Circadian variation of the cell proliferation in the jejunal epithelium of rats at weaning phase

Circadian variation in cell proliferation of the jejunal epithelium of 18-day-old rats was studied using the 2-h arrested metaphase score and crypt isolation method. A continuous decrease in the arrested metaphases occurred from 07.00 h to 13.00 h. From 17.00 h arrested metaphase values increased and were maintained at the higher level during the dark period as showed by Cosinor analyses (P < 0.05). These results indicate that in the young rat there is already a circadian variation in jejunal epithelial cell proliferation as early as 18 days. We can even suggest that the presence of a circadian rhythm at weaning contributes to the steady state of cell proliferation in the intestinal epithelium observed in adult life.     

Kidney accumulation of yellow fluorescent protein after its absorption in the rat intestine

Yellow fluorescent protein (3.6 ng) was administered through a catheter into the Wistar rat intestine lumen. By the method of confocal microscopy it has been established that as soon as in 3 min this protein appears in the epithelial cells of ileum, is absorbed into the blood and accumulated in cells of the nephron proximal segment, not being revealed in the liver, though. The protein accumulation in kidneys continues for several hours. The yellow fluorescent protein is homogeneously distributed in enterocytes, while in epithelial cells of the proximal tubule this protein is localized in vesicles. The data obtained indicate absorption of non-degraded yellow fluorescent protein in the intestine and role of kidney in metabolism not only of endogenous, but also of exogenous proteins.     

Radiation-induced mitotic delay: duration, dose and cell position dependence in the crypts of the small intestine in the mouse

The cells of the proliferative compartment in the crypt of the small intestine undergo a step by step differentiation and/or maturation from stem cells to the functional cells on the villi. The consequent hierarchical organization of the proliferative cell population can be related to the actual position of cells within the crypt. The stem cells are found near the bottom of the crypt with the more mature cells occurring at increasingly higher positions. The sensitivity of proliferative cells in the crypt of small intestine to radiation-induced mitotic delay was investigated at each position within the crypt. Using the stathmokinetic method (vincristine accumulation), the following were noted. The yield of mitotic figures 3 h immediately after irradiation showed a strong cell position dependence with the cells at the base of the crypt being most inhibited and those at the top of the proliferative compartment least affected. The mitotic yields were largely unaffected for the first 15 min suggesting that there is a transition point (Tp) for radiosensitivity which is located about 15 min before metaphase for all crypt cells. Cells located less than 15 min from metaphase are unaffected while those more than 15 min from metaphase are inhibited from further cell cycle progression. After this initial delay all proliferative cells were inhibited in their progression through G2 but some recovered more quickly than others. The ratio of the time of division delay (Td) in stem cells to that in cells at the top of the proliferative compartment was about 3:1. In absolute values Td after 1.0 Gy was about 1 h and 2.8 h, for cells at the top of the crypt and at the base, respectively. After 2.5 Gy the corresponding values were less than 3 h and between 5 and 6 h for the mid-crypt and crypt base respectively. There is thus a dependence on dose for the duration of the mitotic inhibition which for the cells at the top of the crypt is similar to the widely quoted average value 1 h per Gy, but the duration depends strongly on cell position. Thus not all proliferative cells respond in the same way. The duration is shorter the closer the proliferative cells are to their last cell division in the proliferative hierarchy in the crypt and longest for cells situated where the stem cells are to be expected.     

Effect of increasing lipid loads on the ability of the endoplasmic reticulum to transport lipid to the Golgi

