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OBJECTIVE: Mitochondria are found in the extracellular space in rheumatoid arthritis (RA). However, whether mitochondria are a source of autoantigens in RA has not been carefully addressed. Thus, we undertook this study to investigate the presence and significance of antimitochondrial antibodies (AMAs) in patients with RA. METHODS: AMAs were measured in serum samples from 3 cross-sectional cohorts of RA patients (n = 95, n = 192, and n = 117) and healthy individuals (n = 38, n = 72, and n = 50) using a flow cytometry-based assay. Further, AMAs were detected using an anti-mitofusin-1 (anti-MFN-1) IgG enzyme-linked immunosorbent assay and Western blot analysis. A longitudinal inception cohort, followed up for a median of 8 years, was used to study disease progression. RESULTS: AMA levels were elevated in RA patients from all 3 cohorts as compared to healthy individuals (P < 0.001, P < 0.05, and P < 0.01), with a range of 14-26% positivity. Levels of anti-MFN-1 antibodies correlated with AMA levels (r = 0.31, P = 0.006) and were elevated in RA patients as compared to healthy individuals (P < 0.001). The presence of AMAs was associated with erosive disease (P < 0.05) and interstitial lung disease (P < 0.01). Further, AMA levels were found to predict erosive disease (odds ratio [OR] 4.59, P = 0.006) and joint space narrowing (OR 3.08, P = 0.02) independent of anti-citrullinated protein antibodies. Finally, anti-MFN-1 antibodies identified seronegative patients developing erosive disease (OR 9.33; P = 0.02). CONCLUSION: Our findings demonstrate the presence of novel autoantibodies targeting mitochondria in the setting of RA. AMAs were used to stratify patients based on disease phenotype and to predict development of erosive disease, including in patients with seronegative disease. Our results highlight the essential role of mitochondria in the pathogenesis of RA and suggest a possible benefit of therapies targeting mitochondrial-mediated inflammation and clearance in these patients.
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Artritis Reumatoide , Humanos , Estudios Transversales , Autoanticuerpos , Anticuerpos Antiproteína Citrulinada , Ensayo de Inmunoadsorción Enzimática , Péptidos CíclicosRESUMEN
Diffuse alveolar hemorrhage (DAH), although rare, is a life-threatening complication of systemic lupus erythematosus (SLE). Little is known about the pathophysiology of DAH in humans, although increasingly neutrophils, NETosis and inflammatory monocytes have been shown to play an important role in the pristane-induced model of SLE which develops lung hemorrhage and recapitulates many of the pathologic features of human DAH. Using this experimental model, we asked whether endoplasmic reticulum (ER) stress played a role in driving the pathology of pulmonary hemorrhage and what role infiltrating neutrophils had in this process. Analysis of lung tissue from pristane-treated mice showed genes associated with ER stress and NETosis were increased in a time-dependent manner and reflected the timing of CD11b+Ly6G+ neutrophil accumulation in the lung. Using precision cut lung slices from untreated mice we observed that neutrophils isolated from the peritoneal cavity of pristane-treated mice could directly induce the expression of genes associated with ER stress, namely Chop and Bip. Mice which had myeloid-specific deletion of PAD4 were generated and treated with pristane to assess the involvement of PAD4 and PAD4-dependent NET formation in pristane-induced lung inflammation. Specific deletion of PAD4 in myeloid cells resulted in decreased expression of ER stress genes in the pristane model, with accompanying reduction in IFN-driven genes and pathology. Lastly, coculture experiments of human neutrophils and human lung epithelial cell line (BEAS-2b) showed neutrophils from SLE patients induced significantly more ER stress and interferon-stimulated genes in epithelial cells compared to healthy control neutrophils. These results support a pathogenic role of neutrophils and NETs in lung injury during pristane-induced DAH through the induction of ER stress response and suggest that overactivation of neutrophils in SLE and NETosis may underlie development of DAH.
