Identification and characterization of starvation induced msdgc-1 promoter involved in the c-di-GMP turnover

C-di-GMP Bis-(3'-5')-cyclic-dimeric-guanosine monophosphate], a second messenger is involved in intracellular communication in the bacterial species. As a result several multi-cellular behaviors in both Gram-positive and Gram-negative bacteria are directly linked to the intracellular level of c-di-GMP. The cellular concentration of c-di-GMP is maintained by two opposing activities, diguanylate cyclase (DGC) and phosphodiesterase (PDE-A). In Mycobacterium smegmatis, a single bifunctional protein MSDGC-1 is responsible for the cellular concentration of c-di-GMP. A better understanding of the regulation of c-di-GMP at the genetic level is necessary to control the function of above two activities. In this work, we have characterized the promoter element present in msdgc-1 along with the + 1 transcription start site and identified the sigma factors that regulate the transcription of msdgc-1. Interestingly, msdgc-1 utilizes SigA during the initial phase of growth, whereas near the stationary phase SigB containing RNA polymerase takes over the expression of msdgc-1. We report here that the promoter activity of msdgc-1 increases during starvation or depletion of carbon source like glucose or glycerol. When msdgc-1 is deleted, the numbers of viable cells are similar to 10 times higher in the stationary phase in comparison to that of the wild type. We propose here that msdgc-1 is involved in the regulation of cell population density. (C) 2013 Elsevier B.V. All rights reserved.

Backbone-modified amphiphilic cyclic di- and tetrasaccharides

Synthesis of amphiphilic, cyclic di- and tetrasaccharides, which incorporate a methylene moiety at the inter-glycosidic bond, is reported. The amphiphilic properties of the new cyclic tetrasaccharide host were identified through assessing the solubilities of guests in aqueous and in organic solvents. The glycosidic bond stability of the cyclic tetrasaccharide under aqueous acidic condition was also verified.

An Unusual Ring-Contraction/Rearrangement Sequence for Making Functionalized Di- and Triquinanes

A novel ring contraction/rearrangement sequence leading to functionalized 2,8-oxymethano-bridged di- and triquinane compounds is observed in the reaction of various substituted 1-methyl-4-isopropenyl-6-oxabicylo3.2.1]octan-8-ones with Lewis acids. The reaction is novel and is unprecedented for the synthesis of di- and triquinane frameworks.

An Unusual Ring-Contraction/Rearrangement Sequence for Making Functionalized Di- and Triquinanes

A novel ring contraction/rearrangement sequence leading to functionalized 2,8-oxymethano-bridged di- and triquinane compounds is observed in the reaction of various substituted 1-methyl-4-isopropenyl-6-oxabicylo3.2.1]octan-8-ones with Lewis acids. The reaction is novel and is unprecedented for the synthesis of di- and triquinane frameworks.

An Unusual Ring-Contraction/Rearrangement Sequence for Making Functionalized Di- and Triquinanes

A novel ring contraction/rearrangement sequence leading to functionalized 2,8-oxymethano-bridged di- and triquinane compounds is observed in the reaction of various substituted 1-methyl-4-isopropenyl-6-oxabicylo3.2.1]octan-8-ones with Lewis acids. The reaction is novel and is unprecedented for the synthesis of di- and triquinane frameworks.

Glycosidic bond hydrolysis in septanosides: a comparison of mono-, di-, and 2-chloro-2-deoxy-septanosides

A kinetic study of the hydrolytic stabilities of mono-, di-, and 2-chloro-2-deoxy septanosides, under acid-catalysis, is reported herein. A comparison of mono-and diseptanosides, shows that the glycosidic bond in the disaccharide is more stable than the monosaccharide. Further the glycosidic bond at the reducing end hydrolyzes almost twice as faster than that of the non-reducing end of the disaccharide. 2-Chloro-2-deoxy septanoside is found to be the most stable and its glycosidic bond hydrolysis occurs at elevated temperatures only. The orientation of the exo-cyclic hydroxymethyl group and the inductive effect are suggested to play a role in the rates of hydrolysis. (C) 2014 Elsevier Ltd. All rights reserved.

