RESUMEN
The issue of antibiotic resistance in pathogenic microbes is a global concern. This study was aimed to explore in silico and in vitro analysis of the antibacterial efficacy of different natural ligands against bacterial activity. The ligands included in the study were Propolis Neoflavanoide 1, Carvacrol, Cinnamaldehyde, Thymol, p-benzoquinone, and Ciprofloxacin (standard drug S*). The outcomes of molecular docking revealed that Propolis Neoflavaniode-1 showed a highly significant binding energy of - 7.1 and - 7.2 kcal/mol for the two gram-positive bacteria, as compared to the gram-negative bacteria. All ligands demonstrated acute toxicity (oral, dermal), except for Propolis Neoflavanoide 1 and S* drugs, with a confidence score range of 50-60%. Using a molecular dynamic simulation approach, we investigated Propolis Neoflavaniode-1's potential for therapeutic use in more detail. An MD simulation lasting 100 ns was performed using the Desmond Simulation software to examine the conformational stability and steady state of Propolis Neoflavaniode-1 in protein molecule complexes. Additionally, in vitro studies confirmed the antimicrobial activity of Propolis Neoflavaniode 1 by increasing the zone of inhibition against Gram-positive bacteria, p < 0.005 as compared to gram-negative bacteria. This study revealed the promising antibacterial efficacy of Propolis Neoflavaniode 1, demonstrated through robust in silico analyses, minimal toxicity, and confirmed in vitro antimicrobial activity, suggesting its potential as a viable alternative to combat antibiotic resistance.
RESUMEN
BACKGROUND: Anemia is a frequent complication of Crohn's disease (CD). The intestinal iron exporter ferroportin (FPN) is involved in both iron deficiency anemia and the anemia of chronic disease. To examine its role in CD, intestinal FPN expression was studied in subjects with and without CD. METHODS: Duodenal mucosal biopsies from 29 pediatric subjects with CD (n=19) and without CD (n=10) were obtained. FPN protein was measured using Western blot analysis and mRNA was assessed using quantitative real-time polymerase chain reaction (PCR). RESULTS: Intestinal FPN protein was higher in anemic CD subjects than in nonanemic CD subjects (P=0.01), while FPN mRNA levels were not different (P=0.66). In nonanemic CD subjects, erythrocyte sedimentation rate (ESR) (P=0.04), C-reactive protein (CRP) (P=0.03), and interleukin-6 (IL-6) (P=0.01) levels were elevated compared to controls. Nonanemic CD subjects had a lower median FPN protein than nonanemic controls, although it did not reach statistical significance (P=0.07). Median FPN mRNA was similar between groups (P=0.71). Although no correlation between FPN protein and IL-6 was noted, there was a strong negative correlation between serum iron and IL-6, both in subjects with CD (r=-0.88, P<0.0001) and those without anemia (r=-0.58, P=0.02). CONCLUSIONS: Intestinal FPN protein is upregulated in anemic CD subjects, suggesting that iron deficiency or anemia is the driving force regulating FPN levels. A transporter distinct from FPN appears to be involved in the hypoferremia associated with the inflammatory process of CD.
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Anemia Ferropénica/metabolismo , Proteínas de Transporte de Catión/metabolismo , Mucosa Intestinal/metabolismo , Adolescente , Adulto , Anemia Ferropénica/etiología , Anemia Ferropénica/genética , Western Blotting , Proteína C-Reactiva/metabolismo , Estudios de Casos y Controles , Proteínas de Transporte de Catión/genética , Niño , Enfermedad de Crohn/complicaciones , Enfermedad de Crohn/genética , Enfermedad de Crohn/metabolismo , Duodeno/metabolismo , Femenino , Humanos , Masculino , Pronóstico , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
BACKGROUND AND AIM: The regulation of human intestinal lactase-phlorizin hydrolase remains incompletely understood. One kb of pig and 2 kb of rat 5'-flanking sequence controls correct tissue, cell, topographic, and villus LCT expression. To gain insight into human LCT expression, transgenic mouse lines were generated from 3.3 kb of human LPH 5' flanking sequence from a lactase persistent individual fused to a human growth hormone (hGH) reporter bounded by an insulator. METHODS: Four lines were identified in which reporter expression was specifically detectable in the intestine and no other organ, two of which demonstrated hGH expression specific to small and large intestine. Quantitative RT-PCR was carried out on proximal to distal segments of small intestine at fetal days 16.5 and 18.5 and at birth, postnatal days 7 and 28 in line 22. RESULTS: In fetal intestine, hGH expression demonstrated a proximal to distal gradient similar to that in native intestine. There was no significant difference between hGH expression levels at 7 and 28 days in segment 3, the midpoint of the small intestine, where expression of endogenous lactase is maximal at 7 days and declines significantly by 28 days. Distal small intestine displayed high levels of hGH expression in enteroendocrine cells, which were shown to be a subset of the PYY cells. CONCLUSIONS: Thus, a 3.3-kb LPH 5' flanking sequence construct from a lactase persistent individual is able to maintain postnatal expression in transgenic mice post weaning.
