RESUMEN
BACKGROUND: Cases of women with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome developing leiomyomata are rare. A case with mitotically active leiomyomata has not previously been described to our knowledge. CASE: A 43-year-old woman with MRKH syndrome found to have an incidental pelvic mass on imaging studies underwent a diagnostic laparoscopy, followed by resection of leiomyomata and uterine remnant via mini laparotomy. Histopathology revealed focal infarction associated with a mitotically active area in one of the leiomyomata but with no evidence of marked cytologic atypia or hypercellularity. Focal adenomyosis was also noted. CONCLUSION: Studies have shown that mitotically active smooth cell tumors of the uterus having 5-9 mitoses/10 hpf and no cellular atypia have a metastatic rate too low to be regarded as sarcomas. Although the pathology findings in this case are benign with no need for continued surveillance by gynecologic oncology, regular follow-up with a gynecologist annually may be indicated for early diagnosis of recurrence secondary to the uncommon characteristics of this benign tumor, especially in this rare category of patients with Müllerian agenesis. Mitotically active leiomyomata can occur in patients with Müllerian agenesis, but the likelihood that a pelvic mass in a patient with MRKH syndrome is a sarcoma is extremely low.
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Trastornos del Desarrollo Sexual 46, XX/complicaciones , Leiomiomatosis/complicaciones , Conductos Paramesonéfricos/anomalías , Neoplasias Pélvicas/complicaciones , Adenomiosis/complicaciones , Adulto , Anomalías Congénitas , Femenino , Humanos , Laparoscopía , Leiomiomatosis/diagnóstico , Leiomiomatosis/cirugía , Neoplasias Pélvicas/diagnóstico , Neoplasias Pélvicas/cirugíaRESUMEN
We report the design, synthesis, and biological evaluation of imidazopyridine-based peptidomimetics based on the substrate consensus sequence of Akt, an AGC family serine/threonine kinase hyperactivated in over 50% of human tumors. Our ligand-based approach led to the identification of novel substrate mimetic inhibitors of Akt1 featuring an unnatural extended dipeptide surrogate. Compound 11 inhibits Akt isoforms in the sub-micromolar range and exhibits improved proteolytic stability relative to a parent pentapeptide.
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Peptidomiméticos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Piridinas/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Peptidomiméticos/síntesis química , Peptidomiméticos/química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piridinas/síntesis química , Piridinas/química , Relación Estructura-ActividadRESUMEN
Mcl-1, an anti-apoptotic member of the Bcl-2 protein family, is overexpressed in a broad range of human cancers and plays a critical role in conferring resistance to chemotherapy. In the course of screening a natural product-like library of sesquiterpenoid analogs, we identified substituted hexahydronaphthalenes that showed activity against the Mcl-1/BimBH3 interaction in vitro. Here, we describe the synthesis of a small library of analogs and their biological evaluation. The most potent inhibitor in the series (19) exhibits an IC(50) of 8.3 µM by ELISA and disrupts the interaction between endogenously expressed Mcl-1 and Bim in cultured MDA-MB-468 breast cancer cells.
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Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas de la Membrana/antagonistas & inhibidores , Naftalenos/síntesis química , Naftalenos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Antineoplásicos/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Ensayo de Inmunoadsorción Enzimática , Humanos , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Conformación Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Naftalenos/química , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
BACKGROUND: Reports on the usefulness of serum markers for predicting liver necroinflammation are limited. The aim of this study was to determine the serum markers that predict significant inflammation in patients with chronic hepatitis B (CHB) and C (CHC) and normal or mildly elevated serum aminotransferase levels. METHODS: Two hundred twenty-seven patients with CHB or CHC with normal or mildly elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (≤60 IU/L) were enrolled in this study. Significant inflammation was defined as inflammatory grade ≥3 activities using the Batt-Ludwig scoring system. The correlation between liver histology and serum markers of liver inflammation was analysed. RESULTS: Forty-eight (21.1%) and eight patients (3.5%) had grade 3 and 4 inflammation respectively. Univariate analysis revealed that age, platelet coun, and AST, ALT, γ-glutamyl transpeptidase, alkaline phosphatase, hyaluronic acid, haptoglobin, apolipoprotein A1 and procollagen III N-terminal peptide levels were significantly different between the patients with and without significant inflammation. There were no significant differences in the cytokeratin-18 fragment levels between the two groups. On the basis of multivariate analysis, the AST and apolipoprotein A1 levels and stage of fibrosis were highly predictive of significant inflammation. Using AST and apolipoprotein cut-off values ≥44 IU/L and ≤100 ng/ml, respectively, the presence of significant inflammation was predicted with high specificity (96.5%) and with a negative predictive value of 76.3%. CONCLUSION: The AST and apolipoprotein A1 levels were shown to be independent predictors of significant inflammatory activities in patients with CHB and CHC and normal or mildly elevated aminotransferase levels.
