Characterization of carbohydrates using a combination of derivatization, high-performance liquid chromatography and mass spectrometry

Mice trisomic for the distal portion of MMU 16 (Ts65Dn) were examined for differences in jejunal function and plasma amino acids as compared to diploid controls. Eighteen control and 19 Ts65Dn mice were compared for whole-body and intestinal O2 consumption, jejunal glucose uptake, and plasma amino acid concentrations. Ts65Dn mice consumed less (P < 0.02) O2 per gram of fasted body weight. No significant differences were found in either active or passive glucose uptake. Oxygen consumption by jejunal tissue was not different between Ts65Dn and control mice. The apparent energetic efficiency of jejunal active glucose uptake (eta mol ATP expended/eta mol glucose uptake) was significantly higher (115.6 vs. 80.8; P < 0.05) in Ts65Dn mice. Histomorphometric analysis of jejunal mucosa showed that Ts65Dn mice had shorter villus height (P < 0.04) and decreased planar villus circumference (P = 0.05). No differences were found in total jejunal protein (microgram/g) or DNA (mg/g) concentrations. Significantly higher concentrations of plasma tyrosine, phenylalanine, valine, leucine, isoleucine, and citrulline (P < 0.05) were found in Ts65Dn mice. Lower plasma concentrations of hydroxyproline were detected in Ts65Dn mice (P < 0.05). These data suggest that Ts65Dn mice have anomalies in digestive function and amino acid metabolism as compared to normal, diploid controls.     

The fine structural development of preimplantation mouse parthenotes

Crossfostering was performed using lines selected for increased 6-week body weight (H6) and increased 3-to 6-week postweaning gain (M16) and their reciprocal F1 crosses as nurse dams in the selected crossfostering group, and base population controls (C2, ICR) and their reciprocal F1 crosses in the control group. The offspring suckled were H6, M16 and F2 crosses in the selected group, and C2, ICR and their F2 crosses in the control group. Measurements taken on the individual offspring were body weights at birth (WB) and at 12, 21, 31, 42, and 63 days (W12, W21, W31, W42 and W63, respectively) and weight gains between adjacent ages (GB-12, G12-21, G21-31, G31-42 and G42-63, respectively). Least squares constants fitted to populations of genetic and nurse dams were used to calculate specific linear contrasts. Correlated responses to selection in average direct genetic effects were significant and positive for all traits examined in both H6 and M16, while the correlated responses in average maternal genetic effects were negative in M16 and negligible in H6. Selection response was primarily due to average direct genetic effects while the contribution of average maternal genetic effects was of secondary importance. The response in average direct genetic effects was smaller in M16 for postweaning weights (W31, W42 and W63). The correlated responses in average maternal genetic effects were consistently smaller in M16 than in H6. Direct heterosis was significant for all traits except for G12-21 and G42-63 in the control group, whereas maternal heterosis was significant for weight gains at early ages and for body weights. Direct heterosis tended to be larger than maternal heterosis in both selected and control crosses. Percent direct heterosis for body weight was larger in the selected crosses relative to the control crosses through 31 days of age, but the trend was reversed by 63 days. Percent maternal heterosis was consistently larger in the selected crosses.     

Intestinal water and solute absorption studies: comparison of in situ perfusion with chronic isolated loops in rats

The effects of lumenal glucose on jejunal water transport and the influence of glucose-induced water absorption on solute uptake from single-pass perfusions are compared in anesthetized rats in situ and isolated chronic loops in unanesthetized rats in vivo. While the magnitudes of solute membrane permeabilities are consistently higher in the chronic loop system, the effects on water transport and its promotion of jejunal solute uptake are comparable between the two experimental systems. The effect of glucose-induced water absorption on the enhanced/baseline jejunal uptake ratio of the hydrophilic drug, acetaminophen, is greater than that for the lipophilic drug, phenytoin, in both experimental systems. The fact that chronic loop effective solute permeabilities were equivalent to solute membrane permeabilities in situ is consistent with greater lumenal fluid mixing in vivo. In addition, in situ body temperature affects the uptake of phenytoin but not acetaminophen, water, or glucose. This suggests that active and paracellular solute transport is not compromised in situ, while membrane partitioning and diffusion of lipophilic species are more sensitive to experimental conditions.     