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Región de Flanqueo 5'/genética , Intestino Delgado/enzimología , Lactasa-Florizina Hidrolasa/genética , Lactasa-Florizina Hidrolasa/metabolismo , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/metabolismo , Células Epiteliales/citología , Células Epiteliales/enzimología , Femenino , Feto/enzimología , Hormona del Crecimiento/genética , Hormona del Crecimiento/metabolismo , Humanos , Intestino Delgado/citología , Intestino Delgado/embriología , Masculino , Ratones , Ratones TransgénicosRESUMEN
BACKGROUND: Eph receptor tyrosine kinases EphB2 and EphB3, and ephrin-B1 ligand play a critical role in regulating small intestinal epithelial cell migration. Although well studied in developing brain, the expression pattern of Ephs/ephrins has not been delineated in the developing small intestine. AIMS: To examine the gene expression of all known members of Ephs/ephrins during development of mouse small intestine. METHODS: We examined the expression of 21 A- and B-Ephs/ephrins in mouse small intestine or the Caco-2 cell line using reverse-transcription polymerase chain reaction (RT-PCR), quantitative (q)RT-PCR, and immunohistochemical analyses. EphB2-expressing cells from isolated crypts were detected by immunofluorescence and fluorescence-activated cell sorting (FACS) analyses. RESULTS: With the exception of EphA5, all family members were expressed throughout the intestine at all ages examined. Most were uniformly expressed. In contrast, levels of EphA4, EphA8, EphB4, and ephrin-B2 messenger RNA (mRNA) were highest during early fetal development and declined with age. At E15, EphB2 and EphB4 proteins were diffusely expressed in proliferating stratified intestinal epithelial cells. By E18, the proteins had become localized to cell membranes of columnar epithelial cells within intervillus regions, and later were expressed on epithelial cell membranes in adult crypts. EphB2-expressing cells can be specifically isolated from crypt cell fractions. CONCLUSIONS: The current study represents the first analysis of Ephs/ephrins during intestinal development. The elevated expression of EphA4, EphA8, EphB4, and ephrin-B2 during the fetal period of intestinal morphogenesis suggests an important role in development. Continued intestinal expression of other family members implicates a role in differentiation.
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Efrinas/metabolismo , Intestino Delgado/metabolismo , Receptores de la Familia Eph/metabolismo , Factores de Edad , Animales , Células CACO-2 , Diferenciación Celular , Separación Celular , Efrinas/genética , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Humanos , Inmunohistoquímica , Intestino Delgado/embriología , Intestino Delgado/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Morfogénesis , ARN Mensajero/metabolismo , Receptores de la Familia Eph/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
RNA localization is a regulated component of gene expression of fundamental importance in development and differentiation. Several RNA binding proteins involved in RNA localization during development in Drosophila have been identified, of which Y14, Mago, Pumilio, and IMP-1 are known to be expressed in adult mammalian intestine. The present study was undertaken to define the developmental and regional expression of these proteins, as well as Staufen-1, in mouse intestinal cells and in other tissues and cell lines using RT-PCR, and localization using in situ hybridization and immunohistochemistry. Staufen-1, Y14, Mago-m, and Pumilio-1 were expressed in intestinal epithelial cells of both villus and crypt and in Caco-2 and IEC-6 cells. In contrast, expression of IMP-1 was age- and region-specific, showing clear expression in distal fetal and newborn intestine, but very low or no expression in adult. The mRNAs were cytosolic, with more apical than basal expression in enterocytes. Staufen protein showed a similar localization pattern to that of its cognate mRNA. Overall, the data suggest an essential role for these proteins in intestinal cells. Age and regional expression of IMP-1 may indicate a role in regulation of site-specific translation of intestinal genes or in RNA localization.
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Mucosa Intestinal/metabolismo , Transporte de ARN , ARN Mensajero/biosíntesis , Proteínas de Unión al ARN/biosíntesis , Animales , Animales Recién Nacidos , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Hibridación in Situ , Mucosa Intestinal/embriología , Mucosa Intestinal/crecimiento & desarrollo , Ratones , Especificidad de Órganos , Proteínas de Unión al ARN/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Plasmodium, the causative agent of malaria, has to undergo sexual differentiation and development in anopheline mosquitoes for transmission to occur. To isolate genes specifically induced in both organisms during the early stages of Plasmodium differentiation in the mosquito, two cDNA libraries were constructed, one enriched for sequences expressed in differentiating Plasmodium berghei ookinetes and another enriched for sequences expressed in Anopheles stephensi guts containing invading ookinetes and early oocysts. Sequencing of 457 ookinete library clones and 652 early oocyst clones represented 175 and 346 unique expressed sequence tags, respectively. Nine of 13 Plasmodium and four of the five Anopheles novel expressed sequence tags analyzed on Northern blots were induced during ookinete differentiation and mosquito gut invasion. Ancaspase-7, an Anopheles effector caspase, is proteolytically activated during Plasmodium invasion of the midgut. WARP, a gene encoding a Plasmodium surface protein with a von Willebrand factor A-like adhesive domain, is expressed only in ookinetes and early oocysts. An anti-WARP polyclonal antibody strongly inhibits (70-92%) Plasmodium development in the mosquito, making it a candidate antigen for transmission blocking vaccines. The present results and those of an accompanying report (Srinivasan, P., Abraham, E. G., Ghosh, A. K., Valenzuela, J., Ribeiro, J. M. C., Dimopoulos G., Kafatos, F. C., Adams, J. H., and Jacobs-Lorena, M. (2004) J. Biol. Chem. 279, 5581-5587) provide the foundation for further analysis of Plasmodium differentiation in the mosquito and of mosquito responses to the parasite.