Growth response of broiler chickens to inclusion of hydrolyzed porcine mucosa (Palbio) in diets varying in total lysine content.

Growth response of broiler chickens to inclusion of hydrolyzed porcine mucosa (Palbio) in diets varying in total lysine content

Growth response of broilers to lysine levels and hydrolyzed porcine digestive mucosa (Palbio) inclusion in diet from 1 to 21 d of age

Palbio (PAL, Palbio 50 RD, Bioibérica, Spain) is a protein concentrate based on hydrolyzed porcine digestive mucosa dried under a fluid bed system over a soybean carrier, currently used in piglet feeds. The digestibility of PAL is very high and the product may be an excellent source of protein for young chicks. An experiment was conducted with 1,280 straight-run one-d-old Ross 308 chicks to evaluate the growth response of broilers to dietary inclusion of PAL.

Efecto de la incusion de hidrolizado de mucosa digestiva porcina (palbio 50 rd®) y del nivel de lisina del pienso sobre los parametros productivos en pollos de 1 a 21 dias de edad

El hidrolizado de mucosa digestiva de porcino (Palbio 50 RD ® , Bioiéerica, S.A., PAL) se utiliza con resultados óptimos en la alimentación de lechones recién destetados (Lindeman el al. 2000; Corassa et al. 2007). En un trabajo reciente, Mohiti-Asli et al. (2011) observaron que la inclusión de PAL mejoraba los resultados productivos en pollos de engorde a cualquier edad. En este trabajo se demostró que los niveles más recomendables de utilización de PAL teniendo en cuenta razones productivas y económicas, era el 2,5%. En esta investigación se estudio el efecto de la inclusión de 2,5% de PAL en piensos para pollos con niveles crecientes de lisina total (LYS, 1,1 a 1,4%). El objetivo fue estudiar si los efectos beneficiosos del PAL sobre la productividad de los pollos eran independientes o no del nivel de LYS del pienso.

Efecto de la inclusión de un hidrolizado de mucosa digestiva porcina (palbio 50 rd®) en piensos de broilers con niveles diferentes de lisina

La alimentación del pollo broiler durante la primera semana de vida es de creciente importancia debido a que la edad de sacrificio ha disminuido de forma constante en los últimos años. Además, consumos elevados durante la primera semana de vida mejoran el desarrollo del aparato digestivo de las aves, favoreciendo el crecimiento de las vellosidades intestinales y la eficiencia alimenticia (Lilburn, 1998, Noy et al., 2005). En los últimos años, el mercado dispone de nuevos productos de origen animal obtenidos durante el proceso de obtención de la heparina para uso farmacéutico. Uno de estos productos comerciales (Palbio 50 RD, Bioibérica S.A., Palafolls, Barcelona) está formado por la proteína hidrolizada de la mucosa digestiva de porcino limpia de contenidos intestinales, secada mediante un procedimiento especial que incluye la utilización de harina de soja como excipiente. Recientes estudios han demostrado de forma fehaciente el interés de utilizar este ingrediente en piensos de lechones de primera edad (Lindeman, et al., 2000; Corassa et al., 2007) pero los datos existentes en aves son más limitados. El objetivo de esta investigación fue evaluar los efectos de niveles crecientes de este hidrolizado proteico (PAL) sobre la productividad de pollos que recibían piensos con dos niveles diferentes de lisina total (Lys)

Effect of type of dietary carbohydrates on digestion, intestinal barrier and growth performance in 25-d weaned rabbits

