Caracterización del polirribosilribitol fosfato por cromatografía de exclusión molecular de alta eficacia con detección ultravioleta / Characterization of polyribosylribitol phosphate by high efficiency size exclusion chromatography with ultraviolet detection

Haemophilus influenzae tipo b es un importante patógeno del hombre causante de varias de las enfermedades invasivas en niños menores de cinco años, contra el cual fueron autorizadas las vacunas glicoconjugadas a partir del polirribosilribitol fosfato. Quimi-Hib® es la primera y única vacuna contra este patógeno que utiliza el polisacárido obtenido por síntesis química. El Ingrediente Farmacéutico Activo es producido por el Centro de Ingeniería Genética y Biotecnología y se obtiene a partir de su conjugación al toxoide tetánico. En el presente reporte se hizo una caracterización del polirribosilribitol fosfato mediante la técnica de cromatografía de exclusión molecular de alta eficacia con detección ultravioleta a 215 nm. En el estudio se evaluaron tres lotes y se determinó el perfil de elución en una columna SuperdexTM 75 10/300 GL Increase con un porciento de pureza de 77,42 ± 8,97 y una masa molar promedio de 7.381 Da ± 210,93. La principal impureza presente en el polirribosilribitol fosfato es el dimetilsulfóxido, disolvente utilizado en la reacción de activación con el éster N-hidroxisuccinimidilo del ácido β-maleimidopropiónico. El polirribosilribitol fosfato se purificó por filtración con un Amicon Ultra-15 de 2.000 Da hasta una pureza de 99,1 por ciento y se conjugó al toxoide tetánico. El rendimiento de la reacción de conjugación con el polisacárido purificado fue de 30,0 por ciento 1,77 el cual no muestra diferencias significativas con el control que fue 33,7 por ciento ± 3,57 demostrándose que el dimetilsulfóxido no afecta el desempeño de la reacción de conjugación(AU)

Haemophilus influenzae type b is an important human pathogen causing some invasive diseases in children less than five years of age. Glycoconjugate vaccines based on polyribosylribitol phosphate have been licensed against this bacterium. Quimi-Hib® is the first and only vaccine against this pathogen using the chemically synthesized polysaccharide. The Active Pharmaceutical Ingredient is produced by the Center for Genetic Engineering and Biotechnology and is obtained from its conjugation to tetanus toxoid. In the present report a characterization of polyribosylribitol phosphate was performed by high performance molecular exclusion chromatography with ultraviolet detection at 215 nm. Three batches were evaluated in the study and the elution profile was determined on a SuperdexTM 75 10/300 GL Increase column with a purity percentage of 77.42 ± 8.97 and an average molecular weight of 7,381 Da ± 210.93. The main impurity present in polyribosylribitol phosphate was dimethylsulfoxide, the solvent used in the activation reaction with N-hydroxysuccinimidyl ester of β-maleimidopropionic acid. Polyribosylribitol phosphate was purified by filtration using a 2,000 Da cut-off Amicon Ultra-15 to a purity of 99.1 percent and conjugated to tetanus toxoid. The yield of the conjugation reaction with the purified polysaccharide was 30.0 percent ± 1.77 which shows no significant difference with the control which was 33.7 percent ± 3.57 demonstrating that dimethylsulfoxide does not affect the performance of the conjugation reaction(AU)

Effect of cryoprotectant on the motility, viability, fertilization, and DNA integrity of naleh fish Barbonymus sp. (Cyprinidae) sperm / Efeito do crioprotetor na motilidade, viabilidade, fertilização e integridade do DNA no sêmen de peixes naleh Barbonymus sp. (Cyprinidae)

