Biomedical subjects

K Doring

Publications and source records attributed to K Doring.

9 recordsLinked to original sources

PubMed2000

Combinatorial liquid-phase synthesis of

A new method for the synthesis of [1,4]oxazepin-7-ones from readily available aldehydes and alpha-amino alcohols was developed using the Baylis-Hillman reaction as the key step. To determine the scope and limitations of the method, a mixture library was synthesized from six aldehydes and six alpha-amino alcohols on the soluble polymer support poly(ethylene glycol) 5000 monomethyl ether (MeOPEG) via split synthesis and analyzed by GC-EIMS. Those oxazepines that were formed predominantly were resynthesized in a parallel synthesis and fully characterized. Thus, we have shown that split synthesis on MeOPEG can be an efficient method to rapidly screen the substrate spectrum of a newly developed reaction sequence.

Journal Article↗

PubMed2000

Enhanced internal dynamics of a membrane transport protein during substrate translocation.

Conformational changes are essential for the activity of many proteins. If, or how fast, internal fluctuations are related to slow conformational changes that mediate protein function is not understood. In this study, we measure internal fluctuations of the transport protein lactose permease in the presence and absence of substrate by tryptophan fluorescence spectroscopy. We demonstrate that nanosecond fluctuations of alpha-helices are enhanced when the enzyme transports substrate. This correlates with previously published kinetic data from transport measurements showing that millisecond conformational transitions of the substrate-loaded carrier are faster than those in the absence of substrate. These findings corroborate the hypothesis of the hierarchical model of protein dynamics that predicts that slow conformational transitions are based on fast, thermally activated internal motions.

Animals↗

PubMed1998

Fast association rates suggest a conformational change in the MHC class I molecule H-2Db upon peptide binding.

Major histocompatibility complex (MHC) class I molecules bind peptides in the endoplasmic reticulum (ER). For this binding reaction, when performed in vitro, widely differing association rates have been reported. We have expressed empty soluble H-2Db class I molecules in Chinese hamster ovary (CHO) cells and generated complete sets of association, dissociation, and equilibrium constants of unmodified peptides using tritium-labeled peptides and stopped-flow fluorescence spectroscopy. We find that (i) the transition midpoint of temperature denaturation (Tm) of the protein is shifted from 30.5 to 56 degrees C upon the binding of a high-affinity peptide. (ii) With the peptide SV-324-332 (sequence FAPGNYPAL) at 4 degrees C, the dissociation rate constant of 1.02 x 10(-5) s-1 and an equilibrium constant of 8.5 x 10(7) M-1 predict an association rate constant of 870 M-1 s-1 for a simple one-step model of binding. (iii) In contrast, binding of this peptide proceeds much faster, with 1.4 x 10(6) M-1 s-1. These "mismatch kinetics" suggest that peptide binding occurs in several steps, most likely via a conformational rearrangement of the peptide binding groove. The structure of the peptide-class I complex at the time-point of peptide recognition may therefore be different from the equilibrium crystal structures. (iv) Association of modified peptides, in the presence of detergent, or above the Tm of the empty molecule is considerably slower. This might explain why fast on-rates have not been observed in previous studies.

Animals↗

PubMed1997

Nutrient uptake into undifferentiated and differentiated HT-29 cells in culture.

