Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Caveolae are integral part of the smooth muscle membrane and caveolins, the defining proteins of caveolae, act as scaffolding proteins for several G protein-coupled receptor signaling molecules and regulate cellular signaling through direct and indirect interactions with signaling proteins. Caveolin-1 is the predominant isoform in the smooth muscle and drives the formation of caveolae. However, little is known about the role of caveolin-1 in the regulation of excitation-contraction and excitation-transcription coupling in gastrointestinal smooth muscle. In the present study we have characterized muscarinic m2 and m3 receptor signaling in gastric smooth muscle and tested the hypothesis that caveolin-1 positively regulates muscarinic receptor signaling and contractile protein expression in smooth muscle. The role of caveolae/caveolin-1 in the regulation of muscarinic signaling was examined using complementary approaches: a) methyl b-cyclodextrin (MbCD) to deplete cholesterol in dispersed muscle cells, b) caveolin-1 siRNA to suppress caveolin-1 expression in cultured muscle cells, and c) caveolin-1 knockout (KO) mice. RT-PCR, western blot and radioligand binding studies demonstrated the selective expression of m2 and m3 receptor in gastric smooth muscle cells. Carbachol (CCh), acting via m3 receptors caused stimulation of phosphoinositide (PI) hydrolysis, Rho kinase and ZIP kinase activity, and induced phosphorylation of MYPT1 (at Thr696) and MLC20 (at Ser19), and muscle contraction, and acting via m2 receptors caused inhibition of forskolin stimulated cAMP formation. Stimulation of PI hydrolysis, Rho kinase and ZIP kinase activities, phosphorylation of MYPT1 and MLC20 phosphorylation and muscle contraction in response to CCh was attenuated in dispersed cells treated with MbCD or in cultured cells transfected with caveolin-1 siRNA. Similar inhibition of all responses was obtained in gastric muscle cells from caveolin-1 KO mice compared to gastric muscle cells to WT mice. Although, caveolin-1 had no effect on m2 receptor signaling, agonist-induced internalization of m2, but not m3 receptors was blocked in dispersed cells treated with MbCD or in cultured cells transfected with caveolin-1 siRNA. These results suggest that caveolin-1 selectively and positively regulates Gq/13-coupled m3 receptor signaling, Gi-coupled m2 receptor internalization. The expression of contractile proteins, g-actin and caldesmone and the transcription factors SRF and myocardin that regulate the expression of contractile proteins are down regulated, whereas EGF-stimulated EGF receptor phosphorylation and ERK1/2 activity are up-regulated in cells transfected with caveolin-1 siRNA. These results suggest using pharmacological, molecular and genetic approaches provide conclusive evidence that caveolae and caveolin-1 play an important role in orchestrating G protein coupled receptor signaling to have dual pro- excitation-contraction and excitation-transcription coupling, and anti-proliferative role in gastric smooth muscle.