Los objetivos globales de esta tesis han sido estudiar el efecto que los carbohidratos de la dieta ejercen sobre los rendimientos productivos, la barrera intestinal, y la digestión de animales destetados a 25 días de edad. Además se ha estudiado cuál es el mejor periodo para determinar la digestibilidad fecal tras el destete a esta edad. En el primer experimento se estudió el efecto de la fibra neutro detergente soluble (FNDS) sobre la barrera intestinal, digestión, microbiota intestinal y rendimientos productivos de gazapos en gazapos en la fase post-destete. Se diseñaron tres piensos isonutritivos en los que la única fuente de variación fueron los niveles de fibra soluble. A partir de una dieta control (AH) con 103 g/kg de materia seca de FNDS y alfalfa como fuente principal de fibra, se sustituyó la mitad de esta alfalfa por una mezcla de pulpa de remolacha y pulpa de manzana (75:25) en el pienso B-AP y por una mezcla de cascarilla y concentrado de proteína de soja (88:12) en el pienso OH, obteniéndose 131 y 79 g/kg de FNDS sobre materia seca, respectivamente. Los conejos se destetaron a 25 días y fueron alimentados con los piensos experimentales hasta los 35 días de edad, momento en el que se sacrificaron para la determinación de la digestibilidad ileal aparente (DIA) de la materia seca (MS), proteína bruta (PB) y almidón, la morfología de la mucosa, y actividad enzimática en el yeyuno, el tejido linfoide asociado a la mucosa, así como la microbiota intestinal. Para la determinación de la morfología de la mucosa se utilizaron adicionalmente 19 animales lactantes de 35 días de edad. Para el estudio de la tasa de mortalidad, se utilizaron 118 animales más por tratamiento que recibieron los piensos experimentales durante las dos semanas post-destete y posteriormente un pienso comercial hasta los 60 días de edad. Los animales recibieron durante todo el experimento medicación en el agua de bebida (100 ppm de apramicina sulfato y 120 ppm de tilosina tartrato). El nivel de fibra soluble mejoró los parámetros que se utilizaron para la caracterización del estado de la barrera intestinal. Los conejos alimentados con el mayor nivel de FNDS en el pienso presentaron una mayor longitud de los villi (P=0.001), un mayor ratio longitud villi/profundidad de las criptas (8.14; P=0.001), una mayor actividad disacaridásica (8671 μmol de glucosa/g de proteína; P=0.019), así como una mayor digestibilidad ileal (96.8%; P=0.002), observándose una reducción en el flujo ileal de almidón a medida que se incrementó el nivel de fibra soluble en el pienso (1,2 vs 0,5 g/d; P=0.001). Los animales lactantes a 35 días de edad presentaron un ratio longitud de villi/profundidad de las criptas menor que el observado en aquéllos alimentados con el pienso B-AP (6.70), pero superior al de los piensos AH y OH. Niveles inferiores de NDFS tendieron (P=0.074) a incrementar la respuesta inmune de tipo celular (linfocitos CD8+). El pienso también afectó a la producción de IL2 (CD25+; P=0.029; CD5+CD25+; P=0.057), pero sin llegar a establecerse una clara relación con el nivel de fibra soluble. La diversidad de la microbiota intestinal no se vio afectada por el pienso (P ≥ 0.38). Los animales alimentados con las piensos B-AP y AH presentaron una reducción en la frecuencia de detección de Clostridium perfringens tanto en íleon (P=0.062) como en ciego (4.3 vs. 17.6%, P =0.047), comparado con el pienso OH. Además la tasa de mortalidad (118 gazapos/pienso) disminuyó de 14.4% en el pienso OH a 5.1% en el pienso B-AP. Entre los 32 y los 35 días de edad se determinó la digestibilidad fecal aparente (14/pienso) de la materia seca (MS), energía bruta (EB), proteína bruta (PB), fibra neutro detergente (FND), fibra ácido detergente (FAD) y almidón. Este grupo, junto con otros nueve animales por tratamiento se utilizaron para determinar el peso del estómago y el ciego, la concentración cecal de ácidos grasos volátiles (AGV) y amoniaco (NH3), así como las tasas de similitud de la microbiota intestinal. Además se estudiaron los rendimientos productivos (35 animales/tratamiento) de los gazapos durante todo el período de cebo, consumiendo los piensos experimentales desde el destete hasta los 35 días y posteriormente un pienso comercial hasta los 60 días de edad. Niveles crecientes de FNDS mejoraron la digestibilidad fecal de la materia seca (MS) y energía (P<0.001). La inclusión FNDS aumentó de manera lineal el peso del contenido cecal (P=0.001) y el peso del aparato digestivo completo (P=0.008), y en los días previos al sacrificio disminuyó de manera lineal el consumo medio diario (P=0.040). Se observó además, una disminución lineal (P≤0.041) del pH del estómago. No se encontró relación entre el pH, la concentración y proporciones molares de AGV y el nivel de FNDS. El pienso pareció tener un efecto, incluso superior al de la madre, sobre la tasa de similitud de la microbiota, y los efectos fueron mayores a nivel cecal que ileal. La eficacia alimenticia aumentó de manera lineal en un 12% entre piensos extremos tras el destete (25- 39d) y en un 3% en el