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Alanina Transaminasa/sangre , Aspartato Aminotransferasas/sangre , Pruebas Enzimáticas Clínicas , Hepatitis B Crónica/diagnóstico , Hepatitis C Crónica/diagnóstico , Mediadores de Inflamación/sangre , Hígado/enzimología , Adolescente , Adulto , Anciano , Apolipoproteína A-I/sangre , Biomarcadores/sangre , Biopsia , Distribución de Chi-Cuadrado , Femenino , Hepatitis B Crónica/sangre , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/patología , Hepatitis C Crónica/sangre , Hepatitis C Crónica/inmunología , Hepatitis C Crónica/patología , Humanos , Hígado/inmunología , Hígado/patología , Modelos Logísticos , Masculino , Persona de Mediana Edad , Necrosis , Valor Predictivo de las Pruebas , Estudios Prospectivos , República de Corea , Medición de Riesgo , Factores de Riesgo , Índice de Severidad de la Enfermedad , Regulación hacia Arriba , Adulto JovenRESUMEN
The chick anxiety-depression model is a hybrid simulation, which may prove useful as an early preclinical dual pharmacological screen for novel therapeutics. Separate dose-response studies were conducted with seven test compounds that have screened positive for antidepressant effects in rodent depression models and included prasterone (5.0-40.0 mg/kg), memantine (2.5-20.0 mg/kg), ketamine (1.0-10.0 mg/kg), mifepristone (50.0-400.0 mg/kg), DOV216,303 (5.0-20.0 mg/kg), CGP36742 (2.5-15.0 mg/kg), and antalarmin (1.0-30.0 mg/kg). Chicks aged 4-6 days posthatch received test compounds intramuscularly 15 min before social separation, in which distress vocalization rates were recorded. High rates of vocalization in the first phase (0-5 min) of social separation seem to model an anxiety-like state and lower rates of vocalization in the second phase (30-60 min) seem to model a depression-like state. Prasterone, memantine, ketamine, and DOV216,303 attenuated and CPG36742 enhanced the pattern of vocalizations in the first phase. Prasterone, ketamine, mifepristone, DOV216,303, and CPG36742 attenuated and memantine and antalarmin enhanced the pattern of vocalizations in the second phase. This pattern of drug effects parallels what clinical data exist, and highlights two important characteristics of this dual-screening assay. For the compounds tested, this chick model identified phase II and III clinical failures (e.g. memantine and antalarmin) and has the potential to reveal possible contraindications of compounds (i.e. CPG36742) in cases where anxiety symptoms are concomitant with a depressive episode.