Low-affinity potassium uptake system in the archaeon Methanobacterium thermoautotrophicum: overproduction of a 31-kilodalton membrane protein during growth on low-potassium medium

During growth on low-K+ medium (1 mM K+), Methanobacterium thermoautotrophicum accumulated K+ up to concentration gradients ([K+]intracellular/[K+]extracellular) of 25,000- to 50,000-fold. At these gradients ([K+]extracellular of < 20 microM), growth ceased but could be reinitiated by the addition of K+ or Rb+. During K+ starvation, the levels of a protein with an apparent molecular weight of 31,000 increased about sixfold. The protein was associated with the membrane and could be extracted by detergents. Cell suspensions of M. thermoautotrophicum obtained after K+-limited growth catalyzed the transport of both K+ and Rb+ with apparent Km and Vmax values of 0.13 mM and 140 nmol/min/mg, respectively, for K+ and 3.4 mM and 140 nmol/min/mg, respectively, for Rb+. Rb+ competitively inhibited K+ uptake with an inhibitor constant of about 10 mM. Membranes of K+-starved cells did not exhibit K+-stimulated ATPase activity. Immunoblotting with antisera against Escherichia coli Kdp-ATPase did not reveal any specific cross-reactivity against membrane proteins of K+-starved cells. Cells of M. thermoautotrophicum grown at a high potassium concentration (50 mM) catalyzed K+ and Rb+ transport at similar apparent Km values (0.13 mM for K+ and 3.3 mM for Rb+) but at significantly lower apparent Vmax values (about 60 nmol/min/mg for both K+ and Rb+) compared with K+-starved cells. From these data, it is concluded that the archaeon M. thermoautotrophicum contains a low-affinity K+ uptake system which is overproduced during growth on low-K+ medium.     

Phosphate transport in pig proximal small intestines during postnatal development: lack of modulation by calcitriol

The role of calcitriol in the intestinal absorption of inorganic phosphate (Pi) during postnatal development was studied in newborn [<1 week postpartum (pp)], suckling (3-4 weeks pp), and weaned (>6 weeks pp) control piglets (con) and piglets suffering from inherited calcitriol deficiency (def). In addition, a number of def piglets were treated with vitamin D3 (def-D3). Regardless of age, plasma calcitriol concentrations in def piglets were unphysiologically low (16-21 pg/ml) and differed significantly from those in respective con animals (60-69 pg/ml) and vitamin D3-treated def piglets (50-56 pg/ml). However, newborn and suckling def piglets had normal Ca (approximately 3.0 mmol/liter) and Pi (approximately 2.8 mmol/liter) plasma levels. Def piglets became hypocalcemic (1.9 mmol/liter) and hypophosphatemic (1.9 mmol/liter) between 4-6 weeks pp. Treatment with vitamin D3 significantly increased plasma Ca (3.2 mmol/liter) and Pi (2.7 mmol/liter) levels in weaned def animals. Regardless of calcitriol status, net Pi flux rates (active Pi absorption, as determined with the in vitro Ussing-chamber technique) from the upper small intestines was maximal at birth [170-224 nmol/(cm2 x h)] and decreased by approximately 80% during the first week of life before remaining constant [30-50 nmol/(cm2 x h)] during the following development. In weaned def piglets, net Pi flux rates were significantly lower by about 80% compared with those in con animals. Treatment of def piglets with vitamin D3 had no effect in newborn and suckling animals but reconstituted net Pi flux rates to normal values at weaning age. Age-dependent and calcitriol-mediated changes in net Pi flux rates were paralleled by respective maximum velocity values of Na+-dependent Pi uptake across the brush border membrane of the enterocytes (newborn piglets, 1.9-2.2 nmol/(mg protein 10 sec); suckling piglets, 0.4-0.6 nmol/(mg protein x 10 sec); weaned piglets, 0.7, 0.3, and 0.7 nmol/(mg protein x 10 sec) in con, def, and def-D3 animals, respectively). These findings suggest that the apical Pi uptake represents the major rate-limiting step of the overall transepithelial Pi transport. At weaning, Na+/Pi transport across the intestinal brush-border membrane is clearly stimulated by calcitriol, but no significant effects of age or calcitriol on the Km values (0.5-0.7 mmol/liter) were observed. In conclusion, our findings reveal calcitriol-independent mechanisms for active intestinal Pi absorption during the neonatal and suckling periods. The onset of the classical calcitriol-dependent mechanism for active intestinal Pi absorption does not occur until weaning.     