For centuries Aloe vera has been exploited for several verified and unverified medicinal uses such as wound healing, treatment of gastrointestinal ulcers and for its many biological effects including anti-microbial, laxative, anti-inflammatory and immunostimulatory activities. Studies have generally focused on its effects in vitro and in adults. When nursing mothers use Aloe vera extracts, their suckling infants are at risk of indirect exposure to Aloe vera via breast feeding or directly as dietary/health supplements. The gastrointestinal tract (GIT) of the neonate is sensitive to dietary manipulations during the suckling period with long lasting effects that can be irreversible. Thus babies may be at risk if administered Aloe vera extracts directly as dietary supplements or indirectly via breast milk. The main objectives of this study were to evaluate the effects of orally administered aqueous and alcohol extracts of Aloe vera on growth performance, the morphometry and morphology of the gastrointestinal tract and accessory organs, and liver function of suckling rats. Suckling Sprague-Dawley rats (77), males (n=38) and females (n=39) of 6 days old were randomly assigned to one of five treatment groups and given once daily by oral gavage a suspension of lyophilized crude alcohol or aqueous extracts of Aloe vera suspended in distilled water. Group I (control) was gavaged with distilled water (vehicle). Group II received a low dose of the aqueous extract (AqL) at 50mg. kg-1; Group III received a high dose of the aqueous extract (AqH) at 500mg. kg-1; Group IV received a low dose of the alcohol extract (AlcL) at 50mg. kg-1 whilst Group V received a high dose of the alcohol extract (AlcH) at 500mg. kg-1. The extracts and distilled water were 2 administered at a volume of 10ml.kg-1. The pups remained with their dams for the duration of the study and after 8 days on the treatments, the pups were humanely killed to harvest their tissues for measurements and physiological analysis. All data were expressed as mean ± SD and analyzed by one way ANOVA, the values were considered statistically significant when p < 0.05 and then a Bonferroni Post hoc test was applied. The suckling rats fed respectively with high doses of AlcH and AqH had a significantly higher body mass gain than the other groups (p < 0.05, one way ANOVA). Linear growth as measured by tibial length was significantly increased in the AqH group compared to the other groups. There was no significant difference in the mass and relative density of the tibia bones of the rats from the different treatment groups. The differences in growth could not be attributed to circulating concentrations of the somatotrophic hormone, Insulin-like growth factor-1 (IGF-1) which was not significantly different between the groups. The treatments did not result in any significant differences in lengths, and mass of the small and large intestine, however the caecum was significantly enlarged (hypertrophy of muscularis, submucosa and mucosa) in the rats that received the Aloe vera extracts. Although, there was no significant difference in the mass of the rats’ livers, the lipid and glycogen content were significantly higher (p < 0.001) for the AqH group compared to the other groups. Histologically, the hepatocytes showed enlarged nuclei, granular cytoplasm and dilated sinusoids for AqH and AlcH as compared to the control group. An indirect assessment of liver function by measurement of blood concentrations of alkaline phosphatase (ALP) and alanine amino transaminase (ALT) did not reveal a significant difference between the groups. The non fasting concentration of metabolic substrates (glucose and triglycerides) was also not significantly different between the groups. The pups given high doses of the extracts had a significantly greater (p < 0.05) thymus mass (hyperplastic) than the other groups. The short term administration of Aloe vera extracts has shown a growth promoting effect, enhanced hepatic storage of metabolic substrates and hypertrophy of the caecum and thymus of neonatal rats. These effects need to be explored further to enhance animal production and health.

A new model for an adult human gastrointestinal tract (GIT) has been developed for use in internal dose estimations to the wall of the GIT and to the other organs and tissues of the body from radionuclides deposited in the lumenal contents of the five sections of the GIT. These sections were the esophagus, stomach, small intestine, upper large intestine, and the lower large intestine. The wall of each section was separated from its lumenal contents. Each wall was divided into many small regions so that the histologic and radiosensitive variations of the tissues across the wall could be distinguished. The characteristic parameters were determined based on the newest information available in the literature. Each of these sections except the stomach was subdivided into multiple subsections to include the spatiotemporal variations in the shape and characteristic parameters. This new GIT was integrated into an anthropomorphic phantom representing both an adult male and a larger-than-average adult female. The current phantom contains 14 different types of tissue. This phantom was coupled with the MCNP 4C Monte Carlo simulation package. The initial design and coding of the phantom and the Monte Carlo treatment employed in this study were validated using the results obtained by Cristy and Eckerman (1987). The code was used for calculating specific absorbed fractions (SAFs) in various organs and radiosensitive tissues from uniformly distributed sources of fifteen monoenergetic photons and electrons, 10 keV - 4 MeV, in the lumenal contents of the five sections of the GIT. The present studies showed that the average photon SAFs to the walls were significantly different from that to the radiosensitive cells (stem cells) for the energies below 50 keV. Above 50 keV, the photon SAFs were found to be almost constant across the walls. The electron SAF at the depth of the stem cells was a small fraction of the SAF routinely estimated at the contents-mucus interface. Electron studies showed that the “self-dose” for the energies below 300 keV and the “cross-dose” below 2 MeV were only from bremsstrahlung and fluorescent radiations at the depth of the stem cells and were not important.

A new model for an adult human gastrointestinal tract (GIT) has been developed for use in internal dose estimations to the wall of the GIT and to the other organs and tissues of the body from radionuclides deposited in the lumenal contents of the five sections of the GIT. These sections were the esophagus, stomach, small intestine, upper large intestine, and the lower large intestine. The wall of each section was separated from its lumenal contents. Each wall was divided into many small regions so that the histologic and radiosensitive variations of the tissues across the wall could be distinguished. The characteristic parameters were determined based on the newest information available in the literature. Each of these sections except the stomach was subdivided into multiple subsections to include the spatiotemporal variations in the shape and characteristic parameters. This new GIT was integrated into an anthropomorphic phantom representing both an adult male and a larger-than-average adult female. The current phantom contains 14 different types of tissue. This phantom was coupled with the MCNP 4C Monte Carlo simulation package. The initial design and coding of the phantom and the Monte Carlo treatment employed in this study were validated using the results obtained by Cristy and Eckerman (1987). The code was used for calculating specific absorbed fractions (SAFs) in various organs and radiosensitive tissues from uniformly distributed sources of fifteen monoenergetic photons and electrons, 10 keV - 4 MeV, in the lumenal contents of the five sections of the GIT. The present studies showed that the average photon SAFs to the walls were significantly different from that to the radiosensitive cells (stem cells) for the energies below 50 keV. Above 50 keV, the photon SAFs were found to be almost constant across the walls. The electron SAF at the depth of the stem cells was a small fraction of the SAF routinely estimated at the contents-mucus interface. Electron studies showed that the “self-dose” for the energies below 300 keV and the “cross-dose” below 2 MeV were only from bremsstrahlung and fluorescent radiations at the depth of the stem cells and were not important.

