ABSTRACT
Gram-negative bacteria pose a major threat to human health in an era fraught with multi-drug resistant bacterial infections. Despite extensive drug discovery campaigns over the past decades, no new antibiotic target class effective against gram-negative bacteria has become available to patients since the advent of the carbapenems in 1985. Antibiotic discovery efforts against gram-negative bacteria have been hampered by limited intracellular accumulation of xenobiotics, in large part due to the impermeable cell envelope comprising lipopolysaccharide (LPS) in the outer leaflet of the outer membrane, as well as a panoply of efflux pumps. The biosynthesis and transport of LPS are essential to the viability and virulence of most gram-negative bacteria. Thus, both LPS biosynthesis and transport are attractive pathways to target therapeutically. In this review, we summarize the LPS biosynthesis and transport pathways and discuss efforts to find small molecule inhibitors against targets within these pathways.
Subject(s)
Bacterial Outer Membrane Proteins , Bacterial Proteins , Humans , Bacterial Proteins/metabolism , Bacterial Outer Membrane Proteins/metabolism , Bacterial Outer Membrane Proteins/pharmacology , Lipopolysaccharides/pharmacology , Gram-Negative Bacteria/metabolism , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Anti-Bacterial Agents/metabolism , Drug Resistance, MultipleABSTRACT
This mini review describes the current status and challenges regarding institutionalisation of wastewater surveillance systems against COVID-19. Monitoring SARS-CoV-2 in wastewater has been proposed to be a potential tool to understand the actual prevalence of COVID-19 in the community, and it could be an effective approach to monitor the trend during the COVID-19 pandemic. However, challenges to institutionalise wastewater surveillance systems are still abundant and unfolding at a rapid rate given that the international understanding regarding the scientific knowledge and socio-political impacts of COVID-19 are in the developing stages. To better understand the existing challenges and bottlenecks, a comparative study between Japan, Viet Nam, and Indonesia was carried out in the present study. Through gaining a better understanding of common issues as well as issues specific to each country, we hope to contribute to building a robust multistakeholder system to monitor SARS-CoV-2 in wastewater as an effective disease surveillance system for COVID-19.
Subject(s)
COVID-19 , Pandemics , Wastewater , Humans , Indonesia , Japan , SARS-CoV-2 , VietnamABSTRACT
BACKGROUND: Breast cancer originates in breast epithelium and is associated with progressive molecular and morphologic changes. Women with atypical breast ductal epithelial cells have an increased relative risk of breast cancer. In this study, ductal lavage, a new procedure for collecting ductal cells with a microcatheter, was compared with nipple aspiration with regard to safety, tolerability, and the ability to detect abnormal breast epithelial cells. METHODS: Women at high risk for breast cancer who had nonsuspicious mammograms and clinical breast examinations underwent nipple aspiration followed by lavage of fluid-yielding ducts. All statistical tests were two-sided. RESULTS: The 507 women enrolled included 291 (57%) with a history of breast cancer and 199 (39%) with a 5-year Gail risk for breast cancer of 1.7% or more. Nipple aspirate fluid (NAF) samples were evaluated cytologically for 417 women, and ductal lavage samples were evaluated for 383 women. Adequate samples for diagnosis were collected from 111 (27%) and 299 (78%) women, respectively. A median of 13,500 epithelial cells per duct (range, 43-492,000 cells) was collected by ductal lavage compared with a median of 120 epithelial cells per breast (range, 10-74,300) collected by nipple aspiration. For ductal lavage, 92 (24%) subjects had abnormal cells that were mildly (17%) or markedly (6%) atypical or malignant (<1%). For NAF, corresponding percentages were 6%, 3%, and fewer than 1%. Ductal lavage detected abnormal intraductal breast cells 3.2 times more often than nipple aspiration (79 versus 25 breasts; McNemar's test, P<.001). No serious procedure-related adverse events were reported. CONCLUSIONS: Large numbers of ductal cells can be collected by ductal lavage to detect atypical cellular changes within the breast. Ductal lavage is a safe and well-tolerated procedure and is a more sensitive method of detecting cellular atypia than nipple aspiration.