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Células Epiteliales/inmunología , Trampas Extracelulares/inmunología , Hemorragia/inmunología , Neutrófilos/inmunología , Neumonía/inmunología , Alveolos Pulmonares/inmunología , Animales , Modelos Animales de Enfermedad , Células Epiteliales/patología , Femenino , Hemorragia/patología , Humanos , Lupus Eritematoso Sistémico/genética , Ratones , Ratones Endogámicos C57BL , Neutrófilos/patología , Neumonía/etiología , Neumonía/patología , Alveolos Pulmonares/patología , Terpenos/toxicidadRESUMEN
Type I interferon (IFN-I) is implicated in the pathogenesis of systemic lupus erythematosus (SLE) and the closely associated monogenic autoinflammatory disorders termed the "interferonopathies." Recently, the cytosolic DNA sensor cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) and its downstream signaling adaptor stimulator of interferon genes (STING) have been identified as having important, if not central, roles in driving IFN-I expression in response to self-DNA. This review highlights the many ways in which this pathway is regulated in order to prevent self-DNA recognition and underlines the importance of maintaining tight control in order to prevent autoimmune disease. We will discuss the murine and human studies that have implicated the cGAS-STING pathway as being an important contributor to breakdown in tolerance in SLE and highlight the potential therapeutic application of this knowledge for the treatment of SLE.
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Cardiac fibrosis, denoted by the deposition of extracellular matrix, manifests with a variety of diseases such as hypertension, diabetes, and myocardial infarction. Underlying this pathological extracellular matrix secretion is an expansion of fibroblasts. The mouse is now a common experimental model system for the study of cardiovascular remodeling and elucidation of fibroblast responses to cardiac growth and stress is vital for understanding disease processes. Here, using diverse but fibroblast specific markers, we report murine fibroblast distribution and proliferation in early postnatal, adult, and injured hearts. We find that perinatal fibroblasts and endothelial cells proliferate at similar rates. Furthermore, regardless of the injury model, fibroblast proliferation peaks within the first week after injury, a time window similar to the period of the inflammatory phase. In addition, fibroblast densities remain high weeks after the initial insult. These results provide detailed information regarding fibroblast distribution and proliferation in experimental methods of heart injury.
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Fibroblastos/patología , Corazón/crecimiento & desarrollo , Remodelación Ventricular , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colágeno/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Femenino , Fibroblastos/efectos de los fármacos , Corazón/efectos de los fármacos , Isoproterenol/farmacología , Masculino , Ratones Endogámicos C57BL , Infarto del Miocardio/patología , Presión , Receptores Adrenérgicos beta/metabolismo , Remodelación Ventricular/efectos de los fármacosRESUMEN
BACKGROUND: Reconstructing the evolutionary history of nervous systems requires an understanding of their architecture and development across diverse taxa. The spiralians encompass diverse body plans and organ systems, and within the spiralians, annelids exhibit a variety of morphologies, life histories, feeding modes and associated nervous systems, making them an ideal group for studying evolution of nervous systems. RESULTS: We describe nervous system development in the annelid Capitella teleta (Blake JA, Grassle JP, Eckelbarger KJ. Capitella teleta, a new species designation for the opportunistic and experimental Capitella sp. I, with a review of the literature for confirmed records. Zoosymposia. 2009;2:25-53) using whole-mount in situ hybridization for a synaptotagmin 1 homolog, nuclear stains, and cross-reactive antibodies against acetylated α-tubulin, 5-HT and FMRFamide. Capitella teleta is member of the Sedentaria (Struck TH, Paul C, Hill N, Hartmann S, Hosel C, Kube M, et al. Phylogenomic analyses unravel annelid evolution. Nature. 2011;471:95-8) and has an indirectly-developing, lecithotrophic larva. The nervous system of C. teleta shares many features with other annelids, including a brain and a ladder-like ventral nerve cord with five connectives, reiterated commissures, and pairs of peripheral nerves. Development of the nervous system begins with the first neurons differentiating in the brain, and follows a temporal order from central to peripheral and from anterior to posterior. Similar to other annelids, neurons with serotonin-like-immunoreactivity (5HT-LIR) and FMRFamide-like-immunoreactivity (FMRF-LIR) are found throughout the brain and ventral nerve cord. A small number of larval-specific neurons and neurites are present, but are visible only after the central nervous system begins to form. These larval neurons are not visible after metamorphosis while the rest of the nervous system is largely unchanged in juveniles. CONCLUSIONS: Most of the nervous system that forms during larvogenesis in C. teleta persists into the juvenile stage. The first neurons differentiate in the brain, which contrasts with the early formation of peripheral, larval-specific neurons found in some spiralian taxa with planktotrophic larvae. Our study provides a clear indication that certain shared features among annelids - e.g., five connectives in the ventral nerve cord - are only visible during larval stages in particular species, emphasizing the need to include developmental data in ancestral character state reconstructions. The data provided in this paper will serve as an important comparative reference for understanding evolution of nervous systems, and as a framework for future molecular studies of development.