Thesis (Ph. D.)--University of Nevada, Reno, 2002. / Includes bibliographical references. Online version available on the World Wide Web.

There has been little research into the use of water cooling methods for pigs during transport to slaughter under conditions of high ambient temperature. The aim of this study was to examine the effects of water sprinkling pigs before departure from the farm and before unloading at the plant on behaviour during transport, unloading and lairage using live and remote observations, and on pig gastrointestinal tract temperature (GTT) and trailer micro-climate measured by data loggers. Above 23oC, the use of water sprinkling tended to decrease GTT upon arrival and significantly decreased drinking bouts during lairage. There were no detrimental effects of the water sprinkling on unloading behaviours (e.g. slips and falls) or on trailer micro-climate conditions in terms of temperature, humidity or ammonia. Water sprinkling to wet the skin of pigs can therefore be used to cool pigs during transport and lairage under high ambient temperatures.

Introduction: Hirschsprung’s Disease (HD) is the congenital absence of ganglion cells (GCs) within the distal intestine. Our objectives are to determine if apoptosis of myenteric GCs occurs during human development and to determine if myenteric GC apoptosis or injury contributes to HD. Materials and Methods: Apoptosis of myenteric GCs was assessed in archived fetal intestinal tissue (n = 4; 15-41 weeks gestational age) and in HD at the transition zone (TZ) (n = 6) using anti-cleaved caspase-3. Immunohistochemistry for GFAP, CD68, HLA-DR and APP was used to assess the presence of enteric reactive changes. Results: No activated caspase-3 expression was present in the myenteric GCs of the developing human intestine or the TZ of HD. No significant increase in GFAP, CD68, HLA-DR or APP expression was present. Conclusions: Apoptosis does not appear to occur during the development of the human myenteric plexus or, in conjunction with GC injury, in HD.

Introduction: Hirschsprung’s Disease (HD) is the congenital absence of ganglion cells (GCs) within the distal intestine. Our objectives are to determine if apoptosis of myenteric GCs occurs during human development and to determine if myenteric GC apoptosis or injury contributes to HD. Materials and Methods: Apoptosis of myenteric GCs was assessed in archived fetal intestinal tissue (n = 4; 15-41 weeks gestational age) and in HD at the transition zone (TZ) (n = 6) using anti-cleaved caspase-3. Immunohistochemistry for GFAP, CD68, HLA-DR and APP was used to assess the presence of enteric reactive changes. Results: No activated caspase-3 expression was present in the myenteric GCs of the developing human intestine or the TZ of HD. No significant increase in GFAP, CD68, HLA-DR or APP expression was present. Conclusions: Apoptosis does not appear to occur during the development of the human myenteric plexus or, in conjunction with GC injury, in HD.

Ubiquitin (Ub) ligases are important regulatory and signalling molecules, which are involved in majority of cellular processes such as differentiation, DNA repair, and regulation of energetic metabolism or immune response. E3 Ubiquitin ligases are also responsible for pathophysiological changes in the organism and their activity is associated with many human diseases including cancers. This makes E3 Ubiquitin ligases to be new diagnostic markers and interesting pharmaceutical targets. Based on previous studies, these enzymes evince very specific expression in the level of tissues or cell populations. Determination of this specific expression is important for a better understanding of their biological function. In this diploma thesis we systematically screened presence of 370 genes of E3-Ub ligases in gastrointestinal tract under physiological conditions and during acute inflammatory damage of distal colon. Obtained data allowed us to select genes, which can play important role in homeostasis as well as pathophysiology and regeneration of gastrointestinal tract. The screening was based on the expression profiling using qPCR, followed by in situ hybridization to determine the exact localization of the gene expression within tissues. From qPCR analysis was predicted hundred thirty seven candidates for...