Evaluation of the effect of natural peptide 'Urocortin' on corticotrophin releasing factor (CRF) receptor expression in ND7/23 cells

CRF receptors are involved in the stress management of the cells and are believed to have a cytoprotective role in the body. CRF receptors have been reported to be potential drug targets for the treatment of neurodegenerative disorders. The cell line used in the study is ND7/23 (mouse neuroblastoma and rat dorsal root ganglion neuron hybridoma). The aim of the study was to confirm the expression of CRF receptors in ND7/23 cells and to determine if urocortin (Ucn) can enhance the expression of CRF receptors. ND7/23 cells were cultured in RPMI 1640 media and cells grown after the second passage were used for the experiments. RNA was extracted from the cells and amplified by RT-PCR to confirm the presence of CRF receptors. The cells were then subjected to oxidative stress by hydrogen peroxide (0.00375%) and divided into two groups i.e. control and Ucn (10-8 μM) treated. Later RNA was extracted from both group of cells and PCR was performed. Finally, densitometry analysis was conducted on the agarose gel to determine the quantity of PCR product formed. PCR experiment confirmed the expression of both CRF-R1 and CRF-R2 in the cell line, but CRF-R1 was found to be expressed more strongly. Densitometry analysis of the PCR product and calculation of the relative expression of CRF receptors indicated a higher level of expression of CRF receptors in samples treated with Ucn as compared to those that were kept untreated. The results indicate that Ucn may be useful for the management of neuro-degenerative disorders and further studies may be carried out to establish its use as a therapeutic agent.

Receptores de CRF estão envolvidos na gestão do estresse das células e são acreditados para ter um papel de cito-proteção no organismo. Os receptores do CRF têm sido relatados como alvos potenciais de fármacos para o tratamento de doenças neurodegenerativas. A linhagem celular utilizada no estudo é ND7/23 (neuroblastoma de camundongo e hibridoma de raíz dorsal do neurônio ganglionar de rato). O objetivo do estudo foi confirmar o que a expressão de receptores de CRF em células ND7/23 determinar se urocortina (Ucn) pode aumentar a expressão de receptores de CRF. Cultivaram-se células ND7/23 em meio RPMI 1640 e as células que cresceram após a segunda passagem foram usadas para os experimentos. O RNA foi extraído células e amplificado por RT-PCR para confirmar a presença de receptores de CRF. As células foram, então, submetidas a estresse oxidativo por peróxido de hidrogênio (0.00375 %) e divididas em dois grupos, ou seja, controle e tratadas com UCN (10-8 µM). Em seguida, o RNA foi extraído de ambos os grupo de células e realizou-se o PCR. Finalmente, realizou-se análise densitométrica em gel de agarose para determinar a quantidade de produto formado por PCR. O PCR confirmou a expressão de CRF-R1 e CRF-R2 na linhagem celular, mas o CRF-R1 expresso mais fortemente. A análise densitométrica do produto de PCR e o cálculo da expressão relativa de receptores de CRF indicaram um nível mais elevado de expressão de receptores de CRF em amostras tratadas com Ucn, em comparação com aqueles sem tratamento. Os resultados indicam que a Ucn pode ser útil no tratamento de doenças neurodegenerativas e mais estudos podem ser realizados para estabelecer seu uso como agente terapêutico.

Comparative in vitro antibacterial analysis of different brands of cefixime against clinical isolates of Staphylococcus aureus and Escherichia coli.

The objective of this study was to compare the antibacterial activity of standard and different brands of Cefixime, against standard samples and clinical isolates of E. coli and S. aureus collected from different hospitals. Standard samples and isolates of E. coli and S. aureus were separately cultured in Mueller Hinton broth. After the bacterial incubation, 5 ml solution each of standard Cefixime and its different brands were added to the test tubes containing bacterial culture. Cefixime samples were added in the concentration of 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64 and 128μg/ml to separate test tubes. The cultures were again incubated and then the culture samples were analyzed by UV-spectrophotometer, and minimum inhibitory concentrations of all samples were determined. The analysis and interpretation of results were done by single factor ANOVA. An MIC of 0.75μg/ml and 8μg/ml of standard Cefixime was found for standard E. coli and S. aureous respectively. Standard Cefixime and its six selected brands exhibited a higher MIC range for clinical isolates of S. aureus than the clinical isolates of E. coli. Higher MIC values of standard Cefixime and its brands were observed for clinical isolates of E. coli and S. aureus. Higher MIC values for the clinical isolates of E. coli and S. aureus indicated that both the organisms have developed resistance to Cefixime in comparison to standard microorganisms acquired from ATCC.