período global de cebo con niveles mayores de NDFS. El consumo medio diario durante la fase post-destete y durante todo el período de cebo, tendió a aumen tar (P≤0.079) con niveles mayores de FNDS, sin embargo no se apreció efecto sobre la ganancia media diaria (P≥0.15). En conclusión, el incremento del nivel de fibra soluble en el pienso parece resultar beneficioso para la salud del animal ya que mejora la integridad de la mucosa, y reduce la frecuencia de detección de potenciales patógenos como C. perfringens y Campylobacter spp. Conforme a estos resultados, debería tenerse en cuenta el contenido en fibra soluble en la formulación de piensos de conejos en la fase post-destete. El objetivo del segundo experimento fue determinar el efecto de la fuente de almidón sobre la digestión, la microbiota intestinal y los rendimientos productivos en conejos destetados con 25 días de edad. Se formularon tres piensos isonutritivos en los que se modificaron las principales fuentes de almidón: trigo crudo, trigo cocido y una combinación de trigo y arroz cocido. Dos grupos de 99 y 193 animales se destetaron con 25 días de edad. El primero de ellos se utilizó para la determinación de los parámetros productivos conforme al mismo protocolo seguido en el experimento anterior. El segundo de los grupos se utilizó para la determinación de la digestibilidad fecal de 32 a 35 d, la digestibilidad ileal aparente (DIA) a 35 d, la morfología de la mucosa intestinal, los parámetros de fermentación cecal; así como, la caracterización de la microbiota intestinal. Se utilizaron además dos grupos adicionales de animales 384 (medicados) y 177 (no medicados) para estudiar el efecto de la suplementación con antibióticos en el agua de bebida sobre la mortalidad. El procesado térmico del trigo mejoró ligeramente la digestibilidad ileal del almidón (P=0.020) pero no modificó el flujo final de almidón que alcanzó el ciego, observándose una mayor frecuencia de detección de Campylobacter spp. y Ruminococcus spp. en ciego (P≤0.023), pero sin cambios a nivel ileal. El procesado térmico del trigo no afectó tampoco a los parámetros productivos, la mortalidad, la digestibilidad ileal y fecal o la morfología de la mucosa. La sustitución parcial del trigo cocido por arroz cocido, penalizó la digestibilidad ileal del almidón (P=0.020) e incrementó el flujo ileal de este nutriente al ciego (P=0.007). Sin embargo no afectó a la mortalidad, pese a que se detectaron cambios en la microbiota tanto a nivel ileal como cecal, disminuyendo la frecuencia de detección de Campylobacter spp. (en íleon y ciego), Helicobacter spp. (en íleon) y Ruminococcus spp (en ciego) e incrementando Bacteroides spp. (en ciego) (P≤0.046). El empleo de arroz cocido en las piensos post-destete no tuvieron efectos sobre los parámetros productivos, la mortalidad, la digestibilidad ileal y fecal a excepción del almidón, o la morfología de la mucosa. La suplementación con antibiótico redujo la fre cuencia de detección de la mayoría de las bacterias estudiadas (P≤0.048), sobre todo para Campylobacter spp., Clostridium perfringens y Propionibacterium spp. (P≤0.048), observándose un efecto mayor a nivel ileal que cecal, lo que se asoció a la bajada significativa (P<0.001) de la mortalidad. En conclusión, los resultados de este experimento indican que la fuente de almidón afecta a la microbiota intestinal pero no influiye sobre la salud del animal. En relación al procesado, el uso de trigo cocido junto con arroz cocido no mejora los resultados obtenidos con trigo duro, si bienserían necesarios más experimentos que confirmaran este punto. El último de los experimentos se centró en un aspecto metodológico. Dado que, los conejos destetados presentan un patrón digestivo diferente al de un animal adulto resultado de su inmadurez digestiva, el objetivo buscado era tratar de determinar el mejor procedimiento para la determinación de la digestibilidad fecal en los gazapos en la fase post-destete. Para tal fin se utilizaron 15 animales/tratamiento de tres camadas diferentes que se destetaron con 25 días, suministrándoles un pienso comercial de crecimiento-cebo. Se registró el consumo medio diario y la excreción diaria de heces desde el día 25 hasta el día 40 de edad para la determinación de la digestibilidad de la MS. La camada afectó al consumo medio diario y la excreción de heces (P=0.013 y 0.014, respectivamente), observándose una tendencia (P=0.061) en la digestibilidad. La edad afectó (P<0.001) a todos estos factores, incrementándose de manera más evidente la excreción que la ingestión de materia seca en la primera semana de vida, para aumentar de forma paralela a partir de la segunda. La correlación entre el consumo medio diario fue mayor con la excreción de heces del mismo día que con la del día siguiente, por lo que se utilizó el primero para la determinación de la digestibilidad de la MS (MSd). La MSd disminuyó de manera lineal hasta los 32 días de edad (2.17±0.25 unidades porcentuales por día), mientras que permaneció constante desde los 32 a los 40 días (69.4±0.47%). Por otro lado, la