Generation of two hiPSCs lines of two patients carrying truncating mutations in the dimerization domain of filamin C.

Here we introduce the human induced pluripotent stem cell lines (hiPSCs), HIMRi004-A and HIMRi005-A from dermal fibroblasts of a 48-year-old female (HIMRi004-A) carrying missense mutation that translate to the first described filamin C isoform p.W2710X and from a 56-year-old female (HIMRi005-A) carrying a recently described mutation in the same domain p.Y2704X. Both lines are generated via lentiviral expression of OCT4, SOX2, KLF4 and c-MYC. The lines display a typical embryonic stem cell-like morphology, express pluripotency markers, retain a normal karyotype (46, XX) and have the differentiation capacity in all three germ layers. The two lines can be used to elucidate the pathomechanisms of FLNC myofibrillar myopathies and to develop novel therapeutic options.

Generation of two human iPSC lines (HIMRi002-A and HIMRi003-A) derived from Caveolinopathy patients with rippling muscle disease.

Here we introduce the human induced pluripotent stem cell lines (hiPSCs), HIMRi002-A and HIMRi003-A, generated from cultured dermal fibroblasts of 61-year-old (HIMRi002-A) and 38-year-old (HIMRi003-A) female patients, carrying a known heterozygous pathogenic variant (p.A46T) in the Caveolin 3 (CAV3) gene, via lentiviral expression of OCT4, SOX2, KLF4 and c-MYC. HIMRi002-A and HIMRi003-A display typical embryonic stem cell-like morphology, carry the p.A46T CAV3 gene mutation, express several pluripotent stem cell markers, retain normal karyotype (46, XX) and can differentiate in all three germ layers. We postulate that the HIMRi002-A and HIMRi003-A iPSC lines can be used for the characterization of CAV3-associated pathomechanisms and for developing new therapeutic options.

Impact of Implementing an Elaborated CT Perfusion Protocol for Aneurysmal SAH on Functional Outcome: CTP Protocol for SAH.

BACKGROUND AND PURPOSE: The acute phase of aneurysmal SAH is characterized by a plethora of impending complications with the potential to worsen patients' outcomes. The aim of this study was to evaluate whether an elaborated CTP-based imaging protocol during the acute aneurysmal SAH phase is able to prevent delayed infarctions and contribute to a better outcome. MATERIALS AND METHODS: In 2012, an elaborated CTP-based protocol was implemented for the management of patients with aneurysmal SAH. Retrospective analysis of patients with aneurysmal SAH treated from 2010 to 2013 was performed, comparing the patients treated before (group one, 2010-2011) with those treated after the protocol implementation (group two, 2012-2013) with regard to delayed infarctions and outcome according to the mRS at 3-months' follow-up. RESULTS: A total of 133 patients were enrolled, of whom 57 were included in group 1, and 76, in group 2. There were no significant differences between the groups concerning baseline characteristics. In the multivariate analysis, independent predictors of a good outcome (mRS ≤ 2) were younger age (P < .001), lower World Federation of Neurosurgical Societies grade (P < .001), absence of delayed infarction (P = .01), and management according to the CTP protocol (P = .01). Larger or multiple infarctions occurred significantly more often in group 1 compared with group 2 (88% versus 33% of all delayed infarctions, P = .03). The outcome in group 2 was significantly better compared with group 1 (P = .005). CONCLUSIONS: The findings suggest that implementation of an elaborated CTP protocol is associated with a better outcome. An earlier initiation of further diagnostics and treatment with prevention of large territorial and/or multiple infarctions might have led to this finding.

Zolmitriptan inhibits neurogenic inflammation and pain during electrical stimulation in human skin.

BACKGROUND: Triptans are agonists to 5-HT 1B/D/F receptors, which are present on nociceptive neurons not only within but also beyond the trigeminal system. The aim of this study was to investigate whether zolmitriptan interacts with peptidergic nociceptive afferents in human skin. METHODS: Twenty participants (13 women, median age: 25; interquartile range: 23-26 years) entered the randomized, double-blind, cross-over study. Electrically induced neurogenic flare and pain was assessed after either placebo or zolmitriptan on the ventral thigh. Mechanical pain thresholds were investigated at baseline and after electrical stimulation at the stimulation site. RESULTS: The size of the neurogenic flare (F = 10.9; p = 0.002) as well as electrically induced pain were significantly reduced by zolmitriptan (F = 4.46; p = 0.041). Moreover, electrically induced pinprick hyperalgesia was significantly decreased by zolmitriptan compared with placebo (F = 6.243; p = 0.017). CONCLUSIONS: Triptans may have effects outside of the trigeminal system and reduce electrically evoked neurogenic inflammation and pain in human skin.