Effect of polyamines and cystic fibrosis serum on glucose transport

The effect of plasma or serum from homozygotes and heterozygotes for the cystic fibrosis (CF) gene on the active uptake of 3-0-14C-methyl-D-glucose (3-0-14C-MDG) by rat jejunal epithelium was studied. Furthermore, the role of the polyamine, spermidine, and its products of metabolic degradation on glucose transport were investigated, and a relationship to the pathogenesis of membrane dysfunction in cystic fibrosis was postulated. Glucose transport in everted rat jejunal rings was used in the study. Results were expressed as 3-0-14C-MDG concentration ratio between the intracellular (ICF) and the extracellular fluid spaces (ECF) of the jejunal rings at the end of a 60 min incubation period. The mean ratio obtained from incubations of the rat jejunal rings in medium consisting of Krebs-Ringer-bicarbonate buffer and the labeled sugar was considered as 100% uptake. When plasma or serum, with or without spermidine, was mixed with the medium in a volume ratio of 1:3, a decrease in the active uptake of 3-0-14C-MDG was observed, expressed as percent inhibition. Percent inhibition of 3-0-14C-MDG uptake obtained when the rat jejunal rings were incubated in normal plasma was compared to that obtained with plasma from cystic fibrosis genotypes. It was found that: 1) plasma from 25 homozygous children had greater inhibitory effect on glucose uptake than plasma from 26 normal children; 2) plasma from 9 heterozygous women had greater inhibitory effect than that from 6 normal women; 3) the inhibitory effect of plasma from 3 homozygous children was not influenced by dialysis; 4) the inhibitory effects of paired plasma and serum samples from 9 homozygotes were comparable; 5) spermidine added to the incubating electrolyte solution did not affect glucose transport; 6) the addition of spermidine to reaction mixtures containing normal plasma potentiated the inhibitory effect; and 7) mixing and incubation of fresh bovine serum with reaction mixtures containing plasma from homozygotes decreased the inhibitory effect. The predominant inhibitory effect of plasma or serum from homozygotes and heterozygotes for the CF gene appears to be related to a nondialyzeable molecule(s). It does not seem to reflect the presence of high plasma glucose levels in cystic fibrosis nor to be the result of competitive inhibition between sugars. It does not seem to be the result of sodium or other electrolyte differences. A similar inhibitory effect is acquired by normal plasma after the addition of spermidine. On the other hand, plasma from CF homozygotes loses its inhibitory effect after incubation with fresh bovine serum. These findings may indicate that products of metabolic degradation of spermidine are responsible for the inhibitory effect of glucose transport and suggest the possibility of a role in abnormal polyamine metabolism in the pathogenesis of cystic fibrosis.     

Feeding trans fatty acids to rats has no effect on the intestinal uptake of glucose, fatty acids or cholesterol

Trans fatty acids are produced in the manufacture of margarine, and these hydrogenated fatty acids may have a deleterious effect on the reduction in fasting levels of serum cholesterol anticipated from the feeding of cis polyunsaturated fatty acids. We undertook this study in rats to test the effect of feeding trans fatty acids on the intestinal uptake of glucose, fatty acids and cholesterol. Adult female Wistar rats were fed for 2 weeks semisynthetic, isocaloric diets containing no oleic acid (18:1), cis 18:1 or trans 18:1. There was no difference between the three dietary groups in the animals' food consumption or body weight gain. Rats fed trans 18:1 had an approximately 20% decline in the total weight of the ileum as compared with controls fed no 18:1, and therefore there was also a decline in the percentage of the ileal tissue comprised of mucosa. When comparing rats fed trans 18:1 with those fed cis 18:1 or no 18:1, there was no difference in the uptake of varying concentrations of D-glucose when expressed as nmol.100 mg tissue-1.min-1 or nmol.100 mg mucosal-1.min-1 for jejunum or for ileum. Also, there was no difference in the value of the maximal transport rate (Vmax), Michaelis constant (Km), or the contribution of passive uptake of glucose assessed with L-glucose. There was no diet-associated change in the jejunal or ileal uptake of a medium-chain length fatty acid (lauric acid), a long-chain length saturated fatty acid (palmitic acid), a monounsaturated fatty acid (oleic acid), two polyunsaturated fatty acids (linoleic and linolenic acids), or cholesterol. Thus, we conclude that 2 weeks' feeding of trans fatty acid to rats has no influence on the jejunal or ileal uptake of glucose, fatty acids or cholesterol.     