Subject(s)
Breast Neoplasms/diagnosis , Breast/pathology , Adult , Aged , Aged, 80 and over , Biopsy, Needle , Breast Neoplasms/pathology , Cytodiagnosis , Female , Humans , Middle Aged , Prospective Studies , Therapeutic IrrigationABSTRACT
If detected early, breast cancer is curable. We tested cells collected from the breast ducts by methylation-specific PCR (MSP). Methylated alleles of Cyclin D2, RAR-beta, and Twist genes were frequently detected in fluid from mammary ducts containing endoscopically visualised carcinomas (17 cases of 20), and ductal carcinoma in situ (two of seven), but rarely in ductal lavage fluid from healthy ducts (five of 45). Two of the women with healthy mammograms whose ductal lavage fluid contained methylated markers and cytologically abnormal cells were subsequently diagnosed with breast cancer. Carrying out MSP in these fluid samples may provide a sensitive and powerful addition to mammographic screening for early detection of breast cancer.
Subject(s)
Breast Neoplasms/diagnosis , DNA Methylation , Polymerase Chain Reaction/methods , Breast/cytology , Breast Neoplasms/genetics , Female , Humans , Promoter Regions, Genetic , Sensitivity and Specificity , Therapeutic IrrigationABSTRACT
Small molecule natural products have aided in the discovery and characterization of many proteins critical to the progression and maintenance of the cell cycle. Identification of the direct target of a natural product gives scientists a tool to control a specific aspect of the cell cycle, thus facilitating the study of the cell-cycle machinery.
Subject(s)
Biological Products/pharmacology , Cell Cycle/drug effects , Animals , Apoptosis/drug effects , Apoptosis/physiology , Biological Products/chemistry , Biological Products/physiology , Cell Cycle/physiology , DNA Damage , Molecular Structure , Nucleic Acid Synthesis Inhibitors/chemistry , Nucleic Acid Synthesis Inhibitors/pharmacology , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
BACKGROUND: The marine natural product (+)-discodermolide has potent immunosuppressive activity. It inhibits proliferation of a wide range of human and murine cells, induces cell cycle arrest in the G2 or M phase and was recently shown to stabilize microtubules. Total synthesis of discodermolide has made it possible to generate variants of the compound to study its intracellular function in detail. RESULTS: We have determined that (+)-discodermolide arrests MG63 cells at M phase, and has a stabilizing effect on microtubules. In vitro studies show that discodermolide induces polymerization of purified tubulin in the absence of microtubule-associated proteins, and that it binds to tubulin dimers in microtubules at 1:1 stoichiometry. Discodermolide binds taxol-polymerized microtubules at near stoichiometric level, whereas taxol binds discodermolide-induced microtubules poorly. Competition data show that the binding of microtubules by discodermolide and taxol are mutually exclusive; discodermolide binds with higher affinity than taxol. The results of binding assays carried out in vivo or in cell lysates also suggest that the microtubule network is discodermolide's cellular target. CONCLUSIONS: (+)-Discodermolide causes cell cycle arrest at the metaphase-anaphase transition in mitosis, presumably due to its stabilizing effect on microtubules. In vitro, discodermolide polymerizes purified tubulin potently in the absence of MAPs. It binds microtubules at one molecule per tubulin dimer with a higher affinity than taxol, and the binding of microtubules by discodermolide and taxol are mutually exclusive. In total cell lysates discodermolide displays binding activity that is consistent with its effects on microtubules.