We have previously shown (Mansbach, C. M. and P. J. Nevin, 1998. J. Lipid Res. 39: 963;-968) that after the development of a mass steady state with respect to triacylglycerol absorption in rats, the introduction of radiolabeled trioleoylglycerol, while maintaining the input rate of trioleoylglycerol constant at 135 micromol/h, was followed by a slow (60 min) achievement of a radiolabel steady state in the intestinal endoplasmic reticulum (ER). We hypothesized that this was due to the large input load and that the time to steady state would be shorter at lower lipid loads. Rats were infused intraduodenally with 22.5, 45, 90, or 135 micromol trioleoylglycerol/h for 6 h to obtain a mass steady state in the intestine. [(3)H]trioleoylglycerol was added to the infusate and the ER and Golgi were isolated from the proximal intestine after 5;-60 min of radiolabel infusion. The time required to reach a radiolabel steady state in the ER lengthened from 10 min at the 22.5 micromol/h infusion rate to 60 min at the 135 micromol/h rate. Similar data were obtained for the Golgi. Incubation of the ER with lipase reduced the ER;-triacylglycerol amount by 43% and increased its specific activity by 73%. The amount of [(3)H]TG-dpm in the ER was not reduced unless taurocholate, 10 mm, and colipase were added.We conclude that as the rate of triacylglycerol infusion is increased, TG movement from the ER to the Golgi progressively lengthens until finally all the triacylglycerol infused cannot be transported. A portion of this triacylglycerol is disposed on the cytoplasmic face of the ER and thus able to be attacked by lipase whereas another fraction is sequestered in the ER lumen and immune to lipase attack unless the ER membrane is solubilized.     

Trichostrongylus colubriformis: epithelial cell migration in the proximal and distal small intestine of infected rabbits

In Trichostrongylus colubriformis-infected rabbits, epithelial cell migration rates and cell transit times along the villi were compared by radioautography on histological slides to normal values from noninfected small intestine. Regions of gut with high (upper jejunum) and low (ileum) burdens of worms were both examined. In the control rabbits, the estimated values for the cell migration rates in the proximal and distal parts of gut were respectively 5.8 and 2.8 microns/hr. Seventy-two hours after the thymidine injection, the labeled epithelial cells were near the tip of the villi in the jejunum whereas only 60% of the villous length was labeled in the ileum. In the infected rabbits, the presence of T. colubriformis was associated with a two-fold increase of the cell velocity, in the main site of infection. Although less prominent than in the proximal region, a significant acceleration in the cell migration was also noticed in the ileum. The cell transit time was markedly reduced in the parasitized jejunum, but no variation of this parameter was found in the distal part of gut. These changes in the dynamics of epithelial cells in both regions of the gut appeared to underlie the morphological and enzymological changes of the parasitized mucosa. They particularly contribute to create an adaptive region in the small intestine beyond the main site of infection.     

Regional differences in the appearance of adult-type glycosphingolipids in the small intestine of inbred rats at weaning time

The small intestine of 15- to 33-day-old rats was cut into four segments: duodenum, proximal jejunum, distal jejunum, and ileum. Neutral glycosphingolipids and gangliosides were purified from each segment and analyzed by thin-layer chromatography in order to study the developmental appearance of adult-type glycolipids at each level of the small intestine. Type 1 A-6 glycolipid was first detected in the ileum at 15 days and subsequently in the jejunum and duodenum at 19 days of age. N-Glycolylneuraminic acid was expressed first in the ileum at 17 days, then in the proximal jejunum at 21 days, but only after 29 days in the duodenum. In each region, 6-8 days were required between first detection and full expression of N-glycolylneuraminic acid. The presence of 2-hydroxylated fatty acids in glucosylceramide was found first in the ileum at 19 days, 2-3 days before appearing in the duodenum and proximal jejunum. A period of 2-3 days was necessary to reach full adult-type level of 2-hydroxylated fatty acids in glucosylceramide. These results show that adult-type glycolipids appear earlier in the distal than in the proximal region of the rat small intestine, and that different glycolipids appear at different times and at different rates. The finding that the biochemical differentiation of the whole small intestine expands over a period of 3 days to 2 weeks, depending on the region and the glycolipid, before being fully completed indicates that, in addition to the time lag observed between the distal and the proximal region, the new cells arising from the crypt of Lieberkhün after 15 days of age are not at once fully differentiated.     

The effect of starvation on the control of phosphofructokinase activity in the epithelial cells of the rat small intestine.