Insulinomimetic effect on glucose transport by epidermal growth factor when combined with a major histocompatibility complex class I-derived peptide

Peptides derived from the alpha 1-region of the murine H-2Dk molecule enhance glucose uptake in rat adipose cells above the maximum obtained with insulin stimulation alone (Stagsted, J., Reaven, G. M., Hansen, T., Goldstein, A., and Olsson, L. (1990) Cell 62, 297-307). We now describe that epidermal growth factor (EGF) in combination with the same peptides, Dk-(61-85) and Dk-(62-85), stimulates cellular glucose uptake 5-7 times over the basal level, i.e. to 30-50% of the maximal insulin effect. EGF alone increased glucose uptake by only approximately 50% above basal and the peptide alone by 100% above basal. Maximal effect of EGF and peptide was reached in 10-20 min with 30 microM peptide (EC50 10-15 microM) and 50 nM EGF (EC50 1-2 nM). The effect of EGF and peptide on glucose uptake was additive to that of insulin and peptide until the maximal level attained with insulin and peptide was reached. The combined effect of EGF plus peptide on glucose transport was associated with a recruitment of GLUT4 molecules to the plasma membrane. However, the phosphatidylinositol (PI) kinase which is activated by insulin was not activated by EGF plus peptide. Thus, the effect of EGF plus peptide on glucose uptake seems independent of the activity status of the insulin receptor. 125I-Labeled EGF bound specifically to rat adipose cells with an apparent affinity of approximately 2 nM and Bmax approximately 5 x 10(3). However, the major histocompatibility complex (MHC) peptides did not affect EGF-stimulated internalization of EGF receptor, in contrast to their effect on the insulin receptors. Transforming growth factor alpha had an effect similar to EGF on glucose uptake. Three other peptides derived from other parts of murine MHC class I had no effect on glucose uptake in combination with EGF. Thus, EGF in combination with certain MHC class I-derived peptides is insulinomimetic concerning glucose transport and this effect is independent of the insulin receptor activity.     

Phloretin sensitive active urea absorption in frog skin

This report presents evidence for urea active absorption by isolated skin of Rana esculenta. One of the supporting factors of such evidence is that at a low concentration the urea influx is five times greater than the outflux, in the absence of a chemical gradient. The transport shows a saturation kinetics with an apparent Km = 1.33 mM and is inhibited by un uncoupling agent (FCCP). 5 x 10(-4) M Phloretin, added to the external side, markedly inhibits inward urea transport, whereas it is ineffective when added to the serosal fluid. This provides evidence for a phloretin-sensitive mechanism located at the external side of the epithelium. Phloretin stimulates the sodium active transport; the possible coupling of urea and sodium movement is analysed.     