desviación estándar de la MSd se redujo cuando se incrementó el período de recogida de 2 a 6 días en un 54%. Conforme a los resultados obtenidos, se puede concluir que no es aconsejable comenzar las pruebas de digestibilidad antes de los 32 días de edad y que el número de animales necesario para detectar diferencias significativas entre tratamientos dependerá del período de recogida de heces. ABSTRACT The global aim of this thesis has been to study the effect of dietary carbohydrates on growth, performance, digestion and intestinal barrier in 25-d weaned rabbits. In addition there has also been studied which is the best period to determine the fecal digestibility after weaning. The first experiment focused on the effect of Neutral Detergent Soluble Fibre (NDSF) on gut barrier function, digestion, intestinal microbiota and growth performance n rabbits in the post-weaning period. Three isonutritive diets which only varied in the levels of soluble fiber were formulated such as it described as follows: a control diet (AH) containing 103 g of neutral detergent soluble fiber, including alfalfa as main source of fiber, was replaced by a mixture of beet and apple pulp (75-25) in the B-AP diet and, by a mix of oat hulls and soybean protein concentrate (88:12) in the OH diet, resulting 131 and 79 g of NDFS/kg of dry matter, respectively. Rabbits, weaned at 25 days of age, were fed the experimental diets up to 35 days of age, moment in which they were slaughtered for apparent ileal digestibility (AID) of dry matter (DM), crude protein (CP) and starch, mucosa morphology, sucrose activity, characterization of lamina propria lymphocytes and intestinal microbiota. To assess mucosal morphology, 19 suckling 35-d-old rabbits were also used. For mortality study, besides these animals, 118 additional rabbits per treatment were fed the experimental diets for two weeks period and thereafter received a commercial diet until 60 days of age. Rabbits were water medicated during the whole experimental period (100 ppm de apramicine sulphate and 120 ppm of tylosine tartrate). Level of soluble fiber improved all the parameters used for the characterization of the intestinal barrier condition. Villous height of the jejunal mucosa increased with dietary soluble fiber (P=0.001). Villous height of jejunal mucosa increased with dietary soluble fiber (P = 0.001). Rabbits fed the highest level of soluble fiber (BA-P diet) showed the highest villous height/crypth depth ratio (8.14; P = 0.001), sucrase specific activity (8671 μmol glucose/ g protein; P = 0.019), and the greatest ileal starch digestibility (96.8%; P = 0.002). The opposite effects were observed in rabbits fed decreased levels of soluble fiber (AH and OH diets; 4.70, 5,848 μmol of glucose/g of protein, as average, respectively). The lowest ileal starch digestibility was detected for animal fed OH diet (93.2%). Suckling rabbits of the same age showed a lower villous height/crypt depth ratio (6.70) compared with the B-AP diet group, but this ration was higher that the AH or OH diet groups. Lower levels of soluble fiber tended (P = 0.074) to increase the cellular immune response (CD8+ lymphocytes). Diet affected IL-2 production (CD25+, P = 0.029; CD5+CD25+, P = 0.057), with no clear relationship between soluble fiber and IL-2. The intestinal microbiota biodiversity was not affected by diets (P ≥ 0.38). Animals fed B-AP and AH diets had a reduced cecal frequency of detection compatible with Campylobacter spp. (20.3 vs. 37.8, P = 0.074), and Clostridium perfringens (4.3 vs. 17.6%, P = 0.047), compared with the OH diet group. Moreover, the mortality rates decreased from 14.4 (OH diet) to 5.1% (B-AP diet) with the increased presence of soluble fiber in the diet. Between 32 and 35 days of age, faecal apparent digestibility of dry matter (DM), gross energy (GE), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF) and starch was determined (14/diet). This group, plus another nine rabbits/diet were used to determine weight of stomach and caecum and their contents, cecal fermentation traits and similarity rate (SR) of intestinal microbiota. Besides, growth performance parameters (35 rabbits/diet) were studied during the whole fattening period, in animals consuming the experimental feed after the weaning up to 35 days of age and later on a commercial diet up animals reached 60 days of age. Increasing levels of Neutral Detergent Soluble Fiber improved faecal dry matter and energy digestibility (P<0.001). NDSF inclusion improved linearly weight of the caecal content (P=0.001) and the total gastrointestinal tract (P=0.008), and in the previous days to slaughter a linear decrease of daily feed intake in diet with highest level of soluble fiber was also observed. Stomach pH decreased linearly with increasing levels of NDFS (P≤0.041). No relation between NDSF level on pH, concentration and molar proportion of VFA was found. Treatments appeared to influence the similarity rate of microbiota, even higher to mother effect. These effects were higher in ileum than in caecum. A linear positive