Short hairpin RNA targeting insulin-like growth factor binding protein-3 restores the bioavailability of insulin-like growth factor-1 in diabetic rats

ABSTRACT Purpose To investigate whether intracavernosal injection of short hairpin RNA for IGFBP-3 could improve erectile function in streptozotocin-induced diabetic rats. Materials and methods After 12 weeks of IGFBP-3 short hairpin RNA injection treatment, intracavernous pressure responses to electrical stimulation of cavernous nerves were evaluated. The expression of IGFBP-3 and IGF-1 at mRNA and protein levels were detected by quantitative real-time PCR analysis and Western blot, respectively. The concentration of cavernous cyclic guanosine monophosphate was detected by enzyme-linked immunosorbent assay. Results At 12 weeks after intracavernous administration of IGFBP-3 shRNA, the cavernosal pressure was significantly increased in response to the cavernous nerves stimulation compared to the diabetic group (P<0.05). Cavernous IGFBP-3 expression at both mRNA and protein levels was significantly inhibited. At the same time, cavernous IGF-1 expression was significantly increased in the IGFBP-3 shRNA treatment group compared to the diabetic group (P<0.01). Cavernous cyclic guanosine monophosphate concentration was significantly increased in the IGFBP-3 shRNA treatment group compared to the diabetic group (P<0.01). Conclusions Gene transfer of IGFBP-3 shRNA could improve erectile function via the restoration of cavernous IGF-1 bioavailability and an increase of cavernous cGMP concentration in the pathogenesis of erectile dysfunction in streptozotocin-induced diabetic rats.

Construction and application of a yeast expression system for thymosin a1

We want to construct a yeast expression system for thymosin a1 (Ta1) to make the orally administered Ta1 preparation possible. The whole Ta1 DNA fragment was obtained by PCR. After being digested with restriction enzymes, it was cloned into pYES2 vector. Sequencing was performed to identify the recombinant. The sequence of Ta1 in recombinant coincided with the original one reported in Genbank. When pYES2-Ta1 plasmid was transformed into yeast, galactose instead of glucose was used to induce Ta1 expression. Western blot was performed to identify the quality of the expressed Ta1. Dried yeast containing pYEST2-Ta1 was fed to Balb/c mice whose immunities were inhibited by cyclophosphamide in advance. Synthesized Ta1 peptide was used as positive control and empty yeast was used as negative control. Compared with the negative control group, both dried yeast containing pYEST2-Ta1 and synthesized Ta1 peptide can significantly increase the CD8+ level (22.74±1.09 and 18.77±4.72 vs 7.49±2.14, p<0.01), while both of them had little effect on the CD4+ lymphocytes (61.86±6.94 and 65.91±4.78 vs 57.93±10.40, p>0.05). We concluded that a high effective yeast expression system for Ta1 was constructed successfully and the Ta1 protein expressed by this system can improve CD8+ level in immune inhibited mice.

Cinética del enlazamiento de H33342 a la p-glicoproteína: detección y análisis de curvas de progreso / Kinetic of the binding of H33342 to the p-glycoprotein: detection and analysis of progress curves

El transportador multidrogas P-glicoproteína (Pgp) lleva a cabo el eflujo celular, ATP-dependiente, de muchas drogas hidrofóbicas, productos naturales y péptidos. Se propone que la Pgp contiene dos sitios de transporte, conocidos como los sitios -H y -R por sus preferencias por Hoechst 33342 (H33342) y rodamina 123, respectivamente. Cuando H33342 interactúa con la Pgp purificada, su rendimiento cuántico incrementa debido al ambiente hidrofóbico del bolsillo de enlazamiento –H. En este trabajo, estudiamos el enlazamiento de H33342 a la Pgp empleando experimentos cinéticos en la modalidad de stoppedflow. El curso temporal de la reacción fue seguido por el incremento de la fluorescencia del colorante y analizado con la herramienta computacional DYNAFIT, usando un modelo donde una reacción bimolecular rápida, es seguida por tres isomerizaciones secuenciales. Adicionalmente, bajo condiciones de seudo-primer-orden (exceso del ligando), la reacción presentó cuatro relajaciones caracterizadas por cuatro constantes de tiempo (á`s) y cuatro amplitudes (A¡`s) Estos parámetros fueron analizados utilizando la técnica de la matriz de los operadores de proyección. Esta aproximación aportó, por primera vez, información acerca de las constantes de velocidad y propiedades fluorescentes de los diversos intermediarios formados durante el enlazamiento de H33342 a la Pgp.

The P-glycoprotein multidrug transporter (Pgp) carries out ATP-driven cellular efflux of many different hydrophobic drugs, natural products, and peptides. Pgp is proposed to contain two drug transport sites, known as the H site and the R site for their preference for Hoechst 33342 (H33342) and rhodamine 123, respectively. When H33342 interacts with purified Pgp, its quantum yield is increased due to transfer to a hydrophobic environment within the H binding pocket, as shown by the steady-state fluorescence emission. In this work, we studied the binding of H33342 to Pgp using stopped-flow kinetic experiments. The time course of the reaction was followed by enhancement of dye fluorescence and analyzed by the computational tool DYNAFIT, using the model of a fast bimolecular reaction, followed by a three-step sequential isomerization. Additionally, under pseudo-first-order conditions (excess ligand), the reaction presented five normal modes, characterized by four relaxation times (á`s) and four amplitudes (A¡`s) These parameters were analyzed using the matrix projection operator technique, considering a four-step sequential reaction. This approach provides, for the first time, information about the rate constants and fluorescent properties of the diverse intermediates formed during the binding of H33342 to Pgp.