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PURPOSE: This study examines the effect of component downsizing in a modern total knee arthroplasty (TKA) system on the laxity envelope of the knee throughout flexion. METHODS: A robotic testing system was utilized to measure laxity envelopes in the implanted knee by in the anterior-posterior (AP), medial-lateral (ML), internal-external (IE) and varus-valgus (VV) directions. Five fresh-frozen cadavers were tested with a modern cruciate retaining TKA implantation, a 1-mm thinner polyethylene insert and a femoral component 2 mm smaller in the AP dimension. RESULTS: The downsized tibial insert was more lax throughout the flexion arc with up to 2.0 mm more laxity in the AP direction at full extension, a 43.8% increase over the original implantation. A thinner insert consistently increased laxity throughout the arc of flexion in all degrees of freedom. Downsizing the femoral component resulted in 8.5 mm increase in AP laxity at 90°, a 73.9% increase. In mid-flexion, downsizing the femur produced similar laxity values to the downsized insert in AP, ML, IE and VV directions. CONCLUSION: Downsizing the TKA components had significant effects on laxity throughout flexion. Downsizing a femoral component 2 mm had an equivalent increase in laxity in mid-flexion as downsizing the tibial insert 1 mm. This study quantifies the importance of choosing the appropriate implant component size, having the appropriate size available and the effect of downsizing. The laxity of the implanted knee contributes to how the implant feels to the patient and ultimately the patient's satisfaction with their new knee.
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Artroplastia de Reemplazo de Rodilla/instrumentación , Fémur/cirugía , Inestabilidad de la Articulación/prevención & control , Prótesis de la Rodilla , Tibia/cirugía , Adulto , Artroplastia de Reemplazo de Rodilla/efectos adversos , Fenómenos Biomecánicos , Cadáver , Humanos , Inestabilidad de la Articulación/etiología , Articulación de la Rodilla/cirugía , Rango del Movimiento Articular , RobóticaRESUMEN
Redox control of protein function involves oxidation and reduction of amino acid residues, but the mechanisms and regulators involved are insufficiently understood. Here, we report that in conjunction with Mical proteins, methionine-R-sulfoxide reductase B1 (MsrB1) regulates mammalian actin assembly via stereoselective methionine oxidation and reduction in a reversible, site-specific manner. Two methionine residues in actin are specifically converted to methionine-R-sulfoxide by Mical1 and Mical2 and reduced back to methionine by selenoprotein MsrB1, supporting actin disassembly and assembly, respectively. Macrophages utilize this redox control during cellular activation by stimulating MsrB1 expression and activity as a part of innate immunity. We identified the regulatory role of MsrB1 as a Mical antagonist in orchestrating actin dynamics and macrophage function. More generally, our study shows that proteins can be regulated by reversible site-specific methionine-R-sulfoxidation.