effect of feed efficiency was observed, which increased around 12% in the two weeks post-weaning (25-39d) and 3% in the whole fattening period between extreme diets with highest levels of soluble fiber. Average daily feed intake during the two weeks after weaning and in the whole fattening period, tended (P≤0.079) to increase with highest levels of NDSF; although there were no effect on daily weight gain (≥0.15). In conclusion, an increase of soluble fiber in the feed seems to be beneficial for animal health, due to improve mucose integrity and reduce detection frequency of those poten tial pathogens like C. perfringens and Campylobacter spp. According to these results, level of soluble fiber should be taking care in feed rabbit formulation in the post-weaning period. The objective of the second experiment was to determine the effect of source of starch on digestion, intestinal microbiota and growth performance in twenty-five-day old weaned rabbits. To accomplish with this aim three iso-nutritive diets were formulated with different source of starch: raw wheat, boiled wheat and a combination of boiled wheat and boiled rice. Two groups of 99 and 193 rabbits were weaned at 25 days of age. The first group was used for growth performance determination following the same protocol than in previous experiment. The second group was used to determine faecal digestibility from 32 to 35 d, apparent ileal digestibility (AID) at 35 d, jejunal mucosa morphology, caecal fermentation traits and characterization of intestinal microbiota. For mortality, two additional groups of 384 (medicated) and 177 (not medicated) were used in order to study the effect of antibiotic water supply supplementation. Heat processing of starch slightly improved ileal digestibility of starch (P=0.020) but did not modify the flow of starch to the caecum. An increase in frequency of detection of Campylobacter spp. y Ruminococcus spp. was observed in the caecum (P≤0.023), with no changes at ileal level. Heat processing of wheat did not modify growth performance, mortality, ileal or faecal digestibility and mucosa morphology. Partial substitution of boiled wheat for boiled rice in the diet impaired ileal starch digestibility (P=0.020) and increased the ileal flow of this nutrient to the caecum (P=0.007). However, it did not affect mortality rate, although changes in the ileal and caecal intestinal microbiota were detected, decreasing the frequency of detection of Campylobacter spp. (both ileum and caecum), Helicobacter spp. (at ileum) and Ruminococcus spp (at caecum) and increasing the Bacteroides spp. (at caecum) (P≤0.046). The effect of boiled rice supplementation did not alter growth performance, mortality, ileal or faecal digestibility of other nutrients than starch, and mucosa morphology. Medication of rabbits reduced the ileal frequency of detection of most bacteria studied (P≤0.048), especially for Campylobacter spp., Clostridium perfringens y Propionibacterium spp. (P≤0.048), resulting the effect higher at ileal than caecal level and relating it with a strong reduction of mortality rate (P<0.001). In conclusion, the results of this experiment make think that the source of starch affects the intestinal microbiota but they do not seem to influence animal health. In relation to the effect of heat processed the use of cooked wheat or cooked rice it does not seem to im prove the results obtained with hard wheat, but there would be necessary more experiments that were confirming this point. The last experiment focused on a methodological aspect. Considering that, weaned rabbits have a different digestive pattern than older animals due to their digestive immaturity; the fixed objective was to determine the best procedure for faecal digestibility determination in young rabbits in the post-weaning period. Fifteen rabbits from 5 different litters were weaned at 25 days of age and fed with a commercial feed. Feed intake and faeces excretion were recorded daily from 25 to 40 days of age for dry matter digestibility (DMd) determination. Litter affected daily DM intake and excretion (P=0.013 y 0.014, respectively) and tended to affect DMd (P=0.061). Age affected all these factors (P<0.001), but ingestion increased slowly than dry matter excretion during the first week buth they evolved similarly in the second week. The correlation between daily feed intakes was higher with the faeces excretion of the day than with faeces excretion of the next day, and the first values were used to determine daily DMd. The DMd decreased linearly from weaning to 32 d of age (2.17±0.25 percentage units per day), whereas from 32 to 40 d remained constant (69.4±0.47%). On the other hand, average standard deviation of DMd decreased by 54% when the length of collection period increased from 2 to 6d. Consequently to the obtained results, it could be concluded that it would not be advisable to start digestibility trials before the 32 days of age and that the number of animals required to detect a significant difference among means would depend on the collection period.