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Antidepresivos/farmacología , Ansiedad/tratamiento farmacológico , Pollos , Depresión/tratamiento farmacológico , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Animales , Antidepresivos/uso terapéutico , Ensayos Clínicos como Asunto , Relación Dosis-Respuesta a Droga , Masculino , Vocalización Animal/efectos de los fármacosRESUMEN
Aiming to improve the potency and selectivity of scalarane sesterterpenoids, a series of natural and semisynthetic analogues, derived from the cytotoxic naturally abundant sesterterpene heteronemin (1), were evaluated for their in vitro antimicrobial and cytotoxic properties. The new sesterterpenes 16-O-methylsesterstatin 4 (6c), 17, 24-dihydroheteronemin (7a), 16, 25-deacetoxy-17, 24-dihydroheteronemin (7b), and 16-deacetoxy-25-methoxy-17, 24-dihydroheteronemin (7c) were structurally defined via physical data analyses. Scalarane sesterterpenes possessing an unsaturated 1,4-dialdehyde moiety showed potent inhibitory activity against methicillin-resistant Staphylococcus aureus at concentrations that are not significantly cytotoxic to mammalian cells. The structural features for the cytotoxicity of scalarane sesterterpenoids are discussed.
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Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Sesterterpenos/síntesis química , Sesterterpenos/farmacología , Terpenos/síntesis química , Terpenos/farmacología , Animales , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Complejo Mycobacterium avium/efectos de los fármacos , Poríferos/química , Sesterterpenos/química , Terpenos/químicaRESUMEN
A patient who presented with late stage HIV-associated diseases could not be diagnosed by commercial ELISA tests and a Western blot. However, we could amplify proviral DNA of HIV-1. We found a novel GPGGMI motif in the V3 loop, a novel insertion of a proline in the C3 region, and persistent deletion of two amino acids in the vif gene. The patient had been treated with HAART after diagnosis. Forty months after the first amplification of HIV-1 DNA, anti-HIV-1 antibody was confirmed by ELISA and Western blot and, thus, we amplified and sequenced HIV-1 full sequences. Interestingly, the sequence at the TATAA box was TAAAA, although full sequences were not CRF01_AE. The major differences in the level of the HIV-1 gene between the seronegative and seropositive states were changes at the glycosylation site (NXT) next to the inserted proline and many resistance mutations including M184V to antiretroviral drugs occurred. This is the first report on HIV-1 full sequences isolated from seronegative AIDS patients infected with subtype B in Korea.
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Síndrome de Inmunodeficiencia Adquirida/virología , VIH-1/genética , VIH-1/aislamiento & purificación , TATA Box/genética , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida/inmunología , Adulto , Terapia Antirretroviral Altamente Activa , Western Blotting , Farmacorresistencia Viral/genética , Ensayo de Inmunoadsorción Enzimática , Genes vif , Glicosilación , Anticuerpos Anti-VIH/sangre , Proteína gp120 de Envoltorio del VIH/genética , Seropositividad para VIH , VIH-1/clasificación , Humanos , Corea (Geográfico) , Masculino , Datos de Secuencia Molecular , Provirus/genética , Provirus/aislamiento & purificación , Análisis de Secuencia de ADN , Eliminación de SecuenciaRESUMEN
We have previously shown that many of the nef sequences from Korean HIV-1 subtype B carriers were grouped together in phylogenetic tree analyses. To determine whether this pattern was originated from either a single HIV-1-infected person or some biological pressure which directs the HIV-1 genomic mutation by the Korean specific immunological characters, we analyzed nef sequences from HIV-1-infected individuals with different time intervals. Thirty-two out of 46 analyzed patients formed a Korean monophyletic (KM) clade with 93% bootstrapping value. Eighteen patients' nef sequences were analyzed 1-9 years after first analysis. None of the patients shifted their clade from the first clustered clade (KM or non-KM), and all of the re-analyzed isolates were clustered close to the first analyzed clade. Isolates of the KM dade and non-KM clade in nef analysis showed the same pattern as in env analysis. Phylogenetic clustering evidences from both nef and C2/V3 trees strongly support the idea that introduction of the KM clade in subtype B strains originated from a common source. Thus, treatment and development of an AIDS vaccine may be somewhat easier than in other countries with multiple strains of HIV-1.