1. The effect of depriving rats of food for 48 h on the specific activity of phosphofructokinase in the epithelial cells of the small intestine and on the regulatory properties of the enzyme displayed in crude (particle-free) mucosal extracts was studied. 2. The specific activity of phosphofructokinase, measured under optimal conditions at pH8, in the mucosa of fed rats showed a negative aboral gradient along the intestine, decreasing from 15.2 +/- 1.2 units (mumol/min)/g wet wt. in the proximal jejunum to 4.6 +/- 1.2 units/g wet wt. in the terminal ileum. 3. After starvation, the gradient was diminished, but not abolished; the diminution in gradient was due almost exclusively to a decrease in the specific activity of phosphofructokinase in the proximal jejunum by about 30%, there being no change in the terminal ileum. 4. In fed rats, the susceptibility of phosphofructokinase to inhibition by ATP, when assayed in crude mucosal extracts under suboptimal conditions, was independent of length along the small intestine; the ratio of the activity observed at pH 7.0 in the presence of 0.5 mM-fructose 6-phosphate and 2.5 mM-ATP to the optimal activity at pH 8, v0.5/V, was 0.36 +/- 0.05 in the proximal jejunum and 0.42 +/- 0.07 in the terminal ileum. 5. After starvation, the susceptibility of phosphofructokinase to inhibition by ATP was increased and was again found to be independent of length along the small intestine: after starvation, v0.5/V was 0.19 +/- 0.04 and 0.20 +/- 0.07 for the proximal jejunum and the terminal ileum respectively. 6. Re-feeding of previously starved rats on a high-carbohydrate diet overnight for 16 h restored both the specific activities of phosphofructokinase and its susceptibility to inhibition by ATP to normal values for fed rats. 7. The data support the idea that the specific activities and the regulatory properties of phosphofructokinase in the epithelial cells of rat small intestine are mediated by distinct humoral factors. 8. The changes in glucose utilization rate of the jejunum when rats are starved can in principle be accounted for by a combination of changes in the specific activity and in the regulatory properties of mucosal phosphofructokinase.     

Linear growth and poly(beta-hydroxybutyrate) synthesis in response to pulse-wise addition of the growth-limiting substrate to steady-state heterotrophic continuous cultures of Aquaspirillum autotrophicum.

Heterotrophic pyruvate-limited steady-state continuous cultures of the bacterium Aquaspirillum autotrophicum were perturbed with a pulse injection of a small volume of concentrated pyruvate solution. These cultures exhibited an instantaneous change in the growth dynamics, turning from steady state to apparently linear growth. These transient growth-responses had no lag phase and were clearly distinct from unlimited exponential growth according to the initial rates of increase of biomass and substrate disappearance kinetics. A linear accumulation with time of poly(beta-hydroxybutyrate) was observed within the cells. Slopes of these linear responses were negatively correlated with the dilution rate. Physiological bases of linear growth are discussed in the light of the models of H. E. Kubitschek. Poly(beta-hydroxybutyrate) synthesis in the absence of exogenous limitation may serve to protect the cells against a transient metabolic overflow.     

Flow rates of components in digesta of pigs prepared with re-entrant cannulas in the proximal duodenum and terminal ileum, and fed semipurified, hard wheat, and soft wheat diets.

Four pigs prepared with re-entrant cannulas in the proximal duodenum and terminal ileum were used to study flow rates of total digesta, insoluble dry matter, nitrogen, and amino acids entering and leaving the small intestine. The pigs received a semipurified diet, a hard wheat diet, or a soft wheat diet. These were approximately isonitrogenous. A higher rate of passage of digesta through the proximal duodenum and terminal ileum were measured in pigs receiving the hard wheat diet. Peak flow of digesta at the duodenum of all pigs occurred at 1 h post feeding. Peak flow of digesta at the ileum occurred at 9 h post feeding on the soft wheat diet, but somewhat earlier on the hard wheat and semipurified diet. More nitrogen and essential amino acids flowed in the solid fraction of duodenal digesta during the first 2 h post feeding for the wheat diets and 4 h post feeding for the semipurified diet. It was concluded that flow rate of most nutrients from the stomach and through the small intestine of pigs is modified by the composition and texture of the food ingested. It is postulated that efficiency of mixing of digesta with digestive secretions in the stomach is a major factor influencing rate of flow.     

Variation in amino acid transport along the rabbit small intestine. Mutual jejunal carriers of leucine and lysine.