Naleh fish Barbonymus sp. is a commercial freshwater fish, which is indigenous to Aceh, Indonesia. The population of this species has declined over the years as a result of habitat perturbations and overfishing. Hence, the crucial need to develop a cryopreservation method to support breeding programs. This involved the use of a cryoprotectant as an important component. The objective of this study, therefore, was to explore the best cryoprotectant for naleh fish spermatozoa, and a total of five types were tested. These include the DMSO, Methanol, Ethanol, Glycerol, and Ethylene Glycol at a similar concentration of 10%, which were individually combined with 15% egg yolk, and every treatment was performed in three replications. Conversely, Ringer's solution was adopted as an extender, and the sperm was cryopreserved in liquid nitrogen for 15 days. The results showed significant influence on sperm motility and viability, as well as egg fertility of naleh fish (P <0.05), although the DMSO provided the best outcome, compared to others at 47.17%, 50.13%, and 45.67%, respectively. Furthermore, DNA fragmentation had not occurred in the fresh and cryopreserved sperm samples, indicating the protective effect of tested cryoprotectants. It is concluded that the 10% DMSO and 15% egg yolk is the best cryoprotectant for naleh fish spermatozoa.(AU)

O peixe naleh Barbonymus sp. é um peixe comercial de água doce, originário de Aceh, Indonésia. Durante vários anos, as perturbações provocadas no seu habitat e a pesca predatória determinaram o declínio da sua população, cuja preservação deve apoiar-se em um programa de reprodução controlada, com o emprego de espermatozoides criopreservados. O presente trabalho realizou um estudo comparativo de cinco crioprotetores: dimetilsultóxido, metanol, etanol, glicerol e etileno glicol. Todos os crioprotetores foram testados na concentração de 10%, combinados a 15% de gema de ovo. Cada tratamento foi efetuado em triplicatas. A solução de ringer foi utilizada como extensor e o esperma foi criopreservado em nitrogênio líquido por 15 dias. Os resultados obtidos revelaram a existência de influência significante (P<0,05) na viabilidade e motilidade espermática bem como na fertilidade dos ovos do peixe naleh, em que o dimetilsulfóxido apresentou o melhor resultado com os valores de 47,17%, 50,13% e 45,67%, respectivamente. Por outro lado, a fragmentação do DNA não ocorreu nas amostras de esperma fresco e criopreservado, indicando o efeito protetor dos crioprotetores testados. A conclusão obtida foi que o dimetilsulfóxido e 15% de gema de ovo foram o melhor crioprotetor para os espermatozoides do peixe naleh.(AU)

Orthogonal assays for the identification of inhibitors of the single-stranded nucleic acid binding protein YB-1

We have previously shown that high expression of the nucleic acid binding factor YB-1 is strongly associated with poor prognosis in a variety of cancer types. The 3-dimensional protein structure of YB-1 has yet to be determined and its role in transcriptional regulation remains elusive. Drug targeting of transcription factors is often thought to be difficult and there are very few published high-throughput screening approaches. YB-1 predominantly binds to single-stranded nucleic acids, adding further difficulty to drug discovery. Therefore, we have developed two novel screening assays to detect compounds that interfere with the transcriptional activation properties of YB-1, both of which may be generalizable to screen for inhibitors of other nucleic acid binding molecules. The first approach is a cell-based luciferase reporter gene assay that measures the level of activation of a fragment of the promoter by YB-1. The second approach is a novel application of the AlphaScreen system, to detect interference of YB-1 interaction with a single-stranded DNA binding site. These complementary assays examine YB-1 binding to two discrete nucleic acid sequences using two different luminescent signal outputs and were employed sequentially to screen 7360 small molecule compounds leading to the identification of three putative YB-1 inhibitors.

Vitrification of bovine preantral follicles with dimethylsulfoxide and sucrose plus alfa-tocopherol / Vitrificação de folículos pré-antrais bovinos com dimetilxulfoxido e sacarose adicionado de alfa-tocopherol