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Infecciones por VIH/virología , VIH-1/clasificación , Evolución Biológica , Femenino , Genes nef , Infecciones por VIH/epidemiología , Infecciones por VIH/transmisión , VIH-1/genética , Humanos , Corea (Geográfico)/epidemiología , Masculino , Filogenia , Homología de Secuencia de Ácido Nucleico , Conducta SexualRESUMEN
To establish a baseline for monitoring resistance mutation to protease inhibitors (PI), we determined protease(PR) sequences in peripheral blood mononuclear cells obtained from 43 PI-naive Korean HIV-1 infected patients. Interestingly, phylogenetic analysis revealed that 41 of the sequences belonged to subtype B, one belonged to subtype A, and one was unique, not clustering with any subtype. Thirty-one (76%) of the 41 sub-type B sequences formed a subclade within subtype B, a so-called "Korean B cluster." Polymorphisms were observed at 34 (34.3%) of the PR codons. One patient (2.3%) harbored a primary resistance-conferring mutation, L90M along with L63P and A71V, and all 43 strains showed some secondary associated with drug resistance. The percentage of patients with 7, 5, 4, 3, 2, and 1, resistance mutations were 2%, 2%, 14%, 23%,37%, and 9%, respectively. A novel polymorphism in subtype B, L63M was detected in two patients. Another patient showed a gross deletion (257 bp) after codon 91. The average genetic distance of the 41 subtype B sequences to the HXB2 sequence was 3.0% (range, 1.0-5.1%). Six hemophiliacs were infected with a domestic strain of HIV-1 subtype B, while the other two hemophiliacs were infected with nondomestic subtype B and had lived outside Korea. Although this is the first report on the molecular nature of PR in Korea, there is also a need to establish baselines for nonsubtype B HIV-1.
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Infecciones por VIH/virología , Proteasa del VIH/genética , VIH-1/clasificación , Polimorfismo Genético , Adolescente , Adulto , Niño , Femenino , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/epidemiología , Inhibidores de la Proteasa del VIH/uso terapéutico , VIH-1/enzimología , VIH-1/genética , Humanos , Corea (Geográfico)/epidemiología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADNRESUMEN
Most patients with ovarian cancer are diagnosed late in progression and often experience tumor recurrence and relapses due to drug resistance. Surface expression of matrix metalloprotease (MMP)-14 on ovarian cancer cells stimulates a tumor-stromal signaling pathway that promotes angiogenesis and tumor growth. In a cohort of 92 patients, we found that MMP-14 was increased in the serum of women with malignant ovarian tumors. Therefore, we investigated the preclinical efficacy of a MMP-14 monoclonal antibody that could inhibit the migratory and invasive properties of aggressive ovarian cancer cells in vitro. MMP-14 antibody disrupted ovarian tumor-stromal communication and was equivalent to Avastin in suppressing blood vessel growth in mice harboring Matrigel plugs. These effects on angiogenesis correlated with downregulation of several important angiogenic factors. Furthermore, mice with ovarian cancer tumors treated with anti-MMP-14 monotherapy showed a marked and sustained regression in tumor growth with decreased angiogenesis compared with immunoglobulin G (IgG)-treated controls. In a model of advanced peritoneal ovarian cancer, MMP-14-dependent invasion and metastasis was effectively inhibited by intraperitoneal administration of monoclonal MMP-14 antibody. Together, these studies provide a preclinical proof-of-concept for MMP-14 targeting as an adjuvant treatment strategy for advanced ovarian cancer.