The jejuno-ileal variation of amino and imino acid transport across the brush-border membrane of intact rabbit small intestine was studied. For the amino acids tested--beta-alanine, leucine, lysine, MeAIB, proline--and for D-glucose, the rates of transport at constant concentrations increase from very low values in the proximal jejunum to maximum values in the most distal 30 cm of the ileum. The apparent affinity constant for jejunal taurine transport is identical to that of the distal ileum, while the jejunal transport capacity is less than half. In the jejunum, as in the distal ileum, leucine and lysine share both sodium-dependent and sodium-independent carriers. Approx. 50% of the quantitative difference in transport capacity is accounted for by the absence of the beta-alanine carrier in the jejunum. These data indicate that the gradients of transport along the small intestine reflect gradients of transport capacities rather than affinities. In comparison with hamster, man and rat, the rabbit seems unique with respect to the location of transport maximum and the steepness of the gradient along the intestine.     

On correcting the error due to metaphase degeneration in stathmokinetic studies

Metaphases arrested by stathmokinetic agents have a limited life span. This may have to be taken into account in drawing conclusions from experiments whose duration is of the same order as the average metaphase life span, or, in the case of a combined arrest and labelling experiment, if G2 is of long duration. The average life span of arrested metaphases in the tissue under study can be estimated by a simple experiment which isolates the G2 cohort. This enables the investigator to decide whether he can trust his metaphase count without correction for loss due to degeneration. If the proposed experiment requires an accumulation of arrested metaphases over a period longer than the average life span of an arrested metaphase there is a serious risk of under-estimating the metaphase index. We propose a simple method of assessing the magnitude of this risk, and making the necessary correction. Assuming that metaphase degeneration is a random process a corrected index of arrested metaphases can be found for expanding populations multiplying the observed metaphase arrest index by exp [(λ-β) (t-m)], where m is the minimum life span of an arrested metaphase, t is the time point from the onset of the experiment, and β and λ are rates at which metaphases are formed and degenerate, respectively. A slight modification of this formula adapts it to steady state systems.     

The kinetics of villus cell populations in the mouse small intestine

Abstract. The cell population kinetics of the villus epithelium of the mouse have been analysed with respect to the size, flux and time. Microdissection methods were employed to measure the villus cell population size and yielded reproducible, precise results. There was a proximodistal negative size gradient in villus cell population and, in those villi of normal morphology, there was a good correlation with the usual morphometric estimators such as height and row count, although correlation was improved by a product variable consisting of a height multiplied by a width parameter.
Flux onto the villus is the product of the crypt cell production rate, which was measured by a metaphase arrest method using vincristine and crypt microdissection, and the crypt:villus ratio; net villus influx was maximum proximally in the bowel, where the largest villi were found, and decreased distally. The distribution of transit times of labelled cells to the crypt: villus junction and to the villus tip was measured, allowing the measurement of the median villus transit time.
Comparison of the measured villus transit time with the theoretical transit time calculated from the villus influx and population size gave results consistent with a steady state hypothesis. It was found, at each level of the small intestine studied, that the number of epithelial cells on the villus was equivalent to the total number of crypt cells associated with the villus.     

Aboral changes in D-glucose transport by human intestinal brush-border membrane vesicles.

D-Glucose transport was investigated in isolated brush-border membrane vesicles from human small intestine. Characteristics of D-glucose transport from the jejunum were compared with that in the mid and terminal ileum. Jejunal and mid-ileal D-glucose transport was Na+-dependent and electrogenic. The transient overshoot of jejunal D-glucose transport was significantly greater than corresponding values in mid-ileum. The terminal ileum did not exhibit Na+-dependent D-glucose transport, but did exhibit Na+-dependent taurocholate transport. Na+-glucose co-transport activity as measured by tracer-exchange experiments was greatest in the jejunum, and diminished aborally. We conclude that D-glucose transport in man is Na+-dependent and electrogenic in the proximal intestine and directly related to the activity of D-glucose-Na+ transporters present in the brush-border membranes. D-Glucose transport in the terminal ileum resembles colonic transport of D-glucose.     