Location and restoration of function after cerebellar tumor removal-a longitudinal study of children and adolescents.

Sequelae in children following cerebellar tumor removal surgery are well defined, and predictors for poor recovery include lesions of the cerebellar nuclei and the inferior vermis. Dynamic reorganization is thought to promote functional recovery in particular within the first year after surgery. Yet, the time course and mechanisms of recovery within this critical time frame are elusive and longitudinal studies are missing. Thus, a group of children and adolescents (n = 12, range 6-17 years) were followed longitudinally after cerebellar surgery and compared to age- and gender-matched controls (n = 11). Patients were examined (1) within the first days, (2) 3 months, and (3) 1 year after surgery. Each time behavioral tests of balance and upper limb motor function, ataxia rating, and a MRI scan were performed. Data were used for subsequent lesion-symptom mapping of cerebellar function. Behavioral improvements continued beyond 3 months, but were not complete in all patients after 1 year. At that time, remaining deficits were mild. Within the first 3 months, cerebellar lesion volumes were notably reduced by vanishing edema. Reduction in edema affecting the deep cerebellar nuclei but not reduction of total cerebellar lesion volume was a major predictor of early functional recovery. Persistent impairment in balance and upper limb function was linked to permanent lesions of the inferior vermis and the deep cerebellar nuclei.

[The influence of psychosocial factors on mental well-being and physical complaints before and after undergoing an in-patient abortion]. / Psychosoziale Merkmale und subjektives Befinden bei Frauen vor und nach stationärem indikationslosen Schwangerschaftsabbruch.

In this study, 57 women were examined in terms of the influence of different psychosocial factors on their subsequent mental well-being and physical complaints one day before, one day after, and 3 months after undergoing an in-patient abortion. Furthermore a control group of 40 in-patients (women with pregnancy related problems) were included in the study. The results show that prior to the abortion, most women reported a multitude of psychological and physical problems. However, it was also shown that for the majority of the women interviewed, mental well-being and physical complaints improved significantly one day and 3 months after the abortion. While feelings such as relief predominated immediately postoperatively, after 3 months, participants reported feeling cheerful and interested in activities. Further, it was demonstrated that women whose general mood was more pronouncedly anxious-depressive one day prior to operation later (after 3 months) reported many complaints and worse well-being. It appears that these women were not able to experience the abortion as a problem solutions. Finally, the great importance of the quality of their relationship and cohesion was demonstrated in the decision to abort, while pregnancy counselling was found to have no effect.

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.

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.

Effects of ligand binding on the internal dynamics of maltose-binding protein.

Ligand binding to proteins often causes large conformational changes. A typical example is maltose-binding protein (MBP), a member of the family of periplasmic binding proteins of Gram-negative bacteria. Upon binding of maltose, MBP undergoes a large structural change that closes the binding cleft, i.e. the distance between its two domains decreases. In contrast, binding of the larger, nonphysiological ligand beta-cyclodextrin does not result in closure of the binding cleft. We have investigated the dynamic properties of MBP in its different states using time-resolved tryptophan fluorescence anisotropy. We found that the 'empty' protein exhibits strong internal fluctuations that almost vanish upon ligand binding. The measured relaxation times corresponding to internal fluctuations can be interpreted as originating from two types of motion: wobbling of tryptophan side-chains relative to the protein backbone, and orientational fluctuations of entire domains. After binding of a ligand, domain motions are no longer detectable and the fluctuations of some of the tryptophan side-chains become rather restricted. This transformation into a more rigid state is observed upon binding of both ligands, maltose and the larger beta-cyclodextrin. The fluctuations of tryptophan side-chains in direct contact with the ligand, however, are affected in a slightly different way by the two ligands.

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.

Interaction between HLA-DM and HLA-DR involves regions that undergo conformational changes at lysosomal pH.

Antigenic peptide loading of major histocompatibility complex class II molecules is enhanced by lysosomal pH and catalyzed by the HLA-DM molecule. The physical mechanism behind the catalytic activity of DM was investigated by using time-resolved fluorescence anisotropy (TRFA) and fluorescence binding studies with the dye 8-anilino-1-naphthalenesulfonic acid (ANS). We demonstrate that the conformations of both HLA-DM and HLA-DR3, irrespective of the composition of bound peptide, are pH sensitive. Both complexes reversibly expose more nonpolar regions upon protonation. Interaction of DM with DR shields these hydrophobic domains from the aqueous environment, leading to stabilization of the DM and DR conformations. At lysosomal pH, the uncovering of additional hydrophobic patches leads to a more extensive DM-DR association. We propose that DM catalyzes class II peptide loading by stabilizing the low-pH conformation of DR, favoring peptide exchange. The DM-DR association involves a larger hydrophobic surface area with DR/class II-associated invariant chain peptides (CLIP) than with stable DR/peptide complexes, explaining the preferred association of DM with the former. The data support a release mechanism of DM from the DM-DR complex through reduction of the interactive surface, upon binding of class II molecules with antigenic peptide or upon neutralization of the DM-DR complex at the cell surface.