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Anticuerpos Monoclonales/farmacología , Metaloproteinasa 14 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Neovascularización Patológica/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Carga Tumoral/efectos de los fármacos , Animales , Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos/farmacología , Biomarcadores de Tumor/sangre , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Docetaxel , Femenino , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Metaloproteinasa 14 de la Matriz/sangre , Metaloproteinasa 14 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz/administración & dosificación , Ratones , Ratones Desnudos , Invasividad Neoplásica , Metástasis de la Neoplasia , Neovascularización Patológica/genética , Neoplasias Ováricas/genética , Taxoides/farmacología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cooperativity with glucose is a key feature of human glucokinase (GK), allowing its crucial role as a glucose sensor in hepatic and pancreatic cells. We studied the changes in enzyme intrinsic tryptophan fluorescence induced by binding of different ligands to this monomeric enzyme using stopped-flow and equilibrium binding methods. Glucose binding data under pre-steady state conditions suggest that the free enzyme in solution is in a preexisting equilibrium between at least two conformers (super-open and open) which differ in their affinity for glucose (Kd* = 0.17 +/- 0.02 mM and Kd = 73 +/- 18 mM). Increasing the glucose concentration changes the ratio of the two conformers, thus yielding an apparent Kd of 3 mM (different from a Km of 7-10 mM). The rates of conformational transitions of free and GK complexed with sugar are slow and during catalysis are most likely affected by ATP binding, phosphate transfer, and product release steps to allow the kcat to be 60 s-1. The ATP analogue PNP-AMP binds to free GK (super-open) and GK-glucose (open) complexes with comparable affinities (Kd = 0.23 +/- 0.02 and 0.19 +/- 0.08 mM, respectively). However, cooperativity with PNP-AMP observed under equilibrium binding conditions in the presence of glucose (Hill slope of 1.6) is indicative of further complex tightening to the closed conformation. Another physiological modulator (inhibitor), palmitoyl-CoA, binds to GK with similar characteristics, suggesting that conformational changes induced upon ligand binding are not restricted by an active site ligand. In conclusion, our data support control of GK activity and Km through the ratio of distinct conformers (super-open, open, and closed) through either substrate or other ligand binding and/or dissociation.
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Glucoquinasa/metabolismo , Adenilil Imidodifosfato/metabolismo , Catálisis , Glucosa/metabolismo , Humanos , Cinética , Ligandos , Palmitoil Coenzima A/metabolismo , Unión Proteica , Conformación ProteicaRESUMEN
Dipeptidyl peptidase-IV (DPP-IV) is a serine protease with a signature Asp-His-Ser motif at the active site. Our pH data suggest that Gly-Pro-pNA cleavage catalyzed by DPP-IV is facilitated by an ionization of a residue with a pK of 7.2 +/- 0.1. By analogy to other serine proteases this pK is suggestive of His-Asp assisted Ser addition to the P1 carbonyl carbon of the substrate to form a tetrahedral intermediate. Solvent kinetic isotope effect studies yielded a D2Okcat/Km=2.9+/-0.2 and a D2Okcat=1.7+/-0.2 suggesting that kinetically significant proton transfers contribute to rate limitation during acyl intermediate formation (leaving group release) and hydrolysis. A "burst" of product release during pre steady-state Gly-Pro-pNA cleavage indicated rate limitation in the deacylation half-reaction. Nevertheless, the amplitude of the burst exceeded the enzyme concentration significantly (approximately 15-fold), which is consistent with a branching deacylation step. All of these data allowed us to better understand DPP-IV inhibition by saxagliptin (BMS-477118). We propose a two-step inhibition mechanism wherein an initial encounter complex is followed by covalent intermediate formation. Final inhibitory complex assembly (kon) depends upon the ionization of an enzyme residue with a pK of 6.2 +/- 0.1, and we assigned it to the catalytic His-Asp pair which enhances Ser nucleophilicity for covalent addition. An ionization with a pK of 7.9 +/- 0.2 likely reflects the P2 terminal amine of the inhibitor hydrogen bonding to Glu205/Glu206 in the enzyme active site. The formation of the covalent enzyme-inhibitor complex was reversible and dissociated with a koff of (5.5 +/- 0.4) x 10(-5) s(-1), thus yielding a Ki* (as koff/kon) of 0.35 nM, which is in good agreement with the value of 0.6 nM obtained from steady-state inhibition studies. Proton NMR spectra of DPP-IV showed a downfield resonance at 16.1 ppm. Two additional peaks in the 1H NMR spectra at 17.4 and 14.1 ppm were observed upon mixing the enzyme with saxagliptin. Fractionation factors (phi) of 0.6 and 0.5 for the 17.4 and 14.1 ppm peaks, respectively, are suggestive of short strong hydrogen bonds in the enzyme-inhibitor complex.