HT-29 human colon carcinoma cells in culture have many characteristics of enterocytes, and these cells have been used by others to study intestinal drug and nutrient transport and metabolism. When grown in glucose-containing medium, HT-29 cells are largely undifferentiated (HT-29glu), but when grown in the absence of glucose but in the presence of galactose (HT-29gal), the population of cells is mostly differentiated. This study was undertaken with HT-29glu and HT-29gal cells to study the uptake of palmitic acid (16:0), linoleic acid (18:2), and cholesterol. The relationship between concentration and uptake of 16:0, 18:2, and cholesterol was linear in HT-29glu and HT-29gal cells, with the relative values of the slopes of this relationship being 18:2 > > 16:0 > > cholesterol. The rates of uptake of these lipids were at least three times higher in HT-29gal than in HT-29glu cells. In HT-29glu cells, the relative rates of uptake of the sugars at 32 mM were D-glucose = galactose > fructose > > alpha-methylglucose. Uptake of these sugars was much greater in HT-29gal than in HT-29glu cells. When 100 microM forskolin was added to the incubation medium for 7 days post-confluency, which stimulates the activity of adenylate cyclase and thereby increases the intracellular synthesis of cAMP, there was no effect on the uptake of the lipids or the sugars in either HT-29glu or HT-29gal cells. Thus, (i) differentiated HT-29gal cells transport larger amounts of lipids and sugars than do undifferentiated HT-29glu cells; (ii) forskolin has no effect on the uptake of lipids or sugars in these cells. This human cell culture system may be useful to study the in vitro transport of lipids, to establish the role of cell differentiation on these uptake processes, and to determine the potential role of selected intracellular signals.

Absorption↗

PubMed1995

Age of rat influences isolation of jejunal enterocytes from along the villus.

Aging is associated with alterations in the functions of the intestine. Also, the functions of the intestine vary along the length of the villus. A method is reported for the isolation of four fractions of enterocytes from along the villus of young (3 months, 300-350 g) and old (8 months, 700-750 g) male Wistar rats. Different qualitative patterns are seen when the activities of alkaline phosphatase (AP), invertase (INV), or [3H]methylthymidine incorporation are expressed on the basis of millimoles per gram protein, percentage of highest activity, percentage of total activity, cumulative activity for each fraction, or millimoles per gram protein versus cumulative percentage of total protein. When expressed on the basis of protein, AP is higher in upper (FI) versus lower (FIV) villus fractions, and in each fraction AP is higher in old versus young animals. INV varies little along the villus or between young and old rats; [3H]methylthymidine incorporation is highest in the crypt cells. The ratio mg protein/mg DNA varies along the villus and is twofold higher in young than in old rats. In contrast, when activities are expressed on the basis of DNA, the same qualitative patterns are seen along the villus, but INV and AP are both higher in young than in old rats. In summary (i) the qualitative and quantitative differences in the activities of AP and INV along the villus of old versus young rats vary when the data are expressed on the basis of DNA rather than protein; (ii) the ratio of protein to DNA varies along the villus in young and old animals, and for this reason it is appropriate to report enzyme activity on the basis of enterocyte DNA; and (iii) INV and AP, when expressed on the basis of DNA, are higher in young than in old animals. Thus, the isolation of rat jejunal enterocytes from along the villus is affected by the age of the animals.

Aging↗

PubMed1994

Orally administered immunosuppressants modify intestinal uptake of nutrients in rabbits.

The effect on intestinal nutrient transport of the immunosuppressive drugs cyclosporin A (CsA), cyclosporin G (CsG), and rapamycin (RAP) was determined in New Zealand white rabbits. Rabbits received oral doses of CsA (20 mg/kg/day), CsG (10 mg/kg/day), or RAP (1 mg/kg/day) for 10 days. Animals receiving RAP had decreased food intake and weight gain compared with controls. This correlated with a decrease in both total ileal weight and corresponding mucosal weight. CsA and CsG administration had no effect on food intake, total weight gain, or intestinal weight. Villus surface area was significantly decreased in all groups as compared with controls. Jejunal uptake of D-glucose as well as 1 medium and 4 long chain fatty acids was not affected by drug administration, while both mucosal-to-serosal and net 3-0-methylglucose fluxes were increased (P < 0.05) in the jejunum by all 3 drugs. In the ileum, the rates of uptake of D-glucose as well as stearic and linoleic acids were increased in animals treated with RAP compared with controls. There was an increase in the ileal values of the maximal transport rate (Vmax) and apparent Michaelis constant (Km*) in RAP, and a fall in the Vmax and Km* in CsG. CsG administration resulted in a decreased cholesterol uptake in both jejunum and ileum, and a decreased D-glucose uptake in the ileum compared with controls. These differences in glucose uptake among groups could not be attributed to variations in body, intestinal, or mucosal weights. It is unlikely that the changes observed in CsA- and CsG-treated animals would have nutritional importance, as these animals gained weight normally. In addition, in these animals the changes mainly occurred in the ileum, not in the jejunum, where most glucose is absorbed, and the associated alterations in the values of the Vmax and Km* would lead to reciprocal changes in the rates of uptake of varying luminal concentrations of glucose. In contrast, these changes are likely to be of more importance in RAP-treated animals, since they failed to gain weight normally. The significance of these findings needs to be established in chronically treated animals.

