.

Identification of a novel binding site between HIV type 1 Nef C-terminal flexible loop and AP2 required for Nef-mediated CD4 downregulation.

HIV-1 Nef is an accessory protein necessary for HIV-1 virulence and rapid AIDS development. Nef promotes viral replication and infection by connecting CD4 and several other cell surface receptors to the clathrin adaptor protein AP2, resulting in the internalization and degradation of the receptors interacting with Nef. We investigated how Nef can mediate constitutive receptor endocytosis through the interaction of the dileucine motif in its C-terminal flexible loop (C-loop) with AP2, whereas AP2 binding of the transmembrane receptors usually results in an equilibrated (recycled) endocytosis. Our results indicated that in addition to the dileucine motif, there is a second motif in the Nef C-loop involved in the Nef-AP2 interaction. Nef-mediated CD4 downregulation was impaired when the residue in the hydrophobic region in the Nef C-loop (LL165HPMSLHGM173) was mutated to a basic residue K/R or an acidic residue E/D or to the rigid residue P, or when M168L170, L170H171, or G172M173 was mutated to AA. A pull-down assay indicated that AP2 was not coprecipitated with Nef mutants that did not downregulate CD4. Molecular modeling of the Nef C-terminal flexible loop in complex with AP2 suggests that M168L170 occupies a pocket in the AP2 σ2 subunit. Our data suggest a new model in the Nef-AP2 interaction in which the hydrophobic region in the Nef C-loop with the dileucine (L164L165) motif and M168L170 motif binds to AP2(σ2), while the acidic motif E174 and D175 binds to AP2(α), which explains how Nef through the flexible loop connects CD4 to AP2 for constitutive CD4 downregulation.

Endocytosis and membrane potential are required for HeLa cell uptake of R.I.-CKTat9, a retro-inverso Tat cell penetrating peptide.

Cell-penetrating peptides (CPPs) can enter many types of cells and have become useful tools for introducing a variety of cargo such as exogenous peptides, proteins, and nucleic acids into cultured cells in vitro. Tat CPPs derived from the HIV-1 Tat protein are the most widely used among the arginine-rich CPPs. Even though CPPs hold considerable promise for drug delivery, poor biological stability and high in vivo clearance may limit their effectiveness for delivering cargo. Therefore, we utilize a retro-inverso form of a Tat peptide, R.I.-CKTat9, which is proteolytically stable. In the current study, the cellular entry mechanism of this arginine-rich CPP is investigated. Fluorescently labeled R.I.-CKTat9 entered HeLa cells in a concentration- and energy-dependent manner demonstrating both diffuse and punctate (vesicular) appearance inside the cells. The labeled R.I.-CKTat9 colocalized with labeled transferrin in the punctate structure, suggesting that the peptide enters HeLa cells by clathrin-dependent endocytosis. Incubation of cells with an isotonic/high K(+) buffer (KPBS) or an NH(4)Cl solution abolished the diffuse but not the punctate fluorescence, suggesting that membrane potential plays a critical role. This result also suggests that the flux originates from the endosome, not the extracellular space, and relies on the acidity of the endosome. Impairment of clathrin-mediated endocytosis by RNAi with clathrin heavy chain function and endocytosis inhibitors greatly reduced or completely abolished both diffuse and punctate fluorescence, further supporting a single route of endocytosis and subsequent endosomal escape. Since cells in the mitotic (M) phase shut down endocytosis but maintain plasma membrane potential, this property was used to further confirm the endocytic mechanism. Direct measurement of plasma membrane potential confirmed its persistence in M phase arrested HeLa cells. Consistent with our working hypothesis, these cells did not show any vesicular nor diffuse fluorescence of labeled R.I.-CKTat9, providing compelling evidence for the sequential steps of endocytosis and endosomal escape. Binding of labeled R.I.-CKTat9 to the surface of HeLa cells at 0 degrees C was reduced under the mildly acidic conditions of early endosomes, suggesting an acidity-dependent endosomal escape mechanism. Overall, these results indicate that both endocytosis and membrane potential are required for R.I.-CKTat9 entry into HeLa cells and suggest that translocation occurs at the endosomal membrane.

Lysine 144, a ubiquitin attachment site in HIV-1 Nef, is required for Nef-mediated CD4 down-regulation.

Nef is a HIV-1 accessory protein critical for the replication of the virus and the development of AIDS. The major pathological activity of Nef is the down-regulation of CD4, the primary receptor of HIV-1 infection. The mechanism underlying Nef-mediated CD4 endocytosis and degradation remains incompletely understood. Since protein ubiquitination is the predominant sorting signal in receptor endocytosis, we investigated whether Nef is ubiquitinated. The in vivo ubiquitination assay showed that both HIV-1 and SIV Nef proteins expressed in Jurkat T cells and 293T cells were multiple ubiquitinated by ubiquitin-His. The lysine-free HIV-1 Nef mutant (Delta10K) generated by replacing all 10 lysines with arginines was not ubiquitinated and the major ubiquitin-His attachment sites in HIV-1 Nef were determined to be lysine 144 (di-ubiquitinated) and lysine 204 (mono-ubiquitinated). Lysine-free HIV-1 Nef was completely inactive in Nef-mediated CD4 down-regulation, so was the Nef mutant with a single arginine substitution at K144 but not at K204. A mutant HIV-1 provirion NL4-3 with a single arginine substitution in Nef at K144 was also inactive in Nef-mediated CD4 down-regulation. Lysine-free Nef mutant reintroduced with lysine 144 (DeltaK10 + K144) was shown active in CD4 down-regulation. These data suggest that ubiquitination of Nef, particularly diubiquitination of the lysine 144, is necessary for Nef-mediated CD4 down-regulation.