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Actinas/metabolismo , Macrófagos/metabolismo , Metionina Sulfóxido Reductasas/genética , Metionina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Oxigenasas de Función Mixta/metabolismo , Oxidorreductasas/metabolismo , Animales , Células Cultivadas , Ratones , Ratones Noqueados , Proteínas de Microfilamentos , Oxidación-Reducción , Estrés Oxidativo , Oxidorreductasas/genéticaRESUMEN
The lichen cyanobacterial symbiont Nostoc sp. ATCC 53789 and its close relative Nostoc sp. GSV 224 are prolific producers of natural products, generating >25 derivatives of the cryptophycin class of secondary metabolites. Cryptophycin 1, the prototypic member of the class, is a potent tubulin-depolymerizing agent, and several semisynthetic derivatives are being developed as anticancer therapeutics. Here we provide a detailed characterization of the cryptophycin metabolic pathway by stable-isotope labeling experiments and through cloning, sequencing, and annotating the cryptophycin biosynthetic gene cluster. A comparative secondary metabolomic analysis based on polyketide (PK)/non-ribosomal peptide gene clusters from the phylogenetically related, non-cryptophycin producing cycad symbiont, Nostoc punctiforme ATCC 29133, was used to identify the cryptophycin biosynthetic genes that encompass approximately 40 kb within the lichen symbiont Nostoc sp. ATCC 53789 genome. The pathway encodes a collinear set of enzymes, including three modular PK synthases, two non-ribosomal peptide synthetase modules, and an integrated adenylation/ketoreductase didomain for elaboration of the leucic acid subunit. In addition, genes encoding key tailoring steps, including a FAD-dependent halogenase and CYP450 epoxidase, were identified. The inherent flexibility of the cryptophycin biosynthetic enzymes was harnessed to generate a suite of new analogues by altering the pool of PK starter units and selected amino acid extender groups. Characterization of the cryptophycin CYP450 enabled development of the first stereospecific synthesis of cryptophycin 2, through a tandem chemoenzymatic synthesis from the natural seco-cryptophycin 4 chain elongation intermediate.
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Antibióticos Antineoplásicos , Depsipéptidos , Familia de Multigenes , Nostoc/metabolismo , Péptido Sintasas/genética , Sintasas Poliquetidas/genética , Secuencia de Aminoácidos , Antibióticos Antineoplásicos/biosíntesis , Antibióticos Antineoplásicos/síntesis química , Antibióticos Antineoplásicos/química , Clonación Molecular , Depsipéptidos/biosíntesis , Depsipéptidos/síntesis química , Depsipéptidos/química , Depsipéptidos/genética , Datos de Secuencia Molecular , Nostoc/enzimología , Alineación de SecuenciaRESUMEN
BACKGROUND/AIMS: To examine if serum fibrosis biomarkers could accurately identify the stage of liver disease amongst hepatitis C (HCV) and HIV co-infected patients. METHODS: One hundred and thirty seven HIV/HCV co-infected persons were randomly selected from the Johns Hopkins HIV Clinic cohort. Ninety five had complete testing for fibrosis markers in sera collected at the time of liver biopsy. Biopsies were scored according to Ishak modified histological activity index (F0 no fibrosis to F6 cirrhosis). Fibrosis was evaluated against alanine aminotransferase (ALT), aspartate aminotransferase (AST), AST to platelet ratio (APRI), albumin, total bilirubin, hyaluronic acid (HA) and YKL-40. RESULTS: Sixty nine (73%) had no or minimal portal fibrosis (F0-2) and were compared with remaining subjects (F3-6). Fibrosis scores > or =F3 were found 27 times more often in persons with HA levels >86 ng/ml and 5.5 times more often in persons with HA levels 41-86 ng/ml. Less substantial associations were detected with levels of albumin <3.5 g/dl (OR 4.85) and AST >60 iu (OR 5.91). All 35 subjects who had favorable results of HA, albumin, and AST had minimal fibrosis (F0-2). CONCLUSIONS: Amongst HIV/HCV co-infected patients, serum testing for HA, albumin, and AST (SHASTA Index) was able to accurately stage mild and advanced fibrosis.
