Application of wavelet denoising to improve compression efficiency while preserving integrity of digital micrographs.

Modern microscopy methods require efficient image compression techniques owing to collection of up to thousands of images per experiment. Current irreversible techniques such as JPEG and JPEG2000 are not optimized to preserve the integrity of the scientific data as required by 21 CFR part 11. Therefore, to construct an irreversible, yet integrity-preserving compression mechanism, we establish a model of noise as a function of signal in our imaging system. The noise is then removed with a wavelet shrinkage algorithm whose parameters are adapted to local image structure. We ascertain the integrity of the denoised images by measuring changes in spatial and intensity distributions of registered light in the biological images and estimating changes of the effective microscope MTF. We demonstrate that the proposed denoising procedure leads to a decrease in image file size when a reversible JPEG2000 coding is used and provides better fidelity than irreversible JPEG and JPEG2000 at the same compression ratio. We also demonstrate that denoising reduces image artefacts when used as a pre-filtering step prior to irreversible image coding.

Dietary cholecalciferol and phosphorus influence intestinal mucosa phytase activity in broiler chicks.

1. The role of cholecalciferol and phosphorus in the regulation of intestinal mucosa phytase was investigated in broiler chicks. 2. A total of 144 7-d-old male broiler chicks were grouped by weight into 6 blocks of 4 cages with 6 broiler chicks per cage. Four maize-soybean meal-based mash diets were randomly assigned to cages within each block. The 4 diets consisted of cholecalciferol at 0 or 75 microg/kg and total phosphorus at 3.6 or 7.0 g/kg in a 2 x 2 factorial arrangement. The birds were given the experimental diets for 12 d under conditions which excluded ultraviolet light. 3. Broiler chicks fed on diets with the higher concentration of cholecalciferol had higher Vmax and Km of the mucosa phytase, weight gain, feed intake, feed efficiency and percentage tibia ash, higher ileal digestibility of dry matter, energy, phosphorus (P) and calcium (Ca), and increased retention of dry matter, nitrogen, P, Ca and energy. 4. Broiler chicks receiving diets with the higher P concentration showed lower Vmax and Km of the intestinal mucosa phytase but greater weight gain, feed intake, feed efficiency and percentage tibia ash, higher ileal digestibility of dry matter, energy, P and nitrogen, and increased retention of dry matter, energy, nitrogen and Ca. 5. In conclusion, both dietary P and cholecalciferol influenced the activity of intestinal mucosa phytase.

Estimation of apparent L-amino acid diffusion in porcine jejunal enterocyte brush border membrane vesicles.

There is an overlap of carrier-mediated L-amino acid transport and apparent simple diffusion when measured in intestinal brush border membrane vesicles. Using L-threonine and L-glutamine as representative amino acids, this study was undertaken to estimate apparent simple diffusion of L-amino acids and to establish the effective dosage of HgCl2 for completely blocking carrier-mediated L-amino acid transport in porcine jejunal enterocyte brush border membrane vesicles. Jejunal mucosa was scraped from three pigs weighing 26 kg. Enterocyte brush border membrane vesicles, with an average enrichment of 24-fold in sucrase specific activity, were prepared by Mg2+-precipitation and differential centrifugation. In vitro uptake was measured by the fast filtration manual procedure. HgCl2 blocked the carrier-mediated initial transport of L-threonine and L-glutamine under Na+-gradient condition in a dose-dependent manner. At the minimal concentration of 0.165 micromol HgCl2 mg(-1) protein, carrier-mediated L-threonine and L-glutamine transport was completely inhibited. The apparent L-threonine and L-glutamine diffusion was estimated to be 8.6+/-0.7 and 12.4+/-1.0% of the total uptake at the substrate concentrations of 5 microM (L-threonine) and 50 microM (L-glutamine). Therefore, the treatment of porcine brush border membrane vesicles with a minimum of 0.165 micromol HgCl2 mg(-1) protein completely blocks carrier-mediated L-amino acid transport and enables the direct estimation of apparent L-amino acid diffusion in enterocyte brush border membrane vesicles.

Detection of lysozyme in llama, sheep, and cattle tears.