Cholecystokinin-8 protects central cholinergic neurons against fimbria–fornix lesion through the up-regulation of nerve growth factor synthesis

In this study, we demonstrate that cholecystokinin-8 (CCK-8) induces an increase in both nerve growth factor (NGF) protein and NGF mRNA in mouse cortex and hippocampus when i.p. injected at physiological doses. By using fimbria–fornix-lesioned mice, we have also demonstrated that repeated CCK-8 i.p. injections result in recovery of lesion-induced NGF deficit in septum and restore the baseline NGF levels in hippocampus and cortex. Parallel to the effects on NGF, CCK-8 increases choline acetyltransferase (Chat) activity in forebrain when injected in unlesioned mice and counteract the septo-hippocampal Chat alterations in fimbria–fornix-lesioned mice. To assess the NGF involvement in the mechanism by which CCK-8 induces brain Chat, NGF antibody was administrated intracerebrally to saline- and CCK-8-injected mice. We observe that pretreatment with NGF antibody causes a marked reduction of NGF and Chat activity in septum and hippocampus of both saline- and CCK-8-injected mice. This evidence indicates that the CCK-8 effects on cholinergic cells are mediated through the synthesis and release of NGF. Taken together, our results suggest that peripheral administration of CCK-8 may represent a potential experimental model for investigating the effects of endogenous NGF up-regulation on diseases associated with altered brain cholinergic functions.