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Biomarcadores/sangre , Infecciones por VIH/complicaciones , Hepatitis C/complicaciones , Cirrosis Hepática/metabolismo , Cirrosis Hepática/virología , Adulto , Aspartato Aminotransferasas/sangre , Progresión de la Enfermedad , Femenino , Humanos , Ácido Hialurónico/sangre , Hígado/patología , Cirrosis Hepática/patología , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Sensibilidad y Especificidad , Albúmina Sérica/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
Cryptophycins-1 and 52 (epoxides) were discovered to have in-vitro and in-vivo antitumor activity in the early 1990s. The chlorohydrins of these, Cryptophycins-8 and 55 (also discovered in the early 1990s) were markedly more active, but could not be formulated as stable solutions. With no method to adequately stabilize the chlorohydrins at the time, Cryptophycin-52 (LY 355073) entered clinical trials, producing only marginal antitumor activity. Since that time, glycinate esters of the hydroxyl group of the chlorohydrins have been synthesized and found to provide stability. Three of the most active were compared herein. Cryptophycin-309 (C-309) is a glycinate ester of the chlorohydrin Cryptophycin-296. The glycinate derivative provided both chemical stability and improved aqueous solubility. After the examination of 81 different Cryptophycin analogs in tumor bearing animals, C-309 has emerged as superior to all others. The following %T/C and Log Kill (LK) values were obtained from a single course of IV treatment (Q2d x 5) against early staged SC transplantable tumors of mouse and human origin: Mam 17/Adr [a pgp (+) MDR tumor]: 0%T/C, 3.2 LK; Mam 16/C/Adr [a pgp (-) MDR tumor]: 0%T/C, 3.3 LK; Mam 16/C: 0%T/C, 3.8 LK; Colon 26: 0%T/C, 2.2 LK; Colon 51: 0%T/C, 2.4 LK; Pancreatic Ductal Adenocarcinoma 02 (Panc 02): 0%T/C, 2.4 LK; Human Colon HCT15 [a pgp (+) MDR tumor]: 0%T/C, 3.3 LK; Human Colon HCT116: 0%T/C, 4.1 LK. One additional analog, Cryptophycin-249 (C-249, the glycinate of Cryptophycin-8), also emerged with efficacy rivaling or superior to C-309. However, there was sufficient material for only a single C-249 trial in which a 4.0 LK was obtained against the multidrug resistant breast adenocarcinoma Mam-16/C/Adr. C-309 and C-249 are being considered as second-generation clinical candidates.
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Antineoplásicos/farmacología , Depsipéptidos/farmacología , Compuestos Epoxi/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Péptidos Cíclicos/farmacología , Animales , Antineoplásicos/química , Depsipéptidos/química , Ensayos de Selección de Medicamentos Antitumorales , Compuestos Epoxi/química , Ésteres , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Ratones SCID , Trasplante de Neoplasias , Péptidos Cíclicos/química , Relación Estructura-ActividadRESUMEN
Cryptophycin-38 (2), -326 (3), and -327 (4) are three new trace constituents of the terrestrial cyanobacterium Nostoc sp. GSV 224. Cryptophycin-38 is a stereoisomer of cryptophycin-1 (1) and to date is the only naturally occurring analogue that possesses a S,S epoxide group in unit A. Cryptophycin-327 is a geometric isomer that differs from 1 in having a cis Delta(2)-double bond in unit A. Cryptophycin-326 is related to cryptophycin-21, but has two chlorines ortho to the methoxy group in unit B. The relative and absolute stereochemistries of 2 have been related to known cryptophycins by semisynthesis and/or spectral analysis.
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Cianobacterias/química , Péptidos Cíclicos/química , Péptidos Cíclicos/aislamiento & purificación , Cristalografía por Rayos X , Depsipéptidos , Modelos Moleculares , Conformación Molecular , Resonancia Magnética Nuclear Biomolecular , EstereoisomerismoRESUMEN
Two new cytotoxins have been isolated from a species of marine cyanobacterium belonging to the genus Symploca that was collected in Guam. These new compounds, micromide (1) and guamamide (2), were accompanied by the known lipopeptides apramides A (3), B (4), and G (5). The planar structures of both alkaloids were elucidated by standard 2D NMR techniques, and the configurations of the amino acid-derived units in 1 were determined by chiral HPLC. The stereochemistry of the beta-methoxyhexanoic acid in 1 was determined by derivatization with methyl d-mandelate, after acid hydrolysis, and comparison with synthetic standards.
