RESUMEN

To improve animal growth, growth hormone-releasing hormone (GHRH) expression vectors that maintain constant GHRH expression can be directly injected into muscles. To deliver the GHRH expression vectors, biodegradable microspheres have been used as a sustained release system. Although administering GHRH through microspheres is a common practice, the intergenerational effects of this delivery system are unknown. To investigate the intergenerational effects of polylactic-co-glycolic acid (PLGA) encapsulated plasmid-mediated GHRH supplements, pCMV-Rep-GHRH microspheres were injected into pregnant mice. Growth and expression of GHRH were measured in the offspring. RT-PCR and immunohistochemistry reveal GHRH expression 3-21 days post-injection. The proportion of GH-positive cells in the GHRH treated offspring was 48.2% higher than in the control group (P < 0.01). The GHRH treated offspring were 6.15% (P < 0.05) larger than the control offspring. At day 49 post-injection, IGF-I serum levels were significantly higher in the treatment group than in the control group. This study confirms that intramuscular expression of GHRH mediated by PLGA microspheres significantly enhances intergenerational growth.

Asunto(s)

Hormona Liberadora de Hormona del Crecimiento/biosíntesis , Hormona Liberadora de Hormona del Crecimiento/genética , Plásmidos/administración & dosificación , Plásmidos/genética , Animales , Secuencia de Bases , Cartilla de ADN/genética , Preparaciones de Acción Retardada , Sistemas de Liberación de Medicamentos , Femenino , Expresión Génica , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Inmunohistoquímica , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ácido Láctico , Ratones , Microesferas , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Adenohipófisis/crecimiento & desarrollo , Adenohipófisis/metabolismo , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Embarazo , Efectos Tardíos de la Exposición Prenatal , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Aumento de Peso/genética

RESUMEN

The title compound, C(17)H(21)N(3)O(3), has a Z configuration about the N=N double bond. The molecular conformation is stabilized by intramolecular N-H⋯O and O-H⋯N hydrogen bonds.

RESUMEN

The title compound, C(16)H(16)N(2)O(5)·CH(3)OH, was obtained from a condensation reaction of 3,4-dimethoxy-benzaldehyde and 2,4-dihydroxy-benzohydrazide. The non-H atoms of the Schiff base mol-ecule are approximately coplanar (r.m.s. deviation = 0.043 Å) and the dihedral angle between the two benzene rings is 1.6 (1)°. The mol-ecule adopts an E configuration with respect to the C=N double bond. An intra-molecular O-H⋯O hydrogen bond is observed. The Schiff base and methanol mol-ecules are linked into a two-dimensional network parallel to (10) by inter-molecular N-H⋯O, O-H⋯N and O-H⋯O hydrogen bonds.

RESUMEN

Somatostatin (SS) and growth hormone-releasing hormone (GHRH) are synthesized and secreted by the hypothalamus, which can control the synthesis and secretion of the growth hormone (GH) from the hypophysis as well as regulate the GH concentrations in animals and humans. In this article, we describe the regulation of animal growth using plasmid DNA encoding both the GHRH gene and the SS gene fused with the hepatitis B surface antigen (HBsAg) gene. We constructed a series of expression plasmids to express the GHRH and HBsAg-SS fusion genes individually as well as collectively. The fusion gene and GHRH were successfully expressed in Chinese hamster ovary (CHO) cells, as proven by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting tests. Poly D, L-lactide-co-glycolic acid (PLGA) plasmid-encapsulating microspheres were prepared and injected intramuscularly into the leg skeletal muscles of rabbits. Weight gain/day and the levels of insulinlike growth factor-I (IGF-I), SS, and hepatitis B surface antibody (HBsAb) were monitored. During days 30 postinjection, increase in weight gain/day and IGF- I concentration and decrease in SS were observed in treatment groups. From days 15 to 30 postinjection, the weight gain/day significantly increased (P < 0.05) by 129.13%, 106.8%, and 72.82% relative to the control group in the co-expression GHRH and fusion gene (named P-G-HS), fusion gene (named P-HS), and GHRH (named P-G) groups, respectively. And most importantly, the P-G-HS group showed significant weight gain/day (P < 0.05) relative to the P-G and P-HS groups. A significant increase in the IGF-I concentration and decrease in the SS level relative to the control group were also observed. The results indicated that the combination of plasmid-mediated GHRH supplementation and positive immunization against SS led to more robust weight gain/day in rabbits.