Decreased basal, noninsulin-stimulated glucose uptake and metabolism by skeletal soleus muscle isolated from obese-hyperglycemic (ob/ob) mice

Insulin resistance of diaphragms of ob/ob mice has been repeatedly demonstrated previously both in vitro and in vivo. In the present study, transport and metabolism of glucose with and without insulin stimulation were compared in a skeletal muscle more likely than diaphragm or heart to be representative of the overall striated muscle mass, i.e. isolated soleus muscle. Compared with soleus muscle from lean controls, unstimulated lactate release in the presence of exogenous glucose was depressed from 16.2 to 12.3 nmol/60 min per mg wet wt in soleus from ob/ob mutants; glycolysis was decreased from 6.6 to 3.7 and [14C]glucose oxidation to 14CO2 from 0.90 to 0.33 nmol glucose/60 min per mg wet wt. Uptake of 2-deoxyglucose (2-DOG), both with and without insulin, was very much less for soleus from ob/ob than from lean mice, at 2-DOG concentrations ranging from 0.1 to 10 mM, and in mice of 6-15 wk. When 2-DOG concentration was 1 mM, its basal uptake was 0.53 nmol/30 min per mg wet wt for soleus of ob/ob as against 0.96 for soleus of lean mice. The absolute increment due to 1 mU/ml insulin was 0.49 in muscle of ob/ob as against 1.21 in that of lean mice. When the resistance to insulin action was decreased by pretreatment in vivo by either streptozotocin injection or fasting, the decreased basal 2-DOG uptake of subsequently isolated soleus muscle was not improved. Inhibition of endogenous oxidation of fatty acids by 2-bromostearate, while greatly increasing 14CO2 production from [14C]glucose, did not affect basal [5-3H]glucose metabolism or 2-DOG uptake. It is suggested that transport and/or phosphorylation of glucose under basal, unstimulated conditions are depressed in soleus muscle of ob/ob mice, whether or not resistance to insulin and hyperinsulinemia are also present. Although the origin of the decreased basal glucose uptake remains unknown it might be related to a similar decrease in basal glucose uptake by ventromedial hypothalamic cells, an event presumably resulting in a tendency to hyperphagia. Decreased basal glucose uptake by soleus muscle of ob/ob mice might explain the hyperglycemia, and hence partly the hyperinsulinemia and excessive fat deposition of those animals.     

Acute and chronic exposure of rat intestinal mucosa to dextran promotes SGLTI-mediated glucose transport

BACKGROUND: The intestinal handling of dextran, an alpha-1,6-linked glucose polymer, is poor compared with starch, and some ingested dextran might therefore reach the lower small intestine. As luminal sugar up-regulates SGLT1 (sodium-dependent glucose transporter) locally, we report the effects of a dextran-enriched diet on jejunal and ileal brush border membrane (BBM) glucose uptake. METHODS: Rats were maintained on a diet containing 65% maltodextrin or 32.5% maltodextrin + 32.5% dextran (10 kD or 40 kD) for 8-10 days, and the kinetics of phlorizin-sensitive [3H]-glucose uptake by purified BBM vesicles was determined. RESULTS: Ingestion of 40-kD but not 10-kD dextran increased Vmax for jejunal and ileal glucose uptake (+64.3% and +61.8% respectively, both P < 0.02). The transport response to 40-kD dextran was in keeping with lower levels of expired H2 at the end of the feeding period. High-performance liquid chromatography (HPLC) analysis of luminal contents indicated extensive hydrolysis of ingested dextran. Finally, 3-h jejunal exposure to 40-kD dextran in vivo increased the Vmax for glucose uptake by jejunal BBM. CONCLUSION: It is likely that increased SGLT1-mediated glucose uptake after short or longer term mucosal exposure to dextran results from luminal dextran per se or a hydrolysis product. The clinical implications of this up-regulation are discussed.     

A description of glucose uptake in Navicula pelliculosa (Breb) Hilse including a brief comparison with an associated Flavobacterium sp

Navicula pelliculosa and an associated Flavobacterium sp. were isolated from the epiphyton of Scirpus maritimus, an emergent macrophyte growing in a brackish drainage dyke. Both micro-organisms possessed active transport systems for glucose uptake. In N. pelliculosa the transport system was fully induced in the dark in the absence of glucose, and subsequently inactivated when transferred to the light in the absence of the substrate. The presence of glucose during the dark induction period prevented the achievement of maximum specific activity of the transport system, while incubation at a high light intensity with or without the presence of the substrate resulted in a very marked inhibition of glucose uptake. Inhibition in the light was partially offset by blocking photosynthetic electron flow with 3'(3,4 dichlorophenyl)1'1' dimethyl urea. The transport system accumulated 3-O-methyl glucose against a concentration gradient and was highly specific for glucose as there was no competition by most of the other sugars tested. However, 6-deoxyglucose was taken up instead of glucose and this suggested that glucose was transported in a non-phosphorylated state, whereas inhibition of glucose transport activity with dicyclohexylcarbodimide implicated the involvement of an adenosine triphosphatase on the cell membrane. Inhibitors of oxidative phosphorylation tetrachlorosalicylaniline and carbonylcyanide m-chlorophenylhydrazone also inhibited glucose transport activity. The affinity of the diatom for glucose was greater than that shown by the bacterium, but the Km for glucose transport, 1.5x10-5M was too high to allow effective removal of glucose at in situ concentrations.     