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Adamantano/análogos & derivados , Dipéptidos/química , Dipeptidil Peptidasa 4/química , Inhibidores Enzimáticos/química , Adamantano/química , Catálisis , Humanos , Concentración de Iones de Hidrógeno , Cinética , Resonancia Magnética Nuclear Biomolecular , SolventesRESUMEN
Dipeptidyl peptidase-IV is a cell surface protease which plays an important role in glucose homeostasis through proteolytic inactivation of incretin hormones, primarily glucagon like peptide-1 (GLP-1). Substrate N-terminal amino acid (S2-S1) specificity is rather clearly defined, while no substantial information is available on the significance of amino acid interactions towards the C-terminus after the scissile bond (so called prime S1'-S4' or distant S5'-S28' sites). In the present study the increasing length of the peptide towards prime sites (S1'-S4') resulted in approximately 7-fold decrease in Km. Moreover, the Km for GLP-1 cleavage was comparable to that of an S2-S4' peptide, suggesting that few, if any, important enzyme-substrate interactions occur beyond the active site. Effect of substrate length on kcat was less obvious, but kcat/Km showed an increasing trend when His-Ala-pNA (representing the natural two N-terminal residues) was compared to GLP-1. To probe the impact of increasing substrate length on the free energy of activation (as has been suggested for elastase and chymotrypsin) we performed temperature studies. To adequately interpret thermodynamic data we sought to understand what steps limit the kcat expression. Steady-state parameters of the reactions catalyzed by serine proteases are composed of microscopic constants describing binding, acylation, and deacylation steps. Viscosity and pre-steady-state studies suggested that His-Ala-pNA cleavage is limited in the deacylation half-reaction, most likely the product release step. Thus, the free energy of activation, as calculated from the Eyring equation, is underestimated (at least for His-Ala-pNA) and the effect of substrate length on the acylation step (and transition-state stabilization) could not be unambiguously assessed.
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Dipeptidil Peptidasa 4/química , Sitios de Unión , Unión Competitiva , Cromatografía Líquida de Alta Presión , Clonación Molecular , Glucagón/química , Péptido 1 Similar al Glucagón , Humanos , Hidrólisis , Cinética , Modelos Químicos , Fragmentos de Péptidos/química , Unión Proteica , Precursores de Proteínas/química , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Especificidad por Sustrato , Temperatura , Termodinámica , Factores de TiempoRESUMEN
It has been extremely difficult to elicit broadly cross-reactive neutralizing antibodies (Nabs) against human immunodeficiency virus type 1 (HIV-1). In this study, we compared the immunogenic properties of the wild-type and variable loop-deleted HIV-1 envelope glycoproteins. Mice were immunized with recombinant vaccinia viruses expressing either the wild-type or the variable loop-deleted (V1-2, V3, V4, and V1-3) HIV-1(DH12) gp160s. The animals were subsequently boosted with respective recombinant gp120s. All envelope constructs elicited similar levels of gp120-binding antibodies when analyzed by enzyme-linked immunosorbent assay (ELISA). However, the highest neutralizing activity was observed in sera from animals immunized with the wild-type envelope protein, followed by those immunized with DeltaV4 and DeltaV1-2. No neutralizing activity was detected in sera from animals immunized with DeltaV3 or DeltaV1-3. To identify immunogenic epitopes, ELISA was performed with overlapping 15-mer peptides that cover the entire length of gp120. For the wild-type gp120, the immunogenic epitopes mapped primarily to the variable loops V1-2 and to the conserved regions C1 and C5. When they were plotted onto known coordinates of gp120 core crystal structure, the epitopes in the conserved regions mapped predominantly to the inner domain of the protein. By immunizing with variable loop-deleted envelopes, the immune responses could be redirected to other regions of the protein. However, the newly targeted epitopes were neither on the exposed surface of the protein nor on the receptor binding regions. Interestingly, the removal of the V3 loop resulted in loss of immunoreactivity for both V3 and V1/V2 loops, suggesting structural interaction between the two regions. Our results suggest that obtaining broadly reactive Nabs may not be achieved simply by deleting the variable loops of gp120. However, the observation that the immune responses could be redirected by altering the protein composition might allow us to explore alternative strategies for modifying the antigenic properties of HIV-1 envelope glycoprotein.