Subject(s)
Alkanes , Carbamates , Lactones/metabolism , Microtubules/metabolism , Mitosis/drug effects , Paclitaxel/antagonists & inhibitors , 3T3 Cells , Animals , Binding Sites , Cell Line , Humans , Lactones/pharmacology , Mice , Paclitaxel/metabolism , PyronesABSTRACT
BACKGROUND: Cell permeable ligands of low molecular weight can be used to dissect complex cellular processes. During the past several years this approach has been particularly important in the study of intracellular signal transduction. Discodermolide, a marine natural product, appears to inhibit a signaling pathway in immune cells. The structure of natural discodermolide is known, but its absolute stereochemistry is not. We set out to make both enantiomers and to investigate their biological activity. RESULTS: Both enantiomers of discodermolide were prepared by total synthesis. Surprisingly, both enantiomers have biological activity, and their effects seem to be distinct in that they arrest cells at different stages of the cell cycle. A specific binding activity was identified for (+)-discodermolide but not for (-)-discodermolide, and the binding of the two enantiomers was not competitive. CONCLUSIONS: Both enantiomers of discodermolide have antiproliferative activity, but they act by distinct mechanisms and appear to have distinct cellular targets. The natural product is the (+)-enantiomer, which blocks the cell cycle in the G2 or M phase. The (-)-enantiomer blocks cells in S phase. Both may be useful in studies of the regulation of the cell cycle; we have also identified a specific binding activity for (+)-discodermolide, and have provided evidence that it interacts with a functionally relevant receptor.
Subject(s)
Alkanes , Carbamates , Immunosuppressive Agents/metabolism , Immunosuppressive Agents/pharmacology , Lactones/metabolism , Lactones/pharmacology , Animals , Cell Cycle/drug effects , Cell Membrane Permeability/drug effects , Humans , Mice , Pyrones , Tumor Cells, CulturedABSTRACT
The immunosuppressants cyclosporin A (CsA) and FK506 appear to block T-cell function by inhibiting the calcium-regulated phosphatase calcineurin. While multiple distinct intracellular receptors for these drugs (cyclophilins and FKBPs, collectively immunophilins) have been characterized, the functionally active ones have not been discerned. We found that overexpression of cyclophilin A or B or FKBP12 increased T-cell sensitivity to CsA or FK506, respectively, demonstrating that they are able to mediate the inhibitory effects of their respective immunosuppressants in vivo. In contrast, cyclophilin C, FKBP13, and FKBP25 had no effect. Direct comparison of the Ki of each drug-immunophilin complex for calcineurin in vitro revealed that although calcineurin binding was clearly necessary, it was not sufficient to explain the in vivo activity of the immunophilin. Subcellular localization was shown also to play a role, since gene deletions of cyclophilins B and C which changed their intracellular locations altered their activities significantly. Cyclophilin B has been shown previously to be located within calcium-containing intracellular vesicles; its ability to mediate CsA inhibition implies that certain components of the signal transduction machinery are also spatially restricted within the cell.
Subject(s)
Amino Acid Isomerases/metabolism , Calmodulin-Binding Proteins/metabolism , Carrier Proteins/metabolism , Cyclosporine/pharmacology , Phosphoprotein Phosphatases/metabolism , T-Lymphocytes/physiology , Tacrolimus/pharmacology , Amino Acid Isomerases/ultrastructure , Base Sequence , Calcineurin , Carrier Proteins/ultrastructure , Cloning, Molecular , Humans , In Vitro Techniques , Oligodeoxyribonucleotides/chemistry , Peptidylprolyl Isomerase , Protein Sorting Signals , Recombinant Fusion Proteins , Signal Transduction/drug effects , Structure-Activity Relationship , Tacrolimus Binding Proteins , Tumor Cells, CulturedABSTRACT
Thrombin both stimulates phosphoinositide hydrolysis and inhibits adenylyl cyclase in a variety of cell types. Whether the cloned human platelet thrombin receptor accounts for both of these signaling events is unknown. We report that thrombin receptor agonist peptide causes both phosphoinositide hydrolysis and inhibition of adenylyl cyclase in naturally thrombin-responsive CCL-39 cells. To exclude the possibility that the agonist peptide or thrombin itself may activate these pathways via distinct receptors and to circumvent a lack of suitable thrombin receptor-null cells, we utilized a designed "enterokinase receptor," a thrombin receptor with its thrombin cleavage recognition sequence LDPR replaced by DDDDK, the enterokinase cleavage recognition sequence. Transfection of enterokinase-unresponsive cells with this construct conferred both enterokinase-sensitive phosphoinositide hydrolysis and inhibition of adenylyl cyclase. The phosphoinositide hydrolysis response was largely insensitive to pertussis toxin, whereas the adenylyl cyclase response was completely blocked by pertussis toxin. These data show that the cloned thrombin receptor can effect both phosphoinositide hydrolysis and inhibition of adenylyl cyclase via at least two distinct effectors, most likely Gq-like and Gi-like G-proteins.