Quorum Sensing and Biofilm Formation in Mycobacteria: Role of c-di-GMP and Methods to Study This Second Messenger

Bacteria have evolved to survive the ever-changing environment using intriguing mechanisms of quorum sensing (QS). Very often, QS facilitates formation of biofilm to help bacteria to persist longer and the formation of such biofilms is regulated by c-di-GMP. It is a well-known second messenger also found in mycobacteria. Several methods have been developed to study c-di-GMP signaling pathways in a variety of bacteria. In this review, we have attempted to highlight a connection between c-di-GMP and biofilm formation and QS in mycobacteria and several methods that have helped in better understanding of c-di-GMP signaling. (c) 2014 IUBMB Life, 66(12):823-834, 2014

Novel Functions of (p)ppGpp and Cyclic di-GMP in Mycobacterial Physiology Revealed by Phenotype Microarray Analysis of Wild-Type and Isogenic Strains of Mycobacterium smegmatis

The bacterial second messengers (p)ppGpp and bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulate important functions, such as transcription, virulence, biofilm formation, and quorum sensing. In mycobacteria, they regulate long-term survival during starvation, pathogenicity, and dormancy. Recently, a Pseudomonas aeruginosa strain lacking (p) ppGpp was shown to be sensitive to multiple classes of antibiotics and defective in biofilm formation. We were interested to find out whether Mycobacterium smegmatis strains lacking the gene for either (p)ppGpp synthesis (Delta rel(Msm)) or c-di-GMP synthesis (Delta dcpA) would display similar phenotypes. We used phenotype microarray technology to compare the growth of the wild-type and the knockout strains in the presence of several antibiotics. Surprisingly, the Delta rel(Msm) and Delta dcpA strains showed enhanced survival in the presence of many antibiotics, but they were defective in biofilm formation. These strains also displayed altered surface properties, like impaired sliding motility, rough colony morphology, and increased aggregation in liquid cultures. Biofilm formation and surface properties are associated with the presence of glycopeptidolipids (GPLs) in the cell walls of M. smegmatis. Thin-layer chromatography analysis of various cell wall fractions revealed that the levels of GPLs and polar lipids were reduced in the knockout strains. As a result, the cell walls of the knockout strains were significantly more hydrophobic than those of the wild type and the complemented strains. We hypothesize that reduced levels of GPLs and polar lipids may contribute to the antibiotic resistance shown by the knockout strains. Altogether, our data suggest that (p)ppGpp and c-di-GMP may be involved in the metabolism of glycopeptidolipids and polar lipids in M. smegmatis.

Synthesis and evaluation of 2,5-di(4-aryloylaryloxymethyl)-1,3,4-oxadiazoles as anti-cancer agents

A series of 2,5-di(4-aryloylaryloxymethyl)-1,3,4-oxadiazoles 9a-j were obtained via multistep synthesis from hydroxybenzophenones 4a-e. The cytotoxicity of compounds 9a-j was evaluated against human leukemia cell lilies (K562 and CEM). The compounds exhibited moderate to good anti-cancer activity with compounds 9b and 9i having a chloro group exhibiting the best activity (IC50 = 10 mu M). Compound 9i exhibited activity against both the cell lines and 9b only exhibited activity against CEM. Further, a lactate dehydrogenase (LDH) assay and DNA fragmentation studies of the compounds 9a-j were also performed. (C) 2013 Elsevier Masson SAS. All rights reserved.

Relative stabilities of condensed face sharing mono- and di-carboranes: CB20H18 and C2B19H18+

The relative energies of triangular face sharing condensed macro polyhedral carboranes: CB20H18 and C2B19H18+ derived from mono- and di-substitution of carbons in (4) B21H18- is calculated at B3LYP/6-31G* level. The relative energies, H center dot center dot center dot H non-bonding distances, NICS values, topological charge analysis and orbital overlap compatibility connotes the face sharing condensed macro polyhedral mono-carboranes, 8 (4-CB20H18) to be the lowest energy isomer. The di-carba- derivative, (36) 4,4'a-C2B19H18+ with carbons substituted in a different B-12 cage in (4) B21H18- in anti-fashion is the most stable isomer among 28 possibilities. This structure has less non-bonding H center dot center dot center dot H interaction and is in agreement with orbital-overlap compatibility, and these two have the pivotal role in deciding the stability of these clusters. An estimate of the inherent stability of these carboranes is made using near-isodesmic equations which show that CB20H18 (8) is in the realm of the possible. (C) 2015 Elsevier B.V. All rights reserved.

Regulation of Growth, Cell Shape, Cell Division, and Gene Expression by Second Messengers (p)ppGpp and Cyclic Di-GMP in Mycobacterium smegmatis