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Anticuerpos Anti-VIH/biosíntesis , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Ensayo de Inmunoadsorción Enzimática , Epítopos , Proteína gp120 de Envoltorio del VIH/química , Inmunización , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Pruebas de NeutralizaciónRESUMEN
The etiological agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus SARS-CoV. Similar to other coronaviruses, spike (S)-glycoprotein of the virus interacts with a cellular receptor and mediates membrane fusion to allow viral entry into susceptible target cells. Accordingly, S-protein plays an important role in virus infection cycle and is the primary target of neutralizing antibodies. To begin to understand its biochemical and immunological properties, we expressed both full-length and ectodomain of the protein in various primate cells. Our results show that the protein has an electrophoretic mobility of about 160-170 kDa. The protein is glycosylated with high mannose and/or hybrid oligosaccharides, which account for approximately 30 kDa of the apparent protein mass. The detection of S-protein by immunoassays was difficult using human convalescent sera, suggesting that the protein may not elicit strong humoral immune response in virus-infected patients. We were able to pseudotype murine leukemia virus particles with S-protein and produce SARS pseudoviruses. Pseudoviruses infected Vero E6 cells in a pH-independent manner and the infection could be specifically inhibited by convalescent sera. Consistent with low levels of antibodies against S-protein, neutralizing activity was weak with 50% neutralization titers ranging between 1:15 to 1:25. To facilitate quantifying pseudovirus-infected cells, which are stained blue with X-Gal, we devised an automated procedure using an ELISPOT analyzer. The high-throughput capacity of this procedure and the safety of using SARS pseudoviruses should make possible large-scale analyses of neutralizing antibody responses against SARS-CoV.
Asunto(s)
Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/genética , Pruebas de Neutralización/métodos , Síndrome Respiratorio Agudo Grave/diagnóstico , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/aislamiento & purificación , Proteínas del Envoltorio Viral/análisis , Proteínas del Envoltorio Viral/genética , Animales , Anticuerpos Antivirales/análisis , Chlorocebus aethiops , Convalecencia , Vectores Genéticos , Glicosilación , Células HeLa , Humanos , Virus de la Leucemia Murina/genética , Manosa/química , Glicoproteínas de Membrana/química , Peso Molecular , Oligosacáridos/química , Virus Reordenados/aislamiento & purificación , Virus Reordenados/patogenicidad , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Sensibilidad y Especificidad , Síndrome Respiratorio Agudo Grave/sangre , Especificidad de la Especie , Glicoproteína de la Espiga del Coronavirus , Células Vero , Proteínas del Envoltorio Viral/químicaRESUMEN
The oncoprotein hdm2 ubiquitinates p53, resulting in the rapid degradation of p53 through the ubiquitin (Ub)-proteasome pathway. Hdm2-mediated destabilization and inactivation of p53 are thought to play a critical role in a number of human cancers. We have used an in vitro enzyme assay, monitoring hdm2-catalyzed Ub transfer from preconjugated Ub-Ubc4 to p53, to identify small molecule inhibitors of this enzyme. Three chemically distinct types of inhibitors were identified this way, each with potency in the micromolar range. All three types of compounds display selective inhibition of hdm2 E3 ligase activity, with little or no effect on other Ub-using enzymes. Most strikingly, these compounds do not inhibit the autoubiquitination activity of hdm2. Steady-state analysis reveals that all three classes behave as simple reversible inhibitors of the enzyme and that they are noncompetitive with respect to both substrates, Ub-Ubc4 and p53. Studies of the effects of combinations of two inhibitory molecules on hdm2 activity indicate that the three types of compounds bind in a mutually exclusive fashion, suggesting a common binding site on hdm2 for all of these inhibitors. These compounds establish the feasibility of selectively blocking hdm2-mediated ubiquitination of p53 by small molecule inhibitors. Selective inhibitors of hdm2 E3 ligase activity could provide a novel mechanism for the development of new chemotherapeutics for the treatment of human cancers.