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Alcaloides/aislamiento & purificación , Cianobacterias/química , Lipoproteínas/aislamiento & purificación , Oligopéptidos/aislamiento & purificación , Alcaloides/química , Alcaloides/farmacología , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Ensayos de Selección de Medicamentos Antitumorales , Guam , Humanos , Concentración 50 Inhibidora , Células KB , Lipoproteínas/química , Lipoproteínas/farmacología , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Oligopéptidos/química , Oligopéptidos/farmacología , EstereoisomerismoRESUMEN
Nostocyclopeptides A1 and A2 are novel cyclic heptapeptides produced by the terrestrial cyanobacterium Nostoc sp. ATCC53789 that possess a unique imino linkage in the macrocyclic ring. Herein we report the cloning, sequencing, annotation, and biochemical analysis of the 33-kb nostocyclopeptide (ncp) biosynthetic gene cluster, which includes seven open reading frames predicted to be involved in the biosynthesis and transport of these natural products. The genetic architecture and domain organization of the ncpA-B nonribosomal peptide synthetase (NRPS) is co-linear in arrangement with respect to the putative order of the biosynthetic assembly of the cyclic peptide. A reductase domain identified at the C-terminal end of the NRPS NcpB is predicted to catalyze an NAD(P)H-mediated hydride transfer to the heptapeptidyl-S-enzyme intermediate NH(2)-Tyr-Gly-DGln-Ile-Ser-mPro-Leu/Phe-S-NRPS to yield a linear heptapeptide aldehyde that is subsequently captured intramolecularly with the amino group of the N-terminal amino acid residue tyrosine to form a stable imine bond. While a few C-terminal reductases associated with NRPSs have been identified, the ncp reductase is the first to mediate imine macrocyclization involving peptide N- and C-termini. Biochemical analysis of the NcpA1 and NcpB1 adenylation domains coupled with the recent characterization of the (2S,4S)-5-hydroxyleucine dehydrogenase NcpD, which is involved in the biosynthesis of the nonproteinogenic amino acid residue L-4-methylproline from L-leucine, support the involvement of this cluster in nostocyclopeptide biosynthesis.
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Cianobacterias/genética , Familia de Multigenes/genética , Péptido Sintasas/genética , Péptidos Cíclicos/biosíntesis , Clonación Molecular , Cianobacterias/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , Orden Génico , Genes Bacterianos/genética , Datos de Secuencia Molecular , Estructura Molecular , Péptidos Cíclicos/química , Análisis de Secuencia de ADNRESUMEN
Bioassay-guided fractionation of the extract of a species of Lyngbya from Palau has yielded Palau'amide (1), which had an IC(50) value of 13 nM against KB cells. The structure of 1 was elucidated by NMR analysis in a variety of solvents. In particular, a gradient-enhanced band-selective HMBC experiment allowed unambiguous assignment of HMBC correlations to carbon signals that were separated by 0.1 ppm. The relative stereochemistry and the absolute configurations of all but one (C-37) of the nine chiral centers were determined by NMR analysis of the alpha-methoxyphenylacetic acid (MPA) derivatives and chiral HPLC of the degradation products of 1. The assignment of S rather than R stereochemistry at C-37 is discussed in detail.
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Cianobacterias/química , Citotoxinas , Toxinas de Lyngbya , Péptidos Cíclicos , Citotoxinas/química , Citotoxinas/aislamiento & purificación , Citotoxinas/farmacología , Humanos , Concentración 50 Inhibidora , Células KB/efectos de los fármacos , Toxinas de Lyngbya/química , Toxinas de Lyngbya/aislamiento & purificación , Toxinas de Lyngbya/farmacología , Conformación Molecular , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Palau , Péptidos Cíclicos/química , Péptidos Cíclicos/aislamiento & purificación , Péptidos Cíclicos/farmacologíaRESUMEN
[structure: see text]. Tasihalides A and B have been isolated from an assemblage of a marine cyanobacterium, belonging to the genus Symploca, and an unidentified red alga. The gross structures and relative stereochemistries of these diterpenes were elucidated by spectroscopic means. In addition to possessing a novel cage structure, the tasihalides represent the only examples of iodinated diterpenes in nature.
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Cianobacterias/química , Diterpenos/química , Rhodophyta/química , Cromatografía , Cromatografía Líquida de Alta Presión , Diterpenos/análisis , Diterpenos/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Conformación Molecular , Estructura Molecular , Gel de Sílice , Dióxido de Silicio/química , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The structure of lyngbyastatin 3 (1), including the configurations of the two unusual amino acid residues, viz., the 3-amino-2-methylhexanoic acid (Amha) and 4-amino-2,2-dimethyl-3-oxopentanoic acid units (Ibu), has been established by chemical degradation. Analysis of the cyanobacterial samples of lyngbyastatin 3 (1), lyngbyastatin 1 (2), and dolastatin 12 (3) demonstrated that they are mixtures of Ibu epimers [R (major) and S (minor)], whereas the structurally related majusculamide C (4) is a single diastereomer having an S-Ibu unit.