The alarmone (p)ppGpp regulates transcription, translation, replication, virulence, lipid synthesis, antibiotic sensitivity, biofilm formation, and other functions in bacteria. Signaling nucleotide cyclic di-GMP (c-di-GMP) regulates biofilm formation, motility, virulence, the cell cycle, and other functions. In Mycobacterium smegmatis, both (p) ppGpp and c-di-GMP are synthesized and degraded by bifunctional proteins Rel(Msm) and DcpA, encoded by rel(Msm) and dcpA genes, respectively. We have previously shown that the Delta rel(Msm) and Delta dcpA knockout strains are antibiotic resistant and defective in biofilm formation, show altered cell surface properties, and have reduced levels of glycopeptidolipids and polar lipids in their cell wall (K. R. Gupta, S. Kasetty, and D. Chatterji, Appl Environ Microbiol 81:2571-2578, 2015, http://dx.doi.org/10.1128/AEM.03999-14). In this work, we have explored the phenotypes that are affected by both (p) ppGpp and c-di-GMP in mycobacteria. We have shown that both (p) ppGpp and c-di-GMP are needed to maintain the proper growth rate under stress conditions such as carbon deprivation and cold shock. Scanning electron microscopy showed that low levels of these second messengers result in elongated cells, while high levels reduce the cell length and embed the cells in a biofilm-like matrix. Fluorescence microscopy revealed that the elongated Delta rel(Msm) and Delta dcpA cells are multinucleate, while transmission electron microscopy showed that the elongated cells are multiseptate. Gene expression analysis also showed that genes belonging to functional categories such as virulence, detoxification, lipid metabolism, and cell-wall-related processes were differentially expressed. Our results suggests that both (p) ppGpp and c-di-GMP affect some common phenotypes in M. smegmatis, thus raising a possibility of cross talk between these two second messengers in mycobacteria. IMPORTANCE Our work has expanded the horizon of (p) ppGpp and c-di-GMP signaling in Gram-positive bacteria. We have come across a novel observation that M. smegmatis needs (p) ppGpp and c-di-GMP for cold tolerance. We had previously shown that the Delta rel(Msm) and Delta dcpA strains are defective in biofilm formation. In this work, the overproduction of (p) ppGpp and c-di-GMP encased M. smegmatis in a biofilm-like matrix, which shows that both (p) ppGpp and c-di-GMP are needed for biofilm formation. The regulation of cell length and cell division by (p) ppGpp was known in mycobacteria, but our work shows that c-di-GMP also affects the cell size and cell division in mycobacteria. This is perhaps the first report of c-di-GMP regulating cell division in mycobacteria.

Efecto de distintos intervalos de mediciones de leche sobre la estimación de producción de leche total, repetibilidad y la forma de la curva de lactancia en un hato reyna

El presente trabajo se realizó con el objetivo de estudiar el efecto de cuatro intervalos de medición de leche (diario, cada 7, 14 y 28 días) sobre la producción de leche total, la repetibilidad y la forma de la curva de lactancia. Para lo cual se utilizaron los registros productivos y reproductivos del hato criollo Reyna de la Finca San José ubicada en Masatepe, Nicaragua. Se estudiaron 105 lactancia provenientes de 28 vacas durante el periodo de 1982 - 1990. Las características estudiadas fueron PLTOT, repetibilidad y la forma de la curva de lactancia. Todos los análisis estadísticos fueron realizados con el procedimiento de mínimos cuadrados y máxima verosimilitud establecido en el paquete estadístico LSMLMW diante el análisis de varianza se estudió el efecto de cuatro tratamientos (intervalos de medición) sobre la producción de leche total, obteniéndose las siguientes medias de mínimos cuadrados: 1,655.23±77.79 para el tratamiento 1 diciones diarias), 1,655.98±77.79 para el tratamiento 2 diciones cada 7 días), 1,644.64±77.79 para el tratamiento 3 (mediciones cada 14 días) y 1,669.21±77.79 para el tratamiento 4 (mediciones cada 28 días). Encontrándose un efecto no significativo de los diferentes intervalos de medición sobre la producción de leche total. Los valores encontrados para los indices de Repetibilidad para cada uno de los tratamientos fueron de: 0.46±0.11, 0.50±0.11, 0.48±0.11 y 0.50±0.11 para los tratamientos 1, 2, 3, 4 respectivamente Los valores para los parámetros y variables estudias en la curva de la lactancia fueron 3.2±2.5, 0.47±0.32, 0.01± 0.06, 51.81±29.10, 9.45±3.49 y 6.97±1.02 para a, b, c, Tp, Rp y S respectivamente Al estudiar el efecto de los tratamiento sobre las variables y parámetros de la curva de lactancia se determino que solamente la variable (S) que determina la persistencia fue afectada significativamente diferentes intervalos de medición. Analizando el efecto de los intervalos de medición de leche sobre la forma de la curva de lactancia se determinó que dicho efecto es no significativo debido a que la forma de la curva es similar para los cuatro intervalos estudiados.