HIV Nef-mediated CD4 down-regulation is adaptor protein complex 2 dependent.

Nef is a crucial viral protein for HIV to replicate at high titers and in the development of AIDS. One Nef function is down-regulating CD4 from the cell surface, which correlates with Nef-enhanced viral pathogenicity. Nef down-regulates CD4 by linking CD4 to clathrin-coated pits. However, the mechanistic connection between the C-terminal dileucine motif of Nef and the component(s) of the clathrin-coated pits has not been pinpointed. In this report we used two AP-2 complex-specific inhibitors: a dominant negative mutant of Eps15 (Eps15DIII) that binds to the alpha subunit of AP-2 complex and a small interference RNA that is specific for the mu2 subunit of AP-2 complex. We show that both HIV Nef- and SIV Nef-mediated CD4 down-regulations were profoundly blocked by the synergistic effect of Eps15DIII and RNA interference of AP-2 expression. The results demonstrate that HIV/SIV Nef-mediated CD4 down-regulation is AP-2 dependent. We also show that the PMA-induced CD4 down-regulation was blocked by these two inhibitors. Therefore, PMA-induced CD4 down-regulation is also AP-2 dependent. The results demonstrate that, like the tyrosine sorting motif-dependent endocytosis (for which the transferrin receptor and the epidermal growth factor receptor are the two prototypes), dileucine sorting motif-dependent endocytosis of Nef and CD4 are also AP-2 dependent.

CD4 phosphorylation partially reverses Nef down-regulation of CD4.

HIV Nef down-regulates CD4 from the cell surface in the absence of CD4 phosphorylation, whereas PMA down-regulates CD4 through a phosphorylation-dependent pathway. In this study we show that the down-regulation of CD4 in human Jurkat T cells expressing Nef was nearly complete (approximately 95%), whereas that induced by PMA was partial (approximately 40%). Unexpectedly, treating T cells expressing Nef with PMA restored the surface CD4 up to 35% of the steady state level. Both mutating the phosphorylation sites in the CD4 cytoplasmic tail (Ser408 and Ser415) and the use of a protein kinase C inhibitor, bisindolylmaleimide1, abolished the restoration of surface CD4, suggesting that the restoration required CD4 phosphorylation. CD4 and Nef could be cross-linked by a chemical cross-linker, 3,3-dithiobis[sulfosuccinimidyl-propionate], in control T cell membranes, but not in PMA-treated T cell membrane, suggesting that CD4 and Nef interacted with each other in T cells, and the phosphorylation disrupted the CD4-Nef interaction. We propose that this dissociation switches CD4 internalization from the Nef-mediated, nearly complete down-regulation to a phosphorylation-dependent, partial down-regulation, resulting in a net gain of CD4 on the T cell surface.

Lipid raft distribution of CD4 depends on its palmitoylation and association with Lck, and evidence for CD4-induced lipid raft aggregation as an additional mechanism to enhance CD3 signaling.

By mutagenesis, we demonstrated that the palmitoylation of the membrane-proximal Cys(396) and Cys(399)of CD4, and the association of CD4 with Lck contribute to the enrichment of CD4 in lipid rafts. Ab cross-linking of CD4 induces an extensive membrane patching on the T cell surface, which is related to lipid raft aggregation. The lipid raft localization of CD4 is critical for CD4 to induce the aggregation of lipid rafts. The localization of CD4 in lipid rafts also correlates to the ability of CD4 to enhance receptor tyrosine phosphorylation. Thus, our data suggest that CD4-induced aggregation of lipid rafts may play an additional role in CD4 signaling besides its adhesion to MHC molecules and association with Lck.

Fratricide of CD8+ cytotoxic T lymphocytes is dependent on cellular activation and perforin-mediated killing.

CD8+ CTL mediate the destruction of cells displaying foreign peptides in association with class I MHC molecules. Since CD8+ CTL themselves express class I MHC molecules, a phenomenon known as "fratricide" can be elicited by T cells presenting antigens to other CTL. To gain insight into this mechanism, fratricide was induced in a clone of class I-restricted CD8+ CTL by incubating the T cells with their agonist ligand, an octamer peptide derived from chicken ovalbumin. Our results indicate that agonist peptide not only stimulates proliferation and cytolysis of CTL but also initiates signaling pathways that are pertinent to T cell activation, including the mobilization of transcription factors. Also consistent with T cell activation, fratricide induced the transcription and translation of the pro-inflammatory cytokines TNF-alpha and IFN-gamma. Finally, the essential role of perforin, as opposed to Fas/FasL, in fratricide was demonstrated by the selective inhibition of cytolysis with an inhibitor of the perforin pathway, the absence of FasL expression on T cells and the presence of lytic granules visible by electron microscopy. Collectively, these findings reveal that fratricide is mediated by T cell activation and perforin-mediated cytolysis. These results may have implications for the regulation of CD8+ CTL in immune responses.