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Cianobacterias/química , Depsipéptidos , Péptidos Cíclicos/química , Péptidos Cíclicos/aislamiento & purificación , Aminoácidos , Cromatografía Líquida de Alta Presión , Cromatografía de Gases y Espectrometría de Masas , Guam , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Oligopéptidos , EstereoisomerismoRESUMEN
The cloning, sequencing, annotation and biochemical analysis of the nostopeptolide (nos) biosynthetic gene cluster from the terrestrial cyanobacterium Nostoc sp. GSV224 is described. Nostopeptolides A1 and A2 are cyclic peptide-polyketide hybrid natural products possessing nine amino acid residues, a butyric acid group, and an internal acetate-derived unit that are linked by peptide and ester bonds. The nos gene cluster includes eight ORFs encompassing 40 kb and includes most of the genes predicted to be involved in the biosynthesis and transport of this group of nonapeptolides. The genetic architecture and domain organization of the nos synthetase, a mixed non-ribosomal peptide synthetase-polyketide synthase, is co-linear in arrangement with respect to the putative order of the biosynthetic assembly of the lipopeptolide. Biochemical analysis of the NosA1, NosC1 and NosD1 adenylation domains coupled with the recent characterization of the nosE and nosF gene products, which are involved in the biosynthesis of the rare non-proteinogenic amino acid residue L-4-methylproline from L-leucine, support the involvement of this gene cluster in nostopeptolide biosynthesis.
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Cianobacterias/genética , Familia de Multigenes/genética , Péptidos Cíclicos/biosíntesis , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cianobacterias/enzimología , Cianobacterias/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genes Bacterianos/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Péptido Sintasas/genética , Análisis de Secuencia de ADN , Especificidad por SustratoRESUMEN
A new cytotoxic peptide, which displayed an IC(50) value of 0.8 microM against KB cells, has been isolated from the marine cyanobacterium Symploca sp. The planar structure of tasiamide B (1), deduced by 2D NMR experiments, contains the unusual amino acid-derived residue 4-amino-3-hydroxy-5-phenylpentanoic acid (Ahppa). The configuration of 1 was deduced by HPLC analysis of degradation products.
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Cianobacterias/química , Oligopéptidos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Humanos , Concentración 50 Inhibidora , Células KB , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Oligopéptidos/química , PalauRESUMEN
Cryptophycin 52 is a synthetic derivative of Cryptophycin 1, a potent antimicrotubule agent isolated from cyanobacteria. In an effort to increase the potency and water solubility of the molecule, a structure-activity relationship study (SAR) was initiated around the phenyl ring of fragment A. These Cryptophycin 52 analogues were accessed using a Wittig olefination reaction between various triphenylphosphonium salts and a key intermediate aldehyde prepared from Cryptophycin 53. Substitution on the phenyl ring of fragment A was well tolerated, and several of these analogues were equally or more potent than Cryptophycin 52 when evaluated in vitro in the CCRF-CEM leukemia cell line and in vivo against a murine pancreatic adenocarcinoma.
Asunto(s)
Antineoplásicos/síntesis química , Depsipéptidos , Lactamas/síntesis química , Lactonas/síntesis química , Adenocarcinoma/tratamiento farmacológico , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Lactamas/química , Lactamas/farmacología , Lactonas/química , Lactonas/farmacología , Masculino , Ratones , Trasplante de Neoplasias , Neoplasias Pancreáticas/tratamiento farmacológico , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Re-collections of the cyanobacterium Lyngbya sp. have yielded two more members of the lyngbyapeptin and lyngbyabellin families. The gross structures of 15-norlyngbyapeptin A (1) and lyngbyabellin D (3) were deduced through standard 2D NMR techniques, with the absolute configuration of both elucidated through degradation and comparison with commercially available and synthetic standards. Degradation to the alpha-amino acid and NOE correlations determined the absolute and relative configuration of the 4-amino-3-hydroxy-5-methylhexanoic acid unit in 3. Lyngbyabellin D (3) displayed an IC(50) value of 0.1 microM against the KB cell line.