Curva de lactancia para un hato criollo lechero reyna en condiciones de trópico seco

Con el objetivo de caracterizar la curva de lactancia y evaluar los efectos ambientales, año de parto (AAPAR), época de parto (E) y número de parto (NUMPA) asociados con los parámetros de la curva de lactancia, se analizaron registros mensuales (N=241) de vacas criollo Reyna bajo condiciones de trópico seco en la Hacienda "El Pino" Rivas, Nicaragua, los que corresponden a los años 1982-1989. Para la caracterización de la curva de lactancia, se utilizó el modelo propuesto por Wood (1967) de forma linealizada Ln Yx =Ln a + b Ln x - cx, donde Yx es la producción diaria en el periodo x, a representa la producción inicial, b el ascenso al pico, c descenso de producción. A partir del mismo se generaron la persistencia S, rendimiento al pico RP, tiempo al pico TP. La proporción de curvas atípicas encontradas en el presente estudio, fué de 23.21 . Al analizar la curva de lactancia general se observa un a = 1.94t +-0.l7 Kg, b =0.52+-0.04 Kg, c =0.006622+-0.000423 Kg, RP=6.13+-0.11 Kg, TP=89.17+-5.07 días y S = 7.78+-0.13; determinándose una curva sobreestimada producto de un valor para b sumamente grande. En el análisis de varianza se encontró que el factor época fue el que ejerció una mayor influencia sobre las características en estudio. Las medias de mínimos cuadrados y su error estándar según la época de parto, fueron de 2.25+-0.20 Kg, 0.50+-0.04 Kg, 0.007344+-0.0005 Kg para a, b y c respectivamente y para RP, TP y S de 6.38+-0.14 Kq, 67.64+-6.06 días y 7.49+-0.15 en la época 1 (Mayo-Octubre). En la época 2 (Noviembre-Abril) fueron de 1.63+-0.25 Kg para a, 0.52+-0.06 Kg para b, 0.005899+-0.0006 Kg para c y para RP, TP y s de 5.88+-0.17 Kg, l10.7l+-7.53 días y 8.07+-0.09 respectivamente. Los valores estimados para las correlaciones fenotípicas (rs) entre a y b, a y S resultaron de -0.71 y -0.75 respectivamente, y entre PL305 y a, PL305 y c, TP y s b y c, y entre b y S fueron de -0.00, -0.25, 0.66, 0.70 Y 0.84, respectivamente, presentando el resto de las estimaciones correlaciones cercanas a cero.

Caracterización de la curva de lactancia y el comportamiento reproductivo de vaquillas Holstein bajo implementación de medidas de manejo en el período pre-parto

Con el objetivo de caracterizar la curva de lactancia y evaluar el comportamiento reproductivo se analizaron los registros mensuales (N=10) de vaquillas Holstein bajo la implementación y falta de implementación de medidas de manejo pre-parto en la Empresa Agropecuaria “San Ignacio” Mateare, Nicaragua. Para caracterizar la curva de lactancia, se utilizó el modelo de Wood (1967) de forma linealizada Ln Yx = Lna + b Lnx - ex , donde Yx es la producción diaria en el período x, a representa la producción inicial, b el ascenso al pico, c el descenso de producción, a partir del mismo se generaron la Persistencia (S), Rendimiento al pico (RP), Tiempo al pico (TP), también se evaluó el Intervalo parto - concepción (IPC) y el Número de servicios por concepción (NSPC) . Al presentar problemas reproductivos la proporción de curvas atípicas encontradas en este Trabajo fue de 40%. Al analizar la curva de lactancia del grupo experimental se observa un a= 11.00212 kg, b = 0.083528 kg, e = 0.00407148 kg, RP = 12.790 kg, TP = 29.54 días, S = 6.126. El grupo testigo presenta un a = 6.4384 kg, b = 0.193048 kg, e = 0.00466974 kg, RP = 9.49 kg, TP = 34.32 días, S = 6.421. En el análisis de varianza se encontró que el RP tiene diferencia significativa entre los tratamientos a un nivel de significancia (P<0.05). Los parámetros reproductivos IPC y NSPC para el grupo experimental fueron de 243.6 días y 3 servicios respectivamente, el grupo testigo presentó un IPC = 221.8 días y NSPC = 3.6 servicios .

Determinación de los niveles críticos fósforo, potasio y curva de respuesta al nitrógeno, en el cultivo de tabaco (Nicotiana tabacum L)

Durante el cíclo agrícola 1990-1991 se realizaron 38 experimentos en las localidades de Jalapa, Condega y Estelí, con el objetivo de determinar el nivel crítico para el Fósforo y el Potasio, además de la curva de respuesta al Nitrógeno en el cultivo de tabaco. El diseño experimental usado fue el de ensayos dispersos, descritos en el boletín No. 11 de la FAO, en el cual los tratamientos evaluados fueron: 0-0-0, 90-45-0 y 90-45-60. Los mejores resultados obtenidos se dieron con las aplicaciones de 90 y 45 kg/ha de Nitrógeno y Fósforo, tanto para producción como para calidad. Aplicaciones de Potasio en suelos de contenido mayor que 1 meq de k/100 gr de suelo redujeron de manera no significativa los rendimientos y la calidad del tabaco.