The objective of this study was to evaluate the vitrification of bovine preantral follicles with dimethylsulfoxide (D) and sucrose (S) plus α-tocopherol 5mmol/L (T5) or 10mmol/L (T10) and, evaluate the thawed with minimal essential medium (m) with or without sucrose (s). Ovaries of cows were collected from slaughterhouse for the experiment I (n=66) and II (n=51). In the laboratory ovarian fragments were randomly assigned either to fresh control and 8 vitrification treatments (Controle and Dm; Dms, DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Ovarian fragments were placed in vitrification solution (5 min) and immersed in liquid nitrogen (-196°C), after a week, the fragments were thawed and analyzed. In the experiments I, preantral follicles were morphologically observed for histological evaluation, (normal; degenerated and developing of stage). In the experiment II, preantral follicles were mechanically isolated from ovarian tissue and examined with trypan blue, where dead and live corresponded to stained or non-stained. The treatments DSm, DSms and DST10m were effective in preserving the morphology in situ. However, the viability of isolated preantral follicles after vitrification remained high only in treatment DST10m. Thus, DST10m preserves survival rates and morphological integrity during vitrification of bovine preantral follicles.

Os objetivos deste estudo foram avaliar a vitrificação de folículos pré-antrais bovinos com dimetilsulfóxido (D) e sacarose (S) adicionando α-tocoferol 5mmol/L (T5) ou 10mmol/L (T10) e, avaliar o aquecimento com meio essencial mínimo (m) com ou sem sacarose (s). Ovários de fêmeas bovinas foram coletados de abatedouro, para o experimento I (n= 66) e II (n= 51). No laboratório fragmentos ovarianos foram distribuídos aleatoriamente para o controle fresco e 8 tratamentos de vitrificação (Controle e Dm; Dms, a DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Os fragmentos ovarianos foram colocados na solução de vitrificação (5 min) e imersos em nitrogênio líquido (-196°C). Após uma semana os fragmentos foram aquecidos e analisados. No experimento I, folículos pré-antrais foram observados morfologicamente para avaliação histológica (normal, degenerados e estádio de desenvolvimento). No experimento II, folículos pré-antrais foram mecanicamente isolados do tecido ovariano e examinados com o azul de trypan, observando mortos e vivos corados e não corados respetivamente. Os tratamentos a DSm, DSms e DST10m foram eficazes na preservação da morfologia in situ. No entanto, a viabilidade de folículos pré-antrais isolados após a vitrificação manteve-se elevada apenas no tratamento DST10m. Assim, DST10m preservou as taxas de sobrevivência e integridade morfológica durante a vitrificação de folículos pré-antrais bovinos.

Qingfei Xiaoyan Wan, a traditional Chinese medicine formula, ameliorates Pseudomonas aeruginosa-induced acute lung inflammation by regulation of PI3K/AKT and Ras/MAPK pathways

Gram-negative pathogen-induced nosocomial infections and resistance are a most serious menace to global public health. Qingfei Xiaoyan Wan (QF), a traditional Chinese medicine (TCM) formula, has been used clinically in China for the treatment of upper respiratory tract infections, acute or chronic bronchitis and pulmonary infection. In this study, the effects of QF on Pseudomonas aeruginosa-induced acute pneumonia in mice were evaluated. The mechanisms by which four typical anti-inflammatory ingredients from QF, arctigenin (ATG), cholic acid (CLA), chlorogenic acid (CGA) and sinapic acid (SPA), regulate anti-inflammatory signaling pathways and related targets were investigated using molecular biology and molecular docking techniques. The results showed that pretreatment with QF significantly inhibits the release of cytokines (TNF-α and IL-6) and chemokines (IL-8 and RANTES), reduces leukocytes recruitment into inflamed tissues and ameliorates pulmonary edema and necrosis. In addition, ATG was identified as the primary anti-inflammatory agent with action on the PI3K/AKT and Ras/MAPK pathways. CLA and CGA enhanced the actions of ATG and exhibited synergistic NF-κB inactivation effects possibly via the Ras/MAPK signaling pathway. Moreover, CLA is speculated to target FGFR and MEK firstly. Overall, QF regulated the PI3K/AKT and Ras/MAPK pathways to inhibit pathogenic bacterial infections effectively.