OBJECTIVE: To determine whether the tears of llamas, sheep, and cattle contain lysozyme and compare lysozyme concentrations in tears among these species. ANIMALS: 40 llamas, 5 sheep, and 36 cattle. PROCEDURE: Electrophoresis, western blot immunoassay for lysozyme, a spectrophotometric assay to detect tear lysozyme by its ability to lyse a suspension of Micrococcus lysodeiticus, and a microtiter plate colorometric assay were performed. RESULTS: A 13.6-kd protein band was detected by use of electrophoresis and western blot immunoassay in llama and sheep tears but not cattle tears. Results of spectrophotometric assay suggested that llama and sheep tears had high concentrations of lysozyme, whereas cattle tears had low concentrations. Results of the microtiter plate colorometric assay suggested that llama tears had high concentrations of lysozyme, whereas concentrations in sheep and cattle tears were lower. CONCLUSIONS AND CLINICAL RELEVANCE: Lysozyme concentrations in tears may vary among species and this variability may contribute to differing susceptibilities to ocular diseases such as infectious keratoconjunctivitis.

Comparison of tear proteins of llamas and cattle.

OBJECTIVE: To analyze and compare contents of the preocular tear films of llamas and cattle. ANIMALS: 40 llamas and 35 cattle. PROCEDURE: Tear pH was determined by use of a pH meter. Total protein concentration was determined by use of 2 microtiter methods. Tear proteins were separated by use of electrophoresis and molecular weights of bands were calculated. Western blot immunoassay was used to detect IgA, lactoferrin, transferrin, ceruloplasmin, alpha1-antitrypsin, alpha1-amylase, and alpha2-macroglobulin. Enzyme electrophoresis was used to detect proteases. RESULTS: The pH of llama and cattle tears were 8.05 +/- 0.01 and 8.10 +/- 0.01, respectively. For results of both methods, total protein concentration of llama tears was significantly greater than that of cattle tears. Molecular weights of tear protein bands were similar within and between the 2 species, although llama tears had a distinct 13.6-kd band that was not detected in cattle. Lactoferrin, IgA, transferrin, ceruloplasmin, alpha1-antitrypsin, alpha1-amylase, alpha2-macroglobulin, and proteases were detected in both species. CONCLUSIONS AND CLINICAL RELEVANCE: Llama tears have significantly greater total protein concentration than cattle tears, whereas pH is similar between species. Because little variation was detected within species for the number and molecular weight of protein bands, pooling of tears for analysis is justified. Results suggest that lactoferrin, ceruloplasmin, transferrin, alpha1-antitrypsin, alpha2-macroglobulin, alpha1-amylase, and IgA are present in the tears of llamas and cattle.

Low extracellular Ca(2+) activates a transient Cl(-) current in chicken ovarian granulosa cells.

The effects of low Ca(2+) on ion currents in hen ovarian granulosa cells were examined. A fast activating and inactivating transient outward current (TOC) and a slowly activating outward current (SOC) could be observed. In the presence of normal Ca(2+) concentration (2. 5 mM) and with a holding potential of -80 mV, SOC was activated in all cells with command pulses more positive than -20 mV. In 2.5 mM Ca(2+), TOC appeared in 10% of cells at the command pulse of +80 mV and in 60-85% of cells at +100 to +120 mV. In low-Ca(2+) solution and command potential of +80 mV (holding potential of -80 mV), the amplitude of TOC was enhanced in cells that expressed it in normal Ca(2+), and TOC appeared in 43% of the cells that did not express it initially in normal Ca(2+). At both normal and low Ca(2+) levels, TOC decreased as the holding potential became more positive. TOC was reduced in Cl(-)-deficient solution and in the presence of 5-nitro-2-(3-phenylpropylamino)benzoic acid, a Cl(-) channel blocker. These findings suggest that chicken granulosa cells express a Ca(2+)-inactivated TOC carried by Cl(-). This current may serve as a signal for some of the reduced metabolic functions of granulosa cells associated with Ca(2+) deficiency.

Studies on the mechanism of diarrhoea induced by Escherichia coli heat-stable enterotoxin (STa) in newborn calves.