Effect of diversion of bile-pancreatic juice to the ileum on pancreatic secretion and adaptation in the rat

Cannulas were implanted to collect bile and pancreatic juice, and the collected secretions were pumped back into the intestine at the level of the duodenum or the proximal ileum. The effect of 6 days of such treatment on pancreatic secretion and on pancreatic growth was determined. The effect on pancreatic secretion was studied by measuring the pancreatic secretory response to a stimulus, provided by acute diversion of bile-pancreatic juice from the proximal intestine. Trophic effects were studied in a separate group of rats by measuring pancreatic weight, protein content, and chymotrypsin activity after an overnight fast. Stimulated pancreatic secretion was 2.1 times greater for protein output and 3.4 times greater for fluid output in rats with chronic diversion of bile-pancreatic juice to the ileum. Pancreatic weight, protein content, and chymotrypsin activity were increased 2.6, 2.9, and 4.8 times, respectively, by chronic diversion of bile-pancreatic juice to the ileum. These results indicate that pancreatic hypertrophy and hyperplasia reported in rats with bile-pancreatic duct transposition to the ileum are the result of loss of feed-back inhibition from bile-pancreatic juice in the proximal intestine.     

Role of impaired regeneration of the mucosal epithelium in the pathogenesis of acute ulcers of the stomach and small intestine

The method of modelling of acute gastric and small intestinal ulcers by intraperitoneal administration of vincristine at a dose of 0.15-0.30 mg/kg body weight is suggested. The method is based on the property of vincristine to stop mitotic cell division at the stage of metaphase, thus impairing regeneration of the epithelium in the stomach and small intestine. The experiments on 20 dogs have shown that single vincristine administration caused the accumulation of cells with nuclei at the stage of metaphase in the generative areas. Mucosa defects formed at these sites acquired all the features of acute ulcers by the third day. The model demonstrates the pathogenetic role of impaired mucous epithelium regeneration and permits the studies of pathogenesis of acute ulcer formation.     

Identification of a Core Bacterial Community within the Large Intestine of the Horse

The horse has a rich and complex microbial community within its gastrointestinal tract that plays a central role in both health and disease. The horse receives much of its dietary energy through microbial hydrolysis and fermentation of fiber predominantly in the large intestine/hindgut. The presence of a possible core bacterial community in the equine large intestine was investigated in this study. Samples were taken from the terminal ileum and 7 regions of the large intestine from ten animals, DNA extracted and the V1-V2 regions of 16SrDNA 454-pyrosequenced. A specific group of OTUs clustered in all ileal samples and a distinct and different signature existed for the proximal regions of the large intestine and the distal regions. A core group of bacterial families were identified in all gut regions with clear differences shown between the ileum and the various large intestine regions. The core in the ileum accounted for 32% of all sequences and comprised of only seven OTUs of varying abundance; the core in the large intestine was much smaller (5-15% of all sequences) with a much larger number of OTUs present but in low abundance. The most abundant member of the core community in the ileum was Lactobacillaceae, in the proximal large intestine the Lachnospiraceae and in the distal large intestine the Prevotellaceae. In conclusion, the presence of a core bacterial community in the large intestine of the horse that is made up of many low abundance OTUs may explain in part the susceptibility of horses to digestive upset.     

A method to describe enzyme-catalyzed reactions by combining steady state and time course enzyme kinetic parameters

Background

Complete analysis of single substrate enzyme-catalyzed reactions has required a separate use of two distinct approaches. Steady state approximations are employed to obtain substrate affinity and initial velocity information. Alternatively, first order exponential decay models permit simulation of the time course data for the reactions. Attempts to use integrals of steady state equations to describe reaction time courses have so far met with little success.

Methods

Here we use equations based on steady state approximations to directly model time course plots.

Results

Testing these expressions with the enzyme β-galactosidase, which adheres to classical Michaelis–Menten kinetics, produced a good fit between observed and calculated values.

General significance

This study indicates that, in addition to providing information on initial kinetic parameters, steady state approximations can be employed to directly model time course kinetics.Integrated forms of the Michaelis–Menten equation have previously been reported in the literature. Here we describe a method to directly apply steady state approximations to time course analysis for predicting product formation and simultaneously obtain multiple kinetic parameters.     