The antitumor effect of tanshinone IIA on anti-proliferation and decreasing VEGF/VEGFR2 expression on the human non-small cell lung cancer A549 cell line

The effects of tanshinone IIA on the proliferation of the human non-small cell lung cancer cell line A549 and its possible mechanism on the VEGF/VEGFR signal pathway were investigated. The exploration of the interaction between tanshinone IIA and its target proteins provides a feasible platform for studying the anticancer mechanism of active components of herbs. The CCK-8 assay was used to evaluate the proliferative activity of A549 cells treated with tanshinone IIA (2.5-80 μmol/L) for 24, 48 and 72 h, respectively. Flow cytometry was used for the detection of cell apoptosis and cell cycle perturbation. VEGF and VEGFR2 expression were studied by Western blotting. The binding mode of tanshinone IIA within the crystal structure of the VEGFR2 protein was evaluated with molecular docking analysis by use of the CDOCKER algorithm in Discovery Studio 2.1. The CCK-8 results showed that tanshinone IIA can significantly inhibit A549 cell proliferation in a dose- and time-dependent manner. Flow cytometry results showed that the apoptosis rate of tested group was higher than the vehicle control, and tanshinone IIA-treated cells accumulated at the S phase, which was higher than the vehicle control. Furthermore, the expression of VEGF and VEGFR2 was decreased in Western blot. Finally, molecular docking analysis revealed that tanshinone IIA could be stably docked into the kinase domain of VEGFR2 protein with its unique modes to form H-bonds with Cys917 and π-π stacking interactions with Val848. In conclusion, tanshinone IIA may suppress A549 proliferation, induce apoptosis and cell cycle arrest at the S phase. This drug may suppress angiogenesis by targeting the protein kinase domains of VEGF/VEGFR2.

Mouse embryonic hepatic stem cells differentiate into cardiomyocyte-like cells / 中国组织工程研究

BACKGROUND:In recent years, embryonic hepatic stem cel s have attracted more attention, but there are few reports on the potential of embryonic hepatic stem cel s to differentiate into cardiomyocyte-like cel s as wel as the related differentiation conditions. OBJECTIVE:To investigate the moderate condition to induce mice embryonic hepatic stem cel s to differentiate into cardiomyocyte-like cel s in vitro with chemical reagents. METHODS:Dimethylsulfoxide in combination with 5-azacytidine with different concentrations and time were used to induce the embryonic hepatic stem cel s of 13.5 days mice and to observe the differentiation effect. RESULTS AND CONCLUSION:Under in vitro conditions, 0.8%dimethylsulfoxide+5μmol/L 5-azacytidine could induce the mouse embryonic hepatic stem cel s to express the specific markers of myocardial cel s, while increasing the concentration of the inducer and extending the induction time could not improve the induction efficacy.

Comparação de métodos diretos de extração e quantificação dos teores de clorofilas em folhas do capim-Tifton 85 / Comparison of direct methods of extraction and chlorophyll contents quantification in leaves of Tifton 85 bermudagrass

O objetivo deste estudo foi verificar qual é o melhor protocolo para extração das clorofilas a, b e total (a + b) a ser aplicado em estudos com a gramínea forrageira Tifton 85 (Cynodon spp.). Os resultados revelaram que os melhores extratores, tomando-se como base os teores de clorofila total (a + b), foram o N,N-dimetilformamida, o dimetilsulfóxido e a acetona 80 por cento (com as equações propostas por ARNON, 1949). A variação temporal dos teores das clorofilas a, b e total ajustaram-se a um modelo hiperbólico comum a todos os solventes, sendo o período de 48 horas considerado suficiente para uma adequada extração.

This study aimed to assess which is the most appropriate protocol for extraction of chlorophylls a, b and total (a + b) to be applied in studies with Tifton 85 (Cynodon spp.) forage grass. Results revealed that, based on total (a + b) chlorophyll contents, the best extractors were N,N-dimethylformamide, dimethylsulfoxide and acetone 80 percent (using the equations proposed by ARNON, 1949). Temporal changes in the concentrations of chlorophyll a, b and total were well described by a hyperbolic model common to all solvents, being the period of 48 hours considered sufficient for an appropriate extraction.