Enterotoxigenic Escherichia coli (ETEC) produces a heat-stable enterotoxin (STa) that binds to and activates a putative intestinal receptor, guanylate cyclase, causing an increase in the intracellular levels of cyclic guanosine monophosphate (cGMP). Using flow cytometry and 125I-STa binding assays, we studied the distribution of STa-receptors on enterocytes isolated from different segments of the newborn calf's intestinal tract. We also investigated the effect of STa on the intracellular levels of cGMP and ion transport to the intestinal lumen. More STa-receptors were found on enterocytes prepared from the ileum than on enterocytes obtained from the other segments of the intestinal tract. Guanylate cyclase activity was higher in the ileum of STa-challenged calves than in the ileum of control calves. No changes were observed in the guanylate cyclase activity of the other intestinal segments of the STa-challenged and control calves. Na+ levels, as measured by atomic absorption spectroscopy, were significantly increased in the luminal contents of the ileum of STa-challenged calves, whereas serum Cl- levels were significantly lower in the STa-challenged calves than in control calves. This study supports previous observations on the role of guanylate cyclase in the initiation of STa-induced secretory diarrhoea and suggests that Na+/Cl- coupling may be the major mechanism for the loss of ions in the diarrhoeal response that is mostly induced in the ileum of newborn calves.

Characterization of the interaction of Escherichia coli heat-stable enterotoxin (STa) with its putative receptor on the intestinal tract of newborn calves.

Enterotoxigenic Escherichia coli (ETEC) induces severe diarrhea in newborn calves through the elaboration of heat-stable enterotoxin (STa). We investigated the distribution and characteristics of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from anterior jejunum, posterior jejunum, ileum and colon of newborn calves. We found that density of the receptors and their affinity to STa were higher on enterocytes and BBMVs that were derived from the ileum than enterocytes and BBMVs prepared from other segments of the calf intestine. This study suggests that, in newborn calves, the ileum is the major part of the intestinal tract that is affected in the course of secretory diarrhea caused by STa-producing ETEC strains.

Identification of some components of basal lamina of avian ovarian follicle.

Experiments were conducted to identify components of the basal lamina of the ovarian follicle. Pure and intact basal lamina was isolated from preovulatory follicles of the chicken ovary. Some components of the basal lamina could be solubilized with guanidine-HCl (designated Fraction 1) and remaining components with beta-mercaptomethanol containing guanidine-HCl (designated Fraction 2). With Western blot analysis, monoclonal and polyclonal antibodies raised against avian, mammalian, and human proteins recognized proteins in Fractions 1 and 2 of solubilized basal lamina. Thus, antibodies raised against extracellular matrix proteins, laminin, fibronectin, entactin or nidogen, tenascin, heparan sulfate proteoglycan, osteonectin, and Type IV collagen reacted positively with basal lamina proteins. Antibodies raised against the growth factors; epidermal growth factor; acidic and basic fibroblast growth factors; platelet-derived growth factor-AA; transforming growth factor-alpha; transforming growth factor-beta1, -beta2, -beta3, and -beta5; and insulin-like growth factor-I and -II cross-reacted with basal lamina proteins. Similarly, antibodies raised against insulin-like growth factor-binding proteins-2, -3, -4, -5, -6, and -7 cross-reacted with basal lamina proteins. In addition, antibodies generated against matrix metalloproteinases-1, -2, -3, -4, -8, -9, and -13 reacted positively with basal lamina proteins. Furthermore, antibodies produced against tissue inhibitors of matrix metalloproteinases-1, -2, -3, and -4 also reacted positively with basal lamina proteins. Moreover, interleukin-3, granulocyte macrophage-colony-stimulating factor, interferon-gamma antibodies recognized proteins in basal lamina. These observations are consistent with the view that the basal lamina of avian ovarian follicle is a store or source of biologically active molecules, namely growth factors, growth factor-binding proteins, cytokines, matrix metalloproteinases, and their tissue inhibitors. The growth factors could exert major effects on ovarian cell behavior and function, and the enzymes could participate in tissue remodeling during follicular development.

Endothelial-like cells from the bovine placental cotyledon.

A cell-line was established from bovine placental cotyledon. When cultured in M199 with 10% fetal bovine serum, this cell-line had a doubling time of about 18 h. With immunohistochemistry, it was demonstrated that this cell-line expressed vimentin and angiotensin-converting enzyme (ACE). While both molecules are expressed in endothelial cells, ACE is usually considered to be a specific marker for endothelial cells. Furthermore, cells were shown to take up Dil-Ac-LDL (acetylated low-density lipoprotein labeled with 1,1'-dioctadecyl-3,3,3'-tetramethylindo-carbocyanine perchlorate). This characteristic feature has been used to identify endothelial cells. Finally, when cultured on matrigel, this cell-line formed tube-like structures similar to those formed by endothelial cells. Tube-formation on matrigel is a physiological property specific to endothelial cells. In conclusion, these three lines of evidence strongly suggest that this cell-line is endothelial cell in nature. Further studies using an endothelial cell-line from bovine placenta may help to elucidate the cause of bovine placental retention, a major cause for economic loss in bovine industry. Furthermore, an endothelial cell-line could be an important tool in research areas such as tissue remodeling, angiogenesis, and cancer.