Intestinal ion transport: effect of norepinephrine, pilocarpine, and atropine

The effects of parenteral pilocarpine, atropine, and norepinephrine on salt and water transport were studied in jejunum and ileum of anesthetized rats. Pilocarpine increased jejunal transmural PD, reduced absorption of Na, K, HCO3, and H2O, and increased secretion of Cl; in ileum, it caused secretion of Na and H2O, elicited secretion of K, and reduced the absorption of Cl. In both segments, perfusate became more akaline, and there was less of a rise in PCO2. Atropine prevented all changes caused by pilocarpine. Atropine alone increased jejunal absorption of Na and HCO3 and acidity of perfusate, implying that cholinergic nerves influence transport. Norepinephrine augmented jejunal absorption of Na, Cl, and H2O but caused no change in PD. In ileum, norepinephrine increased absorption of Na and Cl, reduced the rise in pH, increased the rise in PCO2 of perfusate, but did not affect net HCO3 movement. With all agents, when Na absorption increased, perfusate became more acidic in jejunum and less alkaline in ileum, evidence of an association between Na and H transport.     

Effects of synthetic human gastrin I on movements on water, electrolytes, and glucose across the human small intestine

The effect of graded doses (0.125, 0.500, 1.00 and 2.00 mug per kg per hr) of intravenous synthetic human gastrin I (SHG) on jejunal transport of water, electrolytes, and glucose from a glucose-saline solution (solution II) was studied in 12 healthy volunteers, using an intestinal perfusion technique with a proximal occluding balloon. SHG when infused at rates of 0.500 mug per kg per hr or greater significantly reduced water and electrolyte absorption; this effect was linearly related to the dose and reached 40 to 60% of basal absorption (and only 10% for glucose) with the highest dose; insorption of sodium and water were significantly decreased by SHG. In a further group of 9 subjects no effect of SHG (2 mug per kg per hr) was found on jejunal absorption from a mannitol-saline solution (solution I) and on ileal absorption from solutions I and II; in 5 additional subjects, SHG did not decrease jejunal transit time of intraluminal fluid. There was no increase in serum thyrocalcitonin during SHG infusion. It is proposed that SHG selectively depresses the glucose-stimulated sodium transport as suggested by the reduction of the rate of net sodium absorption per micromole of glucose absorbed during SHG infusion. Physiological and pathological implications of these findings are discussed, especially in the light of the circulating levels of immunoreactive gastrin achieved during SHG infusion.     

Energy-dependent uptake of calcium by the yeast Schizosaccharomyces pombe

1. In resting cells of the fission yeast Schizosaccharomyces pombe, the uptake of calcium is stimulated by the addition of 90 mM glucose in the presence as in the absence of respiration and inhibited by Antimycin A in the absence of exogenous carbon source. This uptake therefore requires fermentative or respiratory metabolic energy. 2. The calcium uptake by S. pombe exhibits saturation kinetics and high affinity for calcium. At external pH 4.5, the apparent Km is 45 muM ca2+ 400 muM of other divalent cations exert competitive inhibitions of calcium uptake in the following order of affinities: Sr2+ greater than Mn2+ greater than Co2+ greater than Mg2+. Inhibition by KCl is also observed but is of non-competitive type and requires high concentrations of the order of 40 mM. 3. At 30 degrees C, the uptake rate of calcium is about 10-times higher at pH 8925 than at pH 4.0. An extrusion of 45Ca2+, the rate of which is estimated to be lower than one-fifth of the uptake, is observed in the presence of glucose when the external pH is acid. 4. At external pH 4.5, low concentrations of lanthanum chloride, ruthenium red and hexamine cobaltichloride are inhibitory for the uptake of calcium by the yeast cells. 5. In presence of Antimycin A, the uncouplers: NaN3, dinitrophenol, and concentrations of crobonylcyanide m-chlorophenylhydrazone higher than 80 muM inhibit the calcium uptake by glycolysing cells. In the presence of glucose, the K+ ionophore Dio-9 dnhances severalfold the uptake of calcium even at 2 degrees C. 6. It is concluded that S. pombe possess an active transport system for low concentrations of calcium. This transport seems to be dependent on an electric potential (negative inside) across the cellular membrane.     