Effect of sphingosine and phorbol-12-myristate-13-acetate on the growth and dimethylsulfoxide-induced differentiation in the insect trypanosomatid Herpetomonas samuelpessoai

We investigated the effect of two modulators of protein kinase C, sphingosine and phorbol-12-myristate-13-acetate (PMA), on the growth and dimethylsulfoxide (DMSO)-induced differentiation in Herpetomonas samuelpessoai. Sphingosine did not stimulate the transformation of undifferentiated-promastigotes in differentiated-paramastigotes. PMA alone or in association with DMSO increased the number of paramastigotes in comparison to control cells. DMSO inhibited the parasite growth (35 percent) and several unusual morphological features resembling aberrant cell division were observed. Sphingosine did not significantly reduce the growth in contrast to PMA. Collectively, our results demonstrated that the reduction of the proliferation translates in an increase of the differentiation rate in the insect trypanosomatid H. samuelpessoai.

Efecto del dimetilsulfóxido en la respuesta quimioluminiscente y el consumo de oxígeno de neutrófilos humanos activados / Effect of dimethylsulfoxide in the chemiluminescence answer and the consumption of the oxygen of active human neutrophils

El dimetil sulfóxido es un secuestrador inactivante del radical hidroxilo (úOH), e inhibe, de manera proporcional a la dosis, la quimioluminiscencia (QL) de luminol (QLU) y de lucigenina (QLC) en leucocitos polimorfonucleares neutrófilos (PMN) activados con estimulantes solubles y partículas de zimosán opsonizado (ZO). Los resultados indican la inhibición de la QLU en respuesta al ionóforo de calcio A23187 puede deberse al secuestro de úOH por DMSO, mientras que la inhibición de QLC sugiere que el DMSO afecta negativamente a la oxidasa de membrana de PMN. Ello se confirmó al observar el DMSO inhibió el consumo de O2 en PMN activados con FMLP y ZO. Cuando el DMSO se añadió luego de estimulación con FMLP y ZO, no hubo inhibición de la QLU, pero sí de la inducida por A23187. El labado de PMN expuestos a DMSO causó un incremento en la QLU en respuesta a la estimulació con FMLP y ZO. Ello es congruente con la hipótesis de que el DMSO interfiere con la activación de las subunidades de membrana de la oxidasa por las unidades reguladoras citoplasmáticas. Estos resultados implican que el DMSO puede inhibir la QL en fagocitos tanto mediante secuestro de úOH como por interferencia con la producción de superóxido por la oxidasa de membrana.

Dimethylsulfoxide (DMSO), a hydroxyl radical scavenger, exerted a dose dependent inhibition on the luminol and lucigenin-enhanced chemiluminiscent responses of human neutrophils activated with soluble and particulate stimulants. DMSO inhibition of the luminol chemiluminescence induced by calcium ionophore A23187 was probably due to .OH scavenging, whereas inhibition of the lucigenin chemiluminiscence suggested DMSO negatively affects the NADPH-dependent membrane oxidase of neutrophils. In agreement with this, DMSO moderately inhibited O2 consumption in PMN suspensions stimulated with chemotactic peptide and opsonized zymosan. DMSO inhibition of chemotactic peptide and opsonized zymosan-induced luminol chemiluminescence was observed only when added before or in conjunction with stimulants, whereas A23187-induced chemiluminescence was inhibited by DMSO regardless of time of addition. Washing of DMSO-treated PMN resulted in increased luminol enhanced chemiluminescence in response to chemotactic peptide and opsonized zymosan. This is consistent with the idea that DMSO may be interfering with activation of the membrane subunits of the oxidase by translocation and docking of the cytoplasmic, regulatory subunits...