Accuracy of lesion bypass by yeast and human DNA polymerase η

DNA polymerase η (Polη) functions in the error-free bypass of UV-induced DNA lesions, and a defect in Polη in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Both yeast and human Polη replicate through a cis-syn thymine-thymine dimer (TT dimer) by inserting two As opposite the two Ts of the dimer. Polη, however, is a low-fidelity enzyme, and it misinserts nucleotides with a frequency of ≈ 10−2 to 10−3 opposite the two Ts of the TT dimer as well as opposite the undamaged template bases. This low fidelity of nucleotide insertion seems to conflict with the role of Polη in the error-free bypass of UV lesions. To resolve this issue, we have examined the ability of human and yeast Polη to extend from paired and mispaired primer termini opposite a TT dimer by using steady-state kinetic assays. We find that Polη extends from mispaired primer termini on damaged and undamaged DNAs with a frequency of ≈ 10−2 to 10−3 relative to paired primer termini. Thus, after the incorporation of an incorrect nucleotide, Polη would dissociate from the DNA rather than extend from the mispair. The resulting primer-terminal mispair then could be subject to proofreading by a 3′→5′ exonuclease. Replication through a TT dimer by Polη then would be more accurate than that predicted from the fidelity of nucleotide incorporation alone.

Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach.

The structural relationships between interstitial cells of Cajal (ICC), varicose nerve fibers, and smooth muscle cells in the gastrointestinal tract have led to the suggestion that ICC may be involved in or mediate enteric neurotransmission. We characterized the distribution of ICC in the murine stomach and found two distinct classes on the basis of morphology and immunoreactivity to antibodies against c-Kit receptors. ICC with multiple processes formed a network in the myenteric plexus region from corpus to pylorus. Spindle-shaped ICC were found within the circular and longitudinal muscle layers (IC-IM) throughout the stomach. The density of these cells was greatest in the proximal stomach. IC-IM ran along nerve fibers and were closely associated with nerve terminals and adjacent smooth muscle cells. IC-IM failed to develop in mice with mutations in c-kit. Therefore, we used W/W(V) mutants to test whether IC-IM mediate neural inputs in muscles of the gastric fundus. The distribution of inhibitory nerves in the stomachs of c-kit mutants was normal, but NO-dependent inhibitory neuro-regulation was greatly reduced. Smooth muscle tissues of W/W(V) mutants relaxed in response to exogenous sodium nitroprusside, but the membrane potential effects of sodium nitroprusside were attenuated. These data suggest that IC-IM play a critical serial role in NO-dependent neurotransmission: the cellular mechanism(s) responsible for transducing NO into electrical responses may be expressed in IC-IM. Loss of these cells causes loss of electrical responsiveness and greatly reduces responses to nitrergic nerve stimulation.

Structural recovery in lesioned adult mammalian spinal cord by x-irradiation of the lesion site.

Mechanical injury to the adult mammalian spinal cord results in permanent morphological disintegration including severance/laceration of brain-cord axons at the lesion site. We report here that some of the structural consequences of injury can be averted by altering the cellular components of the lesion site with x-irradiation. We observed that localized irradiation of the unilaterally transected adult rat spinal cord when delivered during a defined time-window (third week) postinjury prevented cavitation, enabled establishment of structural integrity, and resulted in regrowth of severed corticospinal axons through the lesion site and into the distal stump. In addition, we examined the natural course of degeneration and cavitation at the site of lesion with time after injury, noting that through the third week postinjury recovery processes are in progress and only at the fourth week do the destructive processes take over. Our data suggest that the adult mammalian spinal cord has innate mechanisms required for recovery from injury and that timed intervention in certain cellular events by x-irradiation prevents the onset of degeneration and thus enables structural regenerative processes to proceed unhindered. We postulate that a radiation-sensitive subgroup of cells triggers the delayed degenerative processes. The identity of these intrusive cells and the mechanisms for triggering tissue degeneration are still unknown.

Estrogen reduces atherosclerotic lesion development in apolipoprotein E-deficient mice.