Administration, Oral↗

PubMed1994

Dietary omega 3 fatty acids and cholesterol modify enterocyte microsomal membrane phospholipids, cholesterol content and phospholipid enzyme activities in diabetic rats.

Diabetes-associated changes in intestinal uptake of nutrients are modified by isocaloric variations in the type of dietary lipids, and are associated with alterations in the phospholipid and fatty acyl content of the intestinal brush border membrane. The present study was designed to test the hypothesis that diet- and diabetes-associated changes in enterocyte microsomal membrane phospholipids are due to variations in the activity of two phospholipid metabolizing enzymes, 1,2-diacylglycerol:CDPcholine cholinephosphotransferase (CPT) and phosphatidylethanolamine methyltransferase (PEMT). Adult female Wistar rats were fed one of four semisynthetic diets--beef tallow low in cholesterol (BT), beef tallow high in cholesterol (BTC), fish oil low in cholesterol (FO) or fish oil high in cholesterol. In half of the animals, diabetes mellitus was produced by injection of streptozotocin. Jejunal and ileal enterocyte microsomes (EMM) were isolated and analyzed for cholesterol and phospholipids, as well as for CPT and PEMT activities. In control animals, feeding FO reduced EMM total phospholipids including phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol. Feeding FO resulted in a greater than 95% reduction in the activity of CPT. Diabetes was associated with increased jejunal EMM total phospholipids including sphingomyelin (SM) and PE, without associated changes in CPT or PEMT. Dietary cholesterol supplementation did not affect EMM total cholesterol or phosphlipid composition in control rats fed BT or FO, but was associated with an increase in EMM cholesterol in diabetic rats fed BT or FO. A decrease in total phospholipids due to a decline in SM, PC and PE in diabetic rats fed FO was not associated with changes in the activities of CPT or PEMT in EMM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

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.

Absorption↗

PubMed1994

Feeding an isocaloric omega-3 fatty acid diet reduces the brush border membrane vesicle uptake of glucose in streptozotocin-diabetic rats.

Glucose uptake is increased into the intestine of diabetic rats, and this adaptation can be modified further by manipulation of the type of fatty acids in the triglycerides in the diet. Jejunal brush border membrane vesicles were used to examine the uptake of D-glucose into the jejunum of non-diabetic control and streptozotocin-diabetic rats fed for two weeks in isocaloric semisynthetic diet enriched with saturated fat (beef tallow) or polyunsaturated omega-3 fatty acids (fish oil). The time-course of uptake of 100 microM glucose demonstrated an overshoot which peaked at approximately 30 seconds and declined thereafter to an equilibrium plateau. In concentration studies, glucose uptake was greater into brush border membrane vesicles of diabetic as compared with control rats. The maximal transport rate (Vmax) was increased approximately 9-fold in diabetics as compared with control rats fed beef tallow (p < 0.05), and was increased approximately 6-fold in diabetic rats fed fish oil. In diabetic rats, feeding fish oil reduced the value of the Vmax by approximately 50% as compared with diabetic rats fed beef tallow. Thus, the enhanced glucose uptake into BBM vesicles of streptozotocin-diabetic rats can be partially corrected by feeding an isocaloric semisynthetic diet enriched with polyunsaturated omega-3 fish oils.

Animals↗