The effects of tannins on nutrient utilisation in the White Pekin duck.

1. Two experiments were conducted to determine the effects of tannins on nutrient utilisation in the White Pekin duck. 2. Experiment 1 was a rapid nutrient balance assay to determine the nitrogen (N) retention and metabolisable energy (ME) of maize, low-tannin sorghum (P-954063) (Sorghum bicolor (L). Moench) and high-tannin sorghum (IS-4225) cultivars for ducks. The assay lasted 120 h, with an initial 24 h food-deprivation period, a 48 h excreta collection period for endogenous losses and a 48 h excreta collection period for ingredient losses. The true metabolisable energy (TMEn) content was lower (P<0.05) in the high-tannin sorghum cultivar (13.85 MJ/kg) than the maize (14.94 MJ/kg) and the low-tannin sorghum cultivar (14.39 MJ/kg). True N retention was lower (P<0.05) for the high-tannin sorghum (0.24 g) than for maize (1.33 g) and low-tannin sorghum (1.1 g). 3. In experiment 2, the brush-border membrane vesicles technique was used to determine whether tannic acid caused inhibition of L-threonine transport across duck intestinal brush-border membrane. The brush-border membrane vesicles were mixed with tannic acid solutions (pH 7.4) to give gradient tannic acid concentrations of 0, 0.05, 0.10, 0.25, 0.50, 1.00 and 2.50%. As a fraction of the control (no tannic acid), the maximal inhibition of L-threonine transport (Imax) under the sodium-gradient condition was 77.10% (P<0.05). Under the sodium-free condition, the maximal inhibition of L-threonine transport (Imax) was 45.15% (P<0.05). 4. These results demonstrated that nutrient utilisation in the White Pekin duck was lower from the high-tannin sorghum cultivar than from the low-tannin sorghum cultivar. The results also suggested that the antinutritive effects of tannins in foodstuffs are due partly to their inhibitory action on intestinal brush-border bound amino acid transporter proteins.

Basal lamina of avian ovarian follicle: influence on morphology of granulosa cells in-vitro.

Experiments were conducted to determine the influence of basal lamina on the morphology of ovarian granulosa cells in vitro. Pure and intact basal lamina was isolated from the large preovulatory follicles of the chicken ovary and designated basal lamina of avian ovarian follicle (BLAOF). Examination of the isolated basal lamina with electron microscope revealed an ultrastructure that is similar to that of basal lamina in the intact ovarian follicle. Pieces of the intact basal lamina were attached to the bottom of 32 mm culture dishes (BLAOF-coated dishes) in which differentiated granulosa cells isolated from the largest preovulatory follicle or undifferentiated granulosa cells isolated from immature small yellow chicken ovarian follicles were cultured; uncoated dishes served as controls. Granulosa cells incubated on intact basal lamina assumed spherical shape, whereas granulosa cells incubated directly on plastic in control dishes became highly flattened. Interestingly, granulosa cells that attached to plastic close to BLAOF (in BLAOF-containing dishes) became rounded. The storage of BLAOF-coated culture dishes at 4 degrees C for 2 years had no apparent effect on its ability of the matrix material to induce changes in granulosa cell shape. Some components of the basal lamina could be solubilized with guanidine-HCl alone (fraction 1; 90-95% of total protein in BLAOF) with the remaining components solubilized with beta-mercaptoethanol containing guanidine-HCl (fraction 2; 5-10% of total protein in BLAOF). Differentiated and undifferentiated chicken granulosa cells became rounded when incubated in fraction 1-pre-coated wells; whereas those incubated directly on plastic in control wells were flattened. Similarly, when fraction 1 of solubilized basal lamina was added as liquid to incubation mixture, it caused both differentiated and undifferentiated granulosa cells to assume spherical shapes. The storage of fraction 1-coated culture dishes at 4 degrees C for 12 or more months had no apparent effect on its ability to influence granulosa cell shape. Fraction 1-induced changes in granulosa cell shape were similar to those observed for complete and intact basal lamina (BLAOF). These findings demonstrate that intact homologous basal lamina (BLAOF) or its solubilized (fluidized) form can induce normal (in vivo) morphology in granulosa cells. It is suggested that BLAOF or its solubilized form can be used to culture cells in experiments designed to examine the influence of the natural basal lamina microenvironment on cellular behavior and function.