Analyses of clinical, pathological and virological features of human rotavirus strain, YO induced gastroenteritis in infant BALB/c mice

Experimental studies of human rotavirus infections in mice are limited and there is lack of information on the quantitative assessment of rotaviral replication and its relationship with histological changes. In the present study, consequences of human rotavirus strain, YO induced gastroenteritis in infant BALB/c mice were analyzed for the occurrence of clinical symptoms, histopathology and virological events. The infected animals developed diarrhea and dehydration and showed accumulation of vacuolated enterocytes with lodging of the rotavirus antigens and shortening of villi in the intestine over a period of 5 days. The ileum was identified as the most susceptible and supportive part of small intestine for perpetuation of rotavirus infection in mice. Rotaviral antigen/RNA in stool and RNA in intestine were detected throughout the clinical disease period. At 48-72?h post inoculation, diarrhea was at the peak (90-95%) in the infected animals with increased load of viral RNA and intense pathological lesions suggesting it as the critical time point in the course of infection. The rising titers of antirotavirus neutralizing antibodies ascertained the replication of human rotavirus strain, YO in mice. These data may contribute to the understanding of pathophysiological, immunological and virological characteristics of rotavirus infections in mice.     

Plato's Cave Algorithm: Inferring Functional Signaling Networks from Early Gene Expression Shadows

Improving the ability to reverse engineer biochemical networks is a major goal of systems biology. Lesions in signaling networks lead to alterations in gene expression, which in principle should allow network reconstruction. However, the information about the activity levels of signaling proteins conveyed in overall gene expression is limited by the complexity of gene expression dynamics and of regulatory network topology. Two observations provide the basis for overcoming this limitation: a. genes induced without de-novo protein synthesis (early genes) show a linear accumulation of product in the first hour after the change in the cell''s state; b. The signaling components in the network largely function in the linear range of their stimulus-response curves. Therefore, unlike most genes or most time points, expression profiles of early genes at an early time point provide direct biochemical assays that represent the activity levels of upstream signaling components. Such expression data provide the basis for an efficient algorithm (Plato''s Cave algorithm; PLACA) to reverse engineer functional signaling networks. Unlike conventional reverse engineering algorithms that use steady state values, PLACA uses stimulated early gene expression measurements associated with systematic perturbations of signaling components, without measuring the signaling components themselves. Besides the reverse engineered network, PLACA also identifies the genes detecting the functional interaction, thereby facilitating validation of the predicted functional network. Using simulated datasets, the algorithm is shown to be robust to experimental noise. Using experimental data obtained from gonadotropes, PLACA reverse engineered the interaction network of six perturbed signaling components. The network recapitulated many known interactions and identified novel functional interactions that were validated by further experiment. PLACA uses the results of experiments that are feasible for any signaling network to predict the functional topology of the network and to identify novel relationships.     

Determinants of regional sucrase-isomaltase expression in adult rat small intestine

Sucrase-isomaltase (SI) expression along the longitudinal and vertical axis of the small intestine was studied by sequentially isolating enterocytes from villus to crypt of rat proximal jejunum and distal ileum. Gradients of sucrase activity were observed with greatest activity occurring in jejunal and villus regions. Along the villus-to-crypt axis, gradients of SI mRNA abundance corresponded with activity. However, along the longitudinal axis no differences in SI mRNA levels were observed, thus not accounting for the observed 3-5-fold difference in SI activities between jejunum and ileum. Comparison of SI immunoprecipitates from jejunal and ileal mucosal scrapings showed significant differences in gel mobilities of the more mature forms, which did not appear to affect SI functional activities. When relative rates of de novo SI protein synthesis were compared, [35S]methionine incorporation into all SI forms was observed to be 3-5-fold greater in jejunum than in ileum at all time points. Because these results suggested differences in regional translational regulation, subcellular distribution of SI mRNA in jejunal and ileal epithelial cells was compared. A greater proportion of jejunal SI mRNA was found to be associated with membrane-bound polyribosomes. We conclude 1) sucrase expression along the villus-to-crypt axis correlates with SI mRNA abundance, 2) post-translational processing of SI differ in ileum and jejunum, but appear not to determine SI expression, and 3) differences in translational processing in distal ileum and proximal jejunum may determine sucrase activity along the longitudinal axis of rat small intestine.