Asunto(s)

Hormona Liberadora de Gonadotropina/fisiología , Antígenos de Superficie de la Hepatitis B/fisiología , Músculo Esquelético/fisiología , Conejos/crecimiento & desarrollo , Somatostatina/fisiología , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Células CHO , Clonación Molecular , Cricetinae , Cricetulus , ADN/química , ADN/genética , Fusión Génica , Hormona Liberadora de Gonadotropina/biosíntesis , Hormona Liberadora de Gonadotropina/genética , Antígenos de Superficie de la Hepatitis B/biosíntesis , Antígenos de Superficie de la Hepatitis B/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ácido Láctico/administración & dosificación , Masculino , Microesferas , Plásmidos/genética , Ácido Poliglicólico/administración & dosificación , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Reacción en Cadena de la Polimerasa , Conejos/fisiología , Somatostatina/biosíntesis , Somatostatina/genética , Transfección/veterinaria

RESUMEN

Biodegradable nanospheres or microspheres have been widely used as a sustained release system for the delivery of bioagents. In the present study, injectable sustained-release growth hormone-releasing factor (GRF) (1-32) microspheres were prepared by a double emulsion-in liquid evaporation process using biodegradable polylactic-co-glycolic acid (PLGA) as the carrier. The entrapment efficiency was 89.79% and the mean particle size was 4.41 mum. The microspheres were injected into mouse tibialis muscle. After 30 days, mice injected with GRF (1-32) microspheres (group I) gained significantly more weight than any other treatment group, including mice injected with the naked plasmid (group II) (10.26 +/- 0.13 vs. 9.09 +/- 0.56; P < 0.05), a mixture of microspheres and plasmid (group III) (10.26 +/- 0.13 vs. 8.57 +/- 0.02; P < 0.05), or saline (IV) (10.26 +/- 0.13 vs. 6.47 +/- 0.26; P < 0.05). In addition, mice treated with the GRF (1-32) microspheres exhibited the highest expression levels of GRF as detected by PCR, RT-PCR, and ELISA (mean 2.56 +/- 0.40, P < 0.05, overall comparison of treatment with groups II, III, and IV). Additionally, rabbits were injected in the tibialis muscle with the same treatments described above. After 30 days, the group treated with GRF (1-32) microspheres gained the most weight. At day 30 postinjection, weight gain in group I was 63.93% higher than group II (plasmid) (877.10 +/- 24.42 vs. 535.05 +/- 26.38; P < 0.05), 108.59% higher than group III (blank MS) (877.10 +/- 24.42 vs. 420.50 +/- 19.39; P < 0.05), and 93.94% higher than group IV (saline) (877.10 +/- 24.42 vs. 452.25 +/- 27.38; P < 0.05). Furthermore, IGF-1 levels in the serum from GRF microsphere-treated group were elevated relative to all other groups. The present results suggest that encapsulation of GRF with PLGA increases GRF gene expression in muscle after local plasmid delivery, and stimulates significantly more weight gain than delivery of the naked plasmid alone.

Asunto(s)

Hormona Liberadora de Hormona del Crecimiento/química , Ácido Láctico/química , Músculo Esquelético/efectos de los fármacos , Ácido Poliglicólico/química , Polímeros/química , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Preparaciones de Acción Retardada , Ensayo de Inmunoadsorción Enzimática , Hormona Liberadora de Hormona del Crecimiento/administración & dosificación , Hormona Liberadora de Hormona del Crecimiento/biosíntesis , Hormona Liberadora de Hormona del Crecimiento/genética , Ácido Láctico/administración & dosificación , Masculino , Ratones , Microesferas , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Plásmidos/genética , Ácido Poliglicólico/administración & dosificación , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Polímeros/administración & dosificación , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Injectable sustained-release pcDNA3-GRF (1-32) microspheres were prepared by double emulsion-in liquid evaporation process,using biodegrable poly lactic-co-glycolic acid as carrier. The enrapment efficiency, mean particle size, drug content thus prepared were 69%, 2.20 microm, 8% and 70% respectively. The result of transfection in vivo showed that after 30 days, accumulative increased body weights on the group injected with pcDNA3-GRF (1-32) microspheres was significantly higher than those group injected with naked plasmid (12.87%), plasmid-empty microspheres (19.72%) and saline (58.58%) respectively. PCR and RT-PCR showed that the expression level of GRF gene on the group injected with pcDNA3-GRF (1-32) microspheres was the highest. GRF gene released by microspheres was still detected after 30 days. In conclusion, pcDNA3-GRF (1-32) microspheres have a controlled release effect and GRF gene could be successfully transfected into muscle cells of mouse by microspheres with higher efficacy and stronger biological function.

Asunto(s)

ADN/administración & dosificación , Hormona Liberadora de Hormona del Crecimiento/genética , Ácido Láctico/administración & dosificación , Microesferas , Músculo Esquelético/metabolismo , Ácido Poliglicólico/administración & dosificación , Animales , Peso Corporal , Crecimiento , Masculino , Ratones , Plásmidos , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Reacción en Cadena de la Polimerasa