Effect of basal lamina on progesterone production by chicken granulosa cells in vitro--influence of follicular development.

Experiments were conducted in vitro to study the regulation of progesterone production in chicken granulosa cells by homologous basal lamina isolated from preovulatory follicles of chicken ovary. The majority of components of the basal lamina (90-95% by weight) were solubilized with guanidine-HCl (and designated fraction 1); the remaining components were solubilized with beta-mercaptoethanol containing guanidine-HCl (and designated fraction 2). The ability of fraction 1 to regulate progesterone production in granulosa cells obtained from the largest (F(1), mature), third largest (F(3), growing), fifth to seventh largest (F(5-7), growing) follicles and a pool of small yellow follicles (SYF, immature) of chicken ovary was assessed. Granulosa cells isolated from SYF follicles were in the least differentiated (undifferentiated) and those obtained from F(1) follicles were in the most differentiated state. The ability of fraction 1 to regulate progesterone production by chicken granulosa cells was influenced both by the state of cell differentiation and the form of the matrix material (whether solid or liquid). When fraction 1 was added as liquid to the incubation mixture, it promoted progesterone production by granulosa cells at all stages of differentiation; however, it caused a greater relative increase in the amount of progesterone produced by undifferentiated (SYF) and differentiating (F(3)) granulosa cells than by differentiated (F(1)) ones. In the presence of the liquid-form of fraction 1, luteinizing hormone (LH) stimulated progesterone production in differentiated (F(1)) and differentiating (F(5-7)) granulosa cells. Similarly, follicle-stimulating hormone (FSH) stimulated progesterone production by differentiating (F(3)) and undifferentiated (SYF) granulosa cells in the presence of the liquid-form of fraction 1 protein. In culture wells that had been pre-coated with fraction 1 (solid-form), progesterone production by less differentiated (SYF, F(5-7)) granulosa cells was enhanced, whereas progesterone production by differentiated (F(1)) cells was reduced. The solid-form of fraction 1 augmented LH-stimulated progesterone production by less differentiated (F(5-7)) granulosa cells however, it attenuated LH-induced progesterone production in differentiated (F(1)) cells. FSH-promoted progesterone production in granulosa cells from immature follicles (SYF) was augmented by solid-form of fraction 1 whereas the effect of FSH on cells obtained from older follicle (F(3)) was suppressed by solid-form of fraction 1. In experiments in which gonadotropin action was attenuated by solid-form of fraction 1, the amount of progesterone produced in the presence of maximally inhibiting concentrations of fraction 1 protein was greater than control values (no fraction 1, no gonadotropin). These results show that basal lamina of the ovarian follicle can regulate progesterone production by granulosa cells. The data demonstrate that the interactions between the components of basal lamina and LH or FSH on granulosa cell function were dependent on the stage of follicular development and were influenced by the form of the matrix material. It is concluded that the basal lamina of the chicken ovarian follicle is biologically active and regulates granulosa cell function.

Ontogenetic development of intestinal digestive functions in White Pekin ducks.

The ontogenetic development of intestinal digestive functions for avian species other than the domesticated chicken are not well documented. Therefore, this study was conducted to resolve the developmental patterns of some intestinal digestive functions in White Pekin ducks. The ducks were killed and their intestines harvested when they were 1, 3, 5 and 7 wk old. Several small intestinal tissue characteristics, sucrase and alkaline phosphatase (ALP) activities of homogenates from the small intestine mucosa were measured, and the small intestinal L-threonine uptake capacities were estimated with brush border membrane vesicles prepared from the corresponding age groups. Between 1 wk (0.37 +/- 0.04 kg) and 7 wk (3.79 +/- 0.06), posthatch ducks exhibited relative body growth rates of 352, 77 and 28% from 1 to 3, 3 to 5 and 5 to 7 wk, respectively. Allometric changes in small intestine weight indicated that the small intestine grew in direct proportion to the duck's metabolic body weight. Total homogenate sucrase activity per unit body weight did not differ (P > 0.05) among the age groups studied. Total homogenate ALP activity per body weight was lower at 3 wk than at 1 wk (P < 0.05) but did not differ (P > 0.05) among 3, 5 and 7 wk-old ducks. The development pattern of L-threonine uptake capacities normalized to body weights paralleled the course of relative body growth rates.