Asunto(s)
Proteínas Nucleares , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas , Ubiquitina/metabolismo , Biotinilación , Proteínas de Unión al Calcio/metabolismo , Catálisis , Complejos de Clasificación Endosomal Requeridos para el Transporte , Humanos , Cinética , Ligasas/metabolismo , Modelos Biológicos , Ubiquitina-Proteína Ligasas Nedd4 , Péptido Sintasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2RESUMEN
An effective vaccine against the human immunodeficiency virus type 1 (HIV-1) will very likely have to elicit both cellular and humoral immune responses to control HIV-1 strains of diverse geographic and genetic origins. We have utilized a pathogenic chimeric simian-human immunodeficiency virus (SHIV) rhesus macaque animal model system to evaluate the protective efficacy of a vaccine regimen that uses recombinant vaccinia viruses expressing simian immunodeficiency virus (SIV) and HIV-1 structural proteins in combination with intact inactivated SIV and HIV-1 particles. Following virus challenge, control animals experienced a rapid and complete loss of CD4(+) T cells, sustained high viral loads, and developed clinical disease by 17 to 21 weeks. Although all of the vaccinated monkeys became infected, they displayed reduced postpeak viremia, had no significant loss of CD4(+) T cells, and have remained healthy for more than 15 months postinfection. CD8(+) T-cell and neutralizing antibody responses in vaccinated animals following challenge were demonstrable. Despite the control of disease, virus was readily isolated from the circulating peripheral blood mononuclear cells of all vaccinees at 22 weeks postchallenge, indicating that immunologic control was incomplete. Virus recovered from the animal with the lowest postchallenge viremia generated high virus loads and an irreversible loss of CD4(+) T-cell loss following its inoculation into a naïve animal. These results indicate that despite the protection from SHIV-induced disease, the vaccinated animals still harbored replication-competent and pathogenic virus.
Asunto(s)
Infecciones por VIH/prevención & control , Infecciones por VIH/virología , VIH-1/genética , Virus de la Inmunodeficiencia de los Simios/genética , Virus Vaccinia/genética , Vacunas Virales/administración & dosificación , Viremia/prevención & control , Animales , Anticuerpos Antivirales/biosíntesis , Linfocitos T CD8-positivos/inmunología , Línea Celular , Infecciones por VIH/inmunología , Depleción Linfocítica , Macaca mulatta , Pruebas de Neutralización , Recombinación Genética , Vacunas Virales/inmunologíaRESUMEN
Amyloid precursor protein (APP) cleaving enzyme (BACE) is the enzyme responsible for beta-site cleavage of APP, leading to the formation of the amyloid-beta peptide that is thought to be pathogenic in Alzheimer's disease (AD). Hence, BACE is an attractive pharmacological target, and numerous research groups have begun searching for potent and selective inhibitors of this enzyme as a potential mechanism for therapeutic intervention in AD. The mature enzyme is composed of a globular catalytic domain that is N-linked glycosylated in mammalian cells, a single transmembrane helix that anchors the enzyme to an intracellular membrane, and a short C-terminal domain that extends outside the phospholipid bilayer of the membrane. Here we have compared the substrate and active site-directed inhibitor binding properties of several recombinant constructs of human BACE. The constructs studied here address the importance of catalytic domain glycosylation state, inclusion of domains other than the catalytic domain, and incorporation into a membrane bilayer on the interactions of the enzyme active site with peptidic ligands. We find no significant differences in ligand binding properties among these various constructs. These data demonstrate that the nonglycosylated, soluble catalytic domain of BACE faithfully reflects the ligand binding properties of the full-length mature enzyme in its natural membrane environment. Thus, the use of the nonglycosylated, soluble catalytic domain of BACE is appropriate for studies aimed at understanding the determinants of ligand recognition by the enzyme active site.