Infusion-Related Toxicities of DMSO Containing Cryopreserved Blood / 대한수혈학회지

PURPOSE: We retrospectively evaluated infusion-related toxicities in the patients transfused with frozen-thawed blood mixed with dimethyl-sulfoxide (DMSO). METHODS: The incidence and severity of infusion-related toxicities in the 25 patients transfused with frozen-thawed blood containing hematopoietic stem cells mixed with 10% DMSO were compaired with those of other 18 patients (11 patients;transfused with ABO-compatible allogeneic marrow blood without DMSO, 7 patients;transfused with platelet concentrates which were frozen with 6% DMSO and washed after thawing to remove DMSO). RESULT: The median transfusion volume of blood containing DMSO was 280cc and that of blood without DMSO was 180cc. The infusion-related toxicities of blood containing DMSO were nausea (96%, 24/25), febrile sensation (88%, 22/25), vomiting (48%, 12/25), hematuria (32%, 8/25), dyspnea (16%, 4/25), convulsion (8%, 2/25), and sudden death (4%, 1/25). That of blood without DMSO was febrile sensation (5.6%, 1/18). The incidence of infusion-related toxicities of blood containing DMSO was significantly higher than that of blood without DMSO (p<0.05). CONCLUSION: It could be suggestive that the infusion-related toxicities of blood containing DMSO was occured more frequently and severely than those of blood without DMSO.

Effects of different membrane proteins and dimethylsulfoxide on neurite growth of cerebellum granule cells / 第三军医大学学报

Objective To observe the effects of different membrane proteins and dimethylsulfoxide on neurite outgrowth of cerebellum granule cells(CGC).Methods Membrane proteins were extracted from the liver,sciatic nerve and brain white matter of adult rats and coated on the cover slips.CGC were dissociated from newborn rats and inoculated on the coated cover slips,while dimethylsulfoxide(DMSO) was added into the CGC suspension.Results The neurite outgrowth was inhibited by membrane protein of brain white mater and the effect was concentration-dependent.Low concentration(

CRYOGENIC PRESERVATION OF ERYTHROCYTES USING DIMETHYLSULFOXIDE (DMSO) / 解放军医学杂志

Erythrocytes were frozen with DMSO of different concentrations (not over 20%) as a cryoprotec-tive agent in freezers of different temperatures.The rate of temperature drop was not controlled. Of the erythrocytes actually frozen, the average recovery rate after thawing was 91.3 per cent (range 87.5~ 95%). The average survival rate of erythrocytes stored at -20℃, -30℃, or -80℃ for up to eight months was 90.9 per cent (range 88.2~ 94.5). As compared with glycerol, which is in common use internationally, DMSO seems to be a better cryoprotective agent.

Protective effect of dimethylsulfoxide on axon degeneration / 第二军医大学学报

Objective:To investigate the protective effect of dimethylsulfoxide(DMSO) on axon degeneration.(Methods: Cultured) rat superior cervical ganglia were treated with DMSO(100%,10 ?l) per well to disconnect axons from the cell bodies.SCGs in DMSO control group were treated with a mixture of DMSO(10 ?l) and medium (2 ml) per well;in positive control group were transfected with herpes simplex virus over-expressing Wld~S protein and the cell body was eliminated;and in blank control group were treated with 10 ?l PBS.The separated axons were fixed with 4% poly formaldehyde at 0,4,8,12 and 24 h after treatment with DMSO for immunostaining with specific antibody to microtubulin.Thus,the changes of axonal structure were investigated.The axonal protein was collected and the degeneration of neurofilament was detected by Western Blotting.Results: In DMSO disconnected group,the axonal morphology and structure showed no obvious change at 12 h post disconnection,but slight degeneration was observed at 24 h post disconnection.The degradation of microtubulin was obviously slowed down and their axonal structures maintained intact 12 h later.The neurofilament could be detected 12 h after disconnection.The above changes in disconnected group were similar to those in positive control group.No obvious protective effects on axonal degeneration were observed in blank and DMSO control groups.Conclusion: Local high concentration of DMSO can delay axonal degeneration,which indicates that DMSO can be used for adjuvant treatment of neurodegenerative diseases.