We have studied the effects of endogenous and exogenous estrogen on atherosclerotic lesions in apolipoprotein E-deficient mice. Female mice ovariectomized (OVX) at weaning displayed increases (P < 0.01) in fatty streak lesions in the proximal aorta and aortic sinus compared with female mice with intact ovarian function. These differences between the OVX and sham controls were apparent in both chow- and "Western-type" diet-fed mice. Moreover, increases in lesion size following OVX occurred without changes in plasma cholesterol. Hormone replacement with subdermal 17-beta-estradiol pellets releasing either 6, 14, or 28 micrograms/day significantly decreased (P < 0.001) atherosclerotic lesion area in both male and OVX female mice. In contrast, neither 17-alpha-estradiol (28 micrograms/day) or tamoxifen (85 micrograms/day) affected lesion progression in OVX female mice. In the Western diet-fed group, exogenous estradiol markedly reduced plasma cholesterol and triglycerides, whereas, in animals fed the chow diet, exogenous estrogen and tamoxifen treatment only decreased plasma and very low density lipoprotein triglycerides. However, lesion area was only weakly correlated with plasma cholesterol and triglycerides, 0.35 and 0.44 tau values, respectively (P < 0.01). In summary, in the apolipoprotein E-deficient mouse 17-beta-estradiol protects against atherosclerotic lesion formation, and this can only be partially explained through effects on plasma lipoprotein levels.

Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.

A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpressed in all chromaffin cells of the adrenal medulla. VMAT2 alone is expressed in histamine-storing enterochromaffin-like cells of the oxyntic mucosa of the stomach. The transport characteristics and pharmacology of each VMAT isoform have been directly compared after expression in digitonin-permeabilized fibroblastic (CV-1) cells, providing information about substrate feature recognition by each transporter and the role of vesicular monoamine storage in the mechanism of action of psychopharmacologic and neurotoxic agents in human. Serotonin has a similar affinity for both transporters. Catecholamines exhibit a 3-fold higher affinity, and histamine exhibits a 30-fold higher affinity, for VMAT2. Reserpine and ketanserin are slightly more potent inhibitors of VMAT2-mediated transport than of VMAT1-mediated transport, whereas tetrabenazine binds to and inhibits only VMAT2. N-methyl-4-phenylpyridinium, phenylethylamine, amphetamine, and methylenedioxymethamphetamine are all more potent inhibitors of VMAT2 than of VMAT1, whereas fenfluramine is a more potent inhibitor of VMAT1-mediated monamine transport than of VMAT2-mediated monoamine transport. The unique distributions of hVMAT1 and hVMAT2 provide new markers for multiple neuroendocrine lineages, and examination of their transport properties provides mechanistic insights into the pharmacology and physiology of amine storage in cardiovascular, endocrine, and central nervous system function.

Differential replication of a single, UV-induced lesion in the leading or lagging strand by a human cell extract: fork uncoupling or gap formation.

We have constructed simian virus 40 minireplicons containing uniquely placed cis,syn-thymine dimers (T <> T) for the analysis of leading- and lagging-strand bypass replication. Assaying for replication in a human cell-free extract through the analysis of full-size labeled product molecules and restriction fragments spanning the T <> T site resulted in the following findings: (i) The primary site of synthesis blockage with T <> T in either the leading or lagging strand was one nucleotide before the lesion. (ii) Replicative bypass of T <> T was detected in both leading and lagging strands. The efficiency of synthesis past T <> T was 22% for leading-strand T <> T and 13% for lagging-strand T <> T. (iii) The lagging-strand T <> T resulted in blocked retrograde synthesis with the replication fork proceeding past the lesion, resulting in daughter molecules containing small gaps (form II' DNA). (iv) With T <> T in the leading-strand template, both the leading and lagging strands were blocked, representing a stalled replication fork. Uncoupling of the concerted synthesis of the two strands of the replication fork was observed, resulting in preferential elongation of the undamaged lagging strand. These data support a model of selective reinitiation downstream from the lesion on lagging strands due to Okazaki synthesis, with no reinitiation close to the damage site on leading strands [Meneghini, R. & Hanawalt, P.C. (1976) Biochim. Biophys. Acta 425, 428-437].