Subject(s)
Adenylyl Cyclase Inhibitors , Cyclic AMP/metabolism , Enteropeptidase/metabolism , Phosphatidylinositols/metabolism , Receptors, Cell Surface/metabolism , Thrombin/pharmacology , Amino Acid Sequence , Animals , Blood Platelets/physiology , Cell Line , Cloning, Molecular , Humans , Hydrolysis , Molecular Sequence Data , Peptides/pharmacology , Rats , Receptors, Cell Surface/genetics , Receptors, Thrombin , Recombinant Proteins/metabolism , Thrombin/metabolism , TransfectionABSTRACT
The human platelet thrombin receptor is activated when thrombin cleaves its receptor's amino-terminal extension to reveal a new amino terminus that functions as a tethered peptide ligand. Exactly how this "agonist peptide domain" remains cryptic within the uncleaved receptor and becomes functional after receptor cleavage is unknown. In this report we define the structural features of the thrombin receptor's agonist peptide domain important for receptor activation. Studies with mutant thrombin receptors have suggested that agonist peptide domain residues 2-6 contained determinants critical for receptor activation, and the synthetic peptide SFLLR-NH2 representing the 1st 5 amino-terminal residues of the agonist peptide domain was sufficient to specify agonist activity. Acetylating or removing the agonist peptide's amino-terminal ammonium group greatly attenuated agonist activity. Agonist peptide residue Phe2 was vital for agonist function; residues Leu4 and Arg5 individually played less important roles. These structure-function relationships held for both platelet activation and activation of the cloned receptor expressed in transfected mammalian cells. Our studies suggest that structures at the extreme amino terminus of the thrombin receptor's agonist peptide domain, in particular the free ammonium group of Ser1 and the phenyl ring of Phe2, are critical for receptor activation and that the agonist function of this domain is expressed when receptor proteolysis unmasks such determinants. In addition to revealing details of the thrombin receptor's proteolytic triggering mechanism, these studies open avenues to the development of drugs targeting the thrombin receptor and to further definition for the role of the thrombin receptor in cellular regulation.
Subject(s)
Peptides/metabolism , Receptors, Cell Surface/metabolism , Thrombin/metabolism , Amino Acid Sequence , Animals , Cell Line , Humans , Hydrolysis , Molecular Sequence Data , Mutagenesis , Phosphatidylinositols/metabolism , Rats , Receptors, Cell Surface/genetics , Receptors, Thrombin , Structure-Activity Relationship , XenopusABSTRACT
The two subunits of the human class I histocompatibility antigen (HLA)-A2 have been expressed at high levels (20-30 mg/liter) as insoluble aggregates in bacterial cells. The aggregates were dissolved in 8 M urea and then refolded to form an HLA-A2-peptide complex by removal of urea in the presence of an antigenic peptide. Two peptides from the matrix protein and nucleoprotein of influenza virus are known to bind to HLA-A2, and both support the refolding of the recombinant HLA-A2 molecule. An additional peptide, a nonamer from the gp120 envelope protein of human immunodeficiency virus type 1, also supported refolding. Yields of purified recombinant HLA-A2 are 10-15%. In the absence of an HLA-A2-restricted peptide, a stable HLA-A2 complex was not formed. Monoclonal antibodies known to bind to native HLA-A2 also bound to the recombinant HLA-A2-peptide complex. Three purified HLA-A2-peptide complexes refolded from bacterially produced protein aggregates crystallize under the identical conditions as HLA-A2 purified from human lymphoblastoid cells. Crystals of the recombinant HLA-A2 molecule in complex with the influenza matrix nonamer peptide, Mp(58-66), diffract to greater than 1.5-A resolution.