Surgical treatment of locally advanced rectal cancer. Options and strategies

To evaluate the contribution of the subsets of T helper lymphocyte (Th) to the development of pulmonary lesion in mycoplasma pneumonia, we compared the pathological findings between Th1 dominant mice (C57BL/6) and Th2 dominant mice (BALB/c) in experimental Mycoplasma pulmonis (M. pulmonis) pneumonia. Mice (ICR, C57BL/6, and BALB/c) were intranasally inoculated with 0.03 ml of a solution containing M. pulmonis (1 x 10(8)) colony forming units per ml. Another M. pulmonis inoculated ICR mice were treated with interleukin-2 (IL2; 4.8 micrograms/day), days 3-9, intracutaneously). All mice were sacrificed at day 14, and the lung specimens were examined. Peribronchial lymphocyte cuffing was more prominent in C57BL/6 mice than that of ICR mice, and intra-alveolar inflammatory-cell infiltration in BALB/c mice was more prominent than in ICR mice. Pathological patterns of the lung in IL-2 treated ICR mice were mimicking those of C57BL/6 mice. These results suggested that pathological patterns of mycoplasma pneumonia in mice might be altered by the imbalance of host T helper lymphocyte subset.     

Algodystrophies of the limbs

1. We studied intestinal glucose transport in pigs during the acute and convalescent phases of an invasive viral enteritis, transmissible gastroenteritis. 2. When diarhoea was severe 40 h after experimental infection, net absorption of glucose, Na+ and water, measured by marker perfusion in the jejunum, was reduced; the enhancement of Na+ and water absorption in response to increasing perfusate glucose concentrations up to 120 mmol/l was diminished compared with the response observed in control and convalescent pigs. 3. Measured in vitro, 40 h after infection, unidirectional fluxes of 3-O-methyl-D-glucose across the jejunal epithelium were reduced and net absorption of the sugar was obliterated. Phlorizin (0.05 mmol/l), which completely inhibited net 3-O-methyl-D-glucose absorption in control tissue, had no significant effect on transmissible gastroenteritis jejunum. 4. Our data suggest that in this invasive viral enteritis, which closely resembles human rotavirus enteritis, glucose absorption is impaired as a result of defects in both active and passive glucose flux. 5. Differences between the mechanisms of viral diarrhoea, demonstrated by our study and those of the enterotoxigenic diarrhoeas, should be taken into consideration in formulating active therapeutic measures for children with acute viral diarrhoea.     