Characterization of the interaction of Escherichia coli heat-stable enterotoxixn (STa) with its intestinal putative receptor in various age groups of mice, using flow cytometry and binding assays.

BACKGROUND AND PURPOSE: Enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) is a major cause of diarrhea in young animals. Age-dependent variation in the density and affinity of the mouse enterocyte receptors specific for STa was investigated. METHODS: Four age groups (2-day-, 1- and 2-week-, and 2-month-old) of Swiss Webster mice were studied (8 to 10 mice/group). Flow cytometry and radiolabeled STa (125I-STa) assays were used as reliable quantitative measures for characterization of STa-enterocyte receptor interaction. RESULTS AND CONCLUSIONS: Interaction of STa with its putative receptor was stronger for enterocytes of 2-day-old mice. Scatchard analysis of 125I-STa-receptor interaction suggested that STa-receptors exist at higher numbers on enterocytes from 2-day-old (7.2 nmol/mg) than older (0.30, 0.36, and 0.40 nmol/mg for 1-week-, 2-week-, and 2-month-old mice, respectively). Additionally, receptors from 2-day-old mice had greater affinity for STa (Kd = 75 nM) than did receptors from older mice (Kd = 125, 1,430, and 1,111 nM for 1-week-, 2-week-, and 2-month-old mice, respectively). Density of STa receptors on enterocytes and their affinity to STa may determine extent of binding and severity of the secretory response, and may explain the high susceptibility of newborn animals and human infants to STa-mediated diarrhea.

Use of flow cytometry to measure the interaction between Escherichia coli heat-stable enterotoxin and its intestinal receptor in mice.

Binding of Escherichia coli heat-stable enterotoxin (STa) to its putative receptor on the brush border membrane of enterocytes is a prerequisite for the induction of diarrhea in infected humans and animals. Humans and animals of different ages vary in their susceptibility to the effect STa, perhaps due to the difference in STa interaction with its intestinal receptor. Flow cytometry was compared to indirect immunofluorescence and 125I-STa binding assays to measure the STa-enterocytes receptor interaction in different age groups of Swiss Webster mice (2-, 7-, 14-day-old). Flow cytometry indicated stronger interaction between STa and its putative receptor on enterocytes from the 2-day-old mice than enterocytes from older mice. 125I-STa-binding assay suggested that the stronger fluorescence intensity on enterocytes from younger mice is due to higher STa receptor density and higher receptor affinity to STa. Flow cytometry is more sensitive quantitative assay to measure the interaction between STa and its intestinal receptor than indirect immunofluorescence microscopy.

Methodological aspects of measuring amino acid uptake in studies with porcine jejunal brush border membrane vesicles.

With L-glutamine, as a representative amino acid this study was undertaken to examine the effects of substrate concentrations on initial and equilibrium amino acid uptake and intravesicular volume determined with porcine jejunal brush border membrane vesicles prepared by Mg2+-aggregation and differential centrifugation. Transport measurements (24 degrees C) were conducted by the rapid filtration manual procedure. Glutamine uptake was shown to occur into an osmotically-active space ranging between 1.09-1.58 microl/mg protein with little non-specific membrane binding. At different concentrations (in parentheses), the duration of initial glutamine uptake in both Na+ gradient and Na+-free conditions was 10 s (0.01 mM), 15 s (0.17 mM), and 20 s (1.9 and 9.4 mM), respectively. Substrate concentrations affected the duration of initial uptake, with lower substrate concentrations giving shorter duration for initial amino acid uptake. At different substrate concentrations (in parentheses), the time required to reach equilibrium glutamine uptake was 5 min (0.01 mM), 10 min (0.17 mM), and 60 min (1.9 and 9.4 mM), respectively. Thus, substrate concentrations also affected the time required to reach equilibrium uptake. The higher the substrate concentration, the longer the incubation time needed to reach equilibrium amino acid uptake. At the glutamine concentrations of 0.01, 0.17, 1.9, and 9.4 mM, the average intravesicular volume was estimated to be 1.58+/-0.21, 1.09+/-0.28, 1.24+/-0.18, and 1.36+/-0.21 microl/mg protein, respectively. Substrate concentrations had no effect (p>0.05) on the intravesicular volume of membrane vesicles. In conclusion, in the experiments on amino acid transport kinetics measured with the rapid filtration manual procedure, the incubation time used for measuring the initial uptake rate should be determined from the time course experiments conducted at the lowest substrate concentration used, whereas the intravesicular volume can be obtained from equilibrium uptake measured at any substrate concentrations.