Subject(s)
HLA-A2 Antigen/chemistry , Peptides/chemistry , Viral Proteins/chemistry , Amino Acid Sequence , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Base Sequence , Cloning, Molecular , Crystallization , Escherichia coli/genetics , HIV Envelope Protein gp120/chemistry , HLA-A2 Antigen/biosynthesis , HLA-A2 Antigen/genetics , Humans , Macromolecular Substances , Molecular Sequence Data , Nucleoproteins/chemistry , Oligodeoxyribonucleotides , Peptides/immunology , Plasmids , Polymerase Chain Reaction , Protein Binding , Protein Conformation , Protein Denaturation , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Restriction Mapping , Viral Matrix Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/immunologyABSTRACT
The abilities of FK506 and rapamycin to block distinct signal transduction pathways are mediated by soluble binding proteins. Previously, a family of these receptors has been recognized that includes a 25 kDa protein, FKBP25. We now report the isolation of a cDNA for FKBP25 from a human hippocampal cDNA library by oligonucleotide screening. The nucleotide sequence reveals an open reading frame that encodes a 224 amino acid polypeptide. Human FKBP25 shows 97% amino acid identity with bovine FKBP25 and 62% homology with human FKBP12.
Subject(s)
Carrier Proteins/genetics , Polyenes/metabolism , Tacrolimus/metabolism , Amino Acid Sequence , Animals , Base Sequence , Carrier Proteins/metabolism , Cattle , Cloning, Molecular , Gene Library , Humans , Immunosuppressive Agents/metabolism , Molecular Sequence Data , Molecular Weight , Oligodeoxyribonucleotides , Oligonucleotide Probes , Open Reading Frames , Sequence Homology, Nucleic Acid , Sirolimus , Tacrolimus Binding ProteinsABSTRACT
Platelet activation by thrombin is critical for hemostasis and thrombosis. Structure-function studies with a recently cloned platelet thrombin receptor suggest that a hirudin-like domain in the receptor's extracellular amino terminal extension is a thrombin-binding determinant important for receptor activation. We now report that a peptide antiserum to this domain is a potent and specific antagonist of thrombin-induced platelet activation. This study demonstrates that the cloned platelet thrombin receptor is necessary for platelet activation by thrombin, and provides a strategy for developing blocking monoclonal antibodies of potential therapeutic value.
Subject(s)
Platelet Activation/drug effects , Receptors, Cell Surface/physiology , Thrombin/pharmacology , Amino Acid Sequence , Animals , Female , Hirudins , Humans , Molecular Sequence Data , Receptors, Thrombin , Xenopus laevisABSTRACT
We recently isolated a cDNA clone encoding a functional platelet thrombin receptor that defined a unique mechanism of receptor activation. Thrombin cleaves its receptor's extracellular amino terminal extension, unmasking a new amino terminus that functions as a tethered peptide ligand and activates the receptor. A novel peptide mimicking this new amino terminus was a full agonist for platelet secretion and aggregation, suggesting that this unusual mechanism accounts for platelet activation by thrombin. Does this mechanism also mediate thrombin's assorted actions on non-platelet cells? We now report that the novel thrombin receptor agonist peptide reproduces thrombin-induced events (specifically, phosphoinositide hydrolysis and mitogenesis) in CCL-39 hamster lung fibroblasts, a naturally thrombin-responsive cell line. Moreover, these thrombin-induced events could be recapitulated in CV-1 cells, normally poorly responsive to thrombin, after transfection with human platelet thrombin receptor cDNA. Our data show that important thrombin-induced cellular events are mediated by the same unusual mechanism of receptor activation in both platelets and fibroblasts, very likely via the same or very similar receptors.