Inactivation of toluene 2-monooxygenase in Burkholderia cepacia G4 by alkynes

High concentrations of acetylene (10 to 50% [vol/vol] gas phase) were required to inhibit the growth of Burkholderia cepacia G4 on toluene, while 1% (vol/vol) (gas phase) propyne or 1-butyne completely inhibited growth. Low concentrations of longer-chain alkynes (C5 to C10) were also effective inhibitors of toluene-dependent growth, and 2- and 3-alkynes were more potent inhibitors than their 1-alkyne counterparts. Exposure of toluene-grown B. cepacia G4 to alkynes resulted in the irreversible loss of toluene- and o-cresol-dependent O2 uptake activities, while acetate- and 3-methylcatechol-dependent O2 uptake activities were unaffected. Toluene-dependent O2 uptake decreased upon the addition of 1-butyne in a concentration- and time-dependent manner. The loss of activity followed first-order kinetics, with apparent rate constants ranging from 0.25 min-1 to 2.45 min-1. Increasing concentrations of toluene afforded protection from the inhibitory effects of 1-butyne. Furthermore, oxygen, supplied as H2O2, was required for inhibition by 1-butyne. These results suggest that alkynes are specific, mechanism-based inactivators of toluene 2-monooxygenase in B. cepacia G4, although the simplest alkyne, acetylene, was relatively ineffective compared to longer alkynes. Alkene analogs of acetylene and propyne-ethylene and propylene-were not inactivators of toluene 2-monooxygenase activity in B. cepacia G4 but were oxidized to their respective epoxides, with apparent Ks and Vmax values of 39.7 microM and 112.3 nmol min-1 mg of protein-1 for ethylene and 32.3 microM and 89.2 nmol min-1 mg of protein-1 for propylene.     

Sustained hyperglycemia in vitro down-regulates the GLUT1 glucose transport system of cultured human term placental trophoblast: a mechanism to protect fetal development?

The trophoblast of human placenta is directly exposed to the maternal circulation. It forms the main barrier to maternal-fetal glucose transport. The present study investigated the effect of sustained hyperglycemia in vitro on the glucose transport system of these cells. Trophoblasts isolated from term placentas and immunopurified were cultured for 24, 48, and 96 h in DMEM containing either 5.5 (normoglycemia) or 25 mmol/l D-glucose (hyperglycemia), respectively. Initial uptake of glucose was measured using 3-O-[14C]methyl-D-glucose. Kinetic parameters were calculated as K(M) = 73 mmol/l and Vmax = 29 fmol s(-1) per trophoblast cell. Uptake rates of cells cultured under hyperglycemic conditions did not differ at exogenous D-glucose concentrations in the physiological range (1, 5.5, 10, and 15 mmol/l), but were significantly decreased by 25% (P<0.05) at diabetes-like concentrations (20 and 25 mmol/l) as compared to normoglycemic conditions. This effect was due to a decrease in Vmax (-50%), whereas K(M) remained virtually unaffected. GLUT1 mRNA levels were lower by 50% (P<0.05; Northern blotting) and GLUT1 protein was reduced by 16% (P<0.05; Western blotting) in trophoblast cells cultured under hyperglycemic vs. normoglycemic conditions. We conclude that prolonged hyperglycemia in vitro reduces trophoblast glucose uptake at substrate concentrations corresponding to blood levels of poorly controlled diabetic gravidas. This effect is due to diminished GLUT1 mRNA and protein expression in the trophoblast.     

Short-term effect of epidermal growth factor (EGF) on sodium and glucose cotransport of isolated jejunal epithelial cells

This study was undertaken to assess the short-term effects of EGF on sodium and glucose uptake, glucose metabolism and Na+/K(+)-ATPase activity in isolated enterocytes of rats. Jejunal cells exposed to EGF had a significantly greater total uptake of sodium compared to controls after 6 min. Kinetic analysis of glucose transport across BBMV's demonstrated similar Km values but a significant increase of the Vmax in vesicles prepared from cells first exposed to EGF as compared to controls. EGF was also associated with a significant increase in glucose metabolism of jejunal enterocytes after 15 min. The activity of Na+/K(+)-ATPase increased in jejunal enterocytes exposed to EGF. The increase in Na+/K(+)-ATPase activity of the cells following EGF exposure was not accompanied by an increase in immunodetectable total or assembled Na+/K(+)-ATPase protein. EGF's effect on enzyme activity was abolished by removing NaCl from the incubation solution, and by preincubating the enterocytes with phlorizin prior to addition of EGF. Preincubation with amiloride did not inhibit the effect of EGF on Na+/K(+)-ATPase. The results confirm that EGF promotes uptake of both sodium and glucose by the jejunal mucosal cells, and suggest the effect of EGF on glucose and sodium is mediated through the brush-border membrane glucose-sodium transporter. The increase in Na+/K(+)-ATPase activity that occurs with EGF appears to be secondary to a rise in intracellular Na+ concentration. The short-term effects of EGF on glucose and sodium transport by the small intestine may have potential therapeutic implications.