Insulin modulates intestinal response of suckling mice to the Escherichia coli heat-stable enterotoxin.

Effect of insulin on the response of suckling mice to the enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) was studied. Four groups (8-10 in each group) of two day old Swiss Webster suckling mice were used. Five, 10, 25, and 50 micrograms of insulin were given orally to half the mice in each group respectively. The rest of the mice in each group were given normal saline as intra-litter controls. After 7 days, the suckling mouse assay for STa was performed on three mice from each insulin-treated and control groups. Enterocyte suspensions were prepared from mice in all groups. Intestinal tissue samples were taken for electron microscopy. Interaction of STa with its putative receptor on the enterocytes was evaluated using indirect immunofluorescence and flow cytometry. The suckling mouse assay revealed a significant increase in the gut weight to body weight ratio in all mice in the insulin treated groups compared to control mice (p < 0.05). Flow cytometry and indirect immunofluorescence analyses suggested that insulin had an upregulatory effect on the STa receptor level. Similarly, insulin was found to increase intestinal brush border membrane differentiation as indicated by the increase in the inward movement of milk particles through the intestinal mucosa. Insulin seems to modify the structure-function of the brush border membrane including the response of suckling mice to STa. This study may provide further insights into the mechanism of STa/receptor interaction in diarrhea in newborn animals and human infants.

Age-dependent variation in the density and affinity of Escherichia coli heat-stable enterotoxin receptors in mice.

Enterotoxigenic strains of Escherichia coli that produce heat-stable enterotoxin (STa), are a major cause of diarrheal disease worldwide. Resistance to diarrheal disease in human infants and newborn animals has been attributed to a gradual turnover in the intestinal brush border membrane receptors to bacterial pili. In this study, we demonstrated age-dependent variation in the density and affinity of the mouse enterocyte receptors specific for STa. Flow cytometry and radiolabeled-STa (125I-STa) assays were used as more reliable quantitative measures for the characterization of STa-enterocyte receptor interaction. These assays indicated a stronger interaction of STa with its putative receptor on the enterocytes of the 2-day-old suckling mice than with enterocytes from 1-week, 2-week and 2-month-old mice. Scatchard plot analysis of 125I-STa-receptor interaction suggested that STa-receptors exist at a higher number on enterocytes from the 2-day-old mice than enterocytes of the older mice. Additionally, receptors from the 2-day-old mice had a greater affinity for STa ligand than receptors from the older mice. Density of STa receptors on enterocytes and their affinity to STa may determine the extent of binding and severity of secretory response. This may further explain the increased susceptibility of newborn animals and human infants to STa-mediated diarrheal disease.

Characterization of brush border membrane-bound alkaline phosphatase activity in different segments of the porcine small intestine.

This study was conducted to characterize enterocyte apical membrane-bound alkaline phosphatase activity in different segments of the porcine small intestine. Duodenal, jejunal, and distal ileal segments were isolated from three 26-kg pigs and enterocyte brush border membrane, enriched between 19- and 24-fold in sucrase specific activity, was prepared by Mg(2+) precipitation and differential centrifugation. With P-nitrophenyl phosphate as substrate, the optimum pH for porcine brush border membrane-bound alkaline phosphatase activity was defined to be 10.5 for all three segments. At the optimal pH, the kinetics of membrane-bound alkaline phosphatase were determined for the three intestinal segments. The affinity of this enzyme (K(m), mM) in the jejunum (0.64 +/- 0.07) was four times greater than that in the duodenum (2.75 +/- 0.59) and the distal ileum (2.71 +/- 1.14). These results indicate that different isomers of membrane-bound alkaline phosphatase might have been expressed in different segments of porcine small intestine. The maximal specific activity (V(max), micromol/mg protein . min) of this enzyme was highest in the duodenal (7.74 +/- 0.95), intermediate in the jejunal (4.31 +/- 0.18), and lowest in the distal ileal (3.53 +/- 0.84) brush border membrane. Therefore, the maximal specific activity of brush border membrane-bound alkaline phosphatase along the intestinal longitudinal axis in growing pigs decreases from the duodenum toward the distal ileum.