Subject(s)
Platelet Membrane Glycoproteins/metabolism , Receptors, Cell Surface/metabolism , Thrombin/metabolism , Amino Acid Sequence , Animals , Cell Division , Cell Line , Cloning, Molecular , Fibroblasts , Humans , Molecular Sequence Data , Oligopeptides/metabolism , Oligopeptides/pharmacology , Phosphatidylinositols/metabolism , Platelet Membrane Glycoproteins/chemistry , Receptors, Cell Surface/chemistry , Receptors, Thrombin , Thrombin/pharmacology , Transfection , Virulence Factors, Bordetella/pharmacologyABSTRACT
Platelet activation by thrombin plays a critical role in hemostasis and thrombosis. Based on structure-activity studies of a cloned platelet thrombin receptor, we designed two "mirror image" antagonists of thrombin and thrombin receptor function. First, "uncleavable" peptides mimicking the receptor domain postulated to interact with thrombin were found to be potent thrombin inhibitors. Second, proteolytically inactive mutant thrombins designed to bind but not cleave the thrombin receptor were found to be specific antagonists of receptor activation by thrombin. The effectiveness of these designed antagonists in blocking thrombin-induced platelet activation suggests a model for thrombin-receptor interaction and possible strategies for the development of novel antithrombotic agents.
Subject(s)
Platelet Activation/drug effects , Receptors, Cell Surface/drug effects , Thrombin/antagonists & inhibitors , Amino Acid Sequence , Binding Sites , Fibrinolytic Agents/pharmacology , Humans , Molecular Sequence Data , Mutation , Receptors, Thrombin , Recombinant Proteins/pharmacology , Structure-Activity Relationship , Thrombin/metabolismABSTRACT
Platelet activation by the coagulation protease thrombin is central to arterial thrombosis, a major cause of morbidity and mortality. We recently isolated a complementary DNA encoding the platelet thrombin receptor. The extracellular amino-terminal extension of this seven transmembrane domain receptor contains the putative thrombin cleavage site LDPR/S which is critical for receptor activation. By replacing this cleavage site with the cleavage site for enterokinase, we have created a functional enterokinase receptor. This result demonstrates that all information necessary for receptor activation is provided by receptor proteolysis. Nanomolar enterokinase concentrations are required to activate this new receptor, in contrast to the picomolar thrombin concentrations that activate wild-type thrombin receptor. We identified a receptor domain critical for thrombin's remarkable potency at its receptor. This domain resembles the carboxyl tail of the leech anticoagulant hirudin and functions by binding to thrombin's anion-binding exosite. Our studies thus define a model for thrombin-receptor interaction. The utility of this model was demonstrated by the design of novel thrombin inhibitors based on receptor peptides.
Subject(s)
Receptors, Cell Surface/chemistry , Thrombin/metabolism , Amino Acid Sequence , Animals , Anions , Binding Sites , Gene Expression , Hirudins/chemistry , Humans , Molecular Sequence Data , Mutagenesis , Oocytes/metabolism , RNA/genetics , RNA, Complementary , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Receptors, Thrombin , Thrombin/pharmacology , XenopusABSTRACT
We isolated a cDNA encoding a functional human thrombin receptor by direct expression cloning in Xenopus oocytes. mRNA encoding this receptor was detected in human platelets and vascular endothelial cells. The deduced amino acid sequence revealed a new member of the seven transmembrane domain receptor family with a large amino-terminal extracellular extension containing a remarkable feature. A putative thrombin cleavage site (LDPR/S) resembling the activation cleavage site in the zymogen protein C (LDPR/I) was noted 41 amino acids carboxyl to the receptor's start methionine. A peptide mimicking the new amino terminus created by cleavage at R41 was a potent agonist for both thrombin receptor activation and platelet activation. "Uncleavable" mutant thrombin receptors failed to respond to thrombin but were responsive to the new amino-terminal peptide. These data reveal a novel signaling mechanism in which thrombin cleaves its receptor's amino-terminal extension to create a new receptor amino terminus that functions as a tethered ligand and activates the receptor.