Pathway of detergent-mediated and peptide ligand-mediated refolding of heterodimeric class II major histocompatibility complex (MHC) molecules.

We investigated the mechanism of refolding and reassembly of recombinant alpha and beta chains of the class II major histocompatibility molecules (MHC-II) HLA-DRB5*0101. Both chains were expressed in the cytosol of Escherichia coli, purified in urea and SDS, and reassembled to functional heterodimers by replacement of SDS by mild detergents, incubation in a redox-shuffling buffer and finally by oxidation and removal of detergent. Refolding was mediated by mild detergents and by peptide ligands. Early stages of structure formation were characterized by circular dichroism, fluorescence, and time-resolved fluorescence anisotropy decay (FAD) spectroscopies. We found that formation of secondary structure was detectable after replacement of SDS by mild detergents. At that stage the alpha and beta chains were still monomeric, the buffer was strongly reducing, and the folding intermediates did not yet interact with peptide ligands. Formation of folding intermediates capable of interacting with peptide ligands was detected after adjusting the redox potential with oxidized glutathione and incubation in mild detergents. We conclude that at that stage a tertiary structure close to the native structure is formed at least locally. The nature and concentration of detergent critically determined the refolding efficiency. We compared detergents with different carbohydrate headgroups, and with aliphatic chains ranging from C6 to C14 in length. For each of the detergents we observed a narrow concentration range for mediating refolding. Surprisingly, detergents with long aliphatic chains had to be used at higher concentrations than short-chain detergents, indicating that increasing the solubility of folding intermediates is not the only function of detergents during a refolding reaction. We discuss structure formation and interactions of detergents with stable folding intermediates. Understanding such interactions will help to develop rational strategies for refolding hydrophobic or oligomeric proteins.

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.

The use of a long-lifetime component of tryptophan to detect slow orientational fluctuations of proteins.

The membrane protein porin and a synthetic polypeptide of 21 hydrophobic residues were inserted into detergent micelles or lipid membranes, and the fluorescence of their single tryptophan residue was measured in the time-resolved and polarized mode. In all cases, the tryptophan fluorescence exhibits a long-lifetime component of about 20 ns. This long-lifetime component was exploited to detect slow orientational motions in the range of tens of nanoseconds via the anisotropy decay. For this purpose, the analysis of the anisotropy has to be extended to account for different orientations of the dipoles of the short- and long-lifetime components. This is demonstrated for porin and the polypeptide solubilized in micelles, in which the longest relaxation time reflects the rotational diffusion of the micelle. When the polypeptide is inserted into lipid membranes, it forms a membrane-spanning alpha-helix, and the slowest relaxation process is interpreted as reflecting orientational fluctuations of the helix.

[Growth of the fetal thyroid gland in the 2nd half of pregnancy--biometric ultrasound studies]. / Wachstum der fetalen Schilddrüse in der II. Schwangerschaftshälfte-Sonographisch-biometrische Untersuchungen.

PURPOSE: Determination of normal values for fetal thyroid size in the 2nd half of pregnancy. METHOD: In 211 pregnant women (completed 20th to 41st week for pregnancy) without thyroid disease the fetal thyroid was measured and characterized by 5 parameters: right lobe transverse and p.a. diameter, left lobe transverse and p.a. diameter, right lobe and left lobe transverse including trachea. The level of measurement was a horizontal section of the neck with circular image of the trachea (centrally) and with the carotids as lateral limiting markers. RESULTS: The trend lines and regression coefficients of the parameters are shown in relation to week of pregnancy. The transverse diameter of both thyroid lobes including the trachea grows from 10 mm (20th week of pregnancy) to 20 mm (40th week of pregnancy). The transverse and p.a. diameters of either lobe double in the 2nd half of pregnancy. CONCLUSION: The normal values presented can help in early diagnosis of fetal thyroid hypertrophy.

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.

A long lifetime component in the tryptophan fluorescence of some proteins.

The tryptophan fluorescence of two membrane proteins (outer membrane protein A and lactose permease), a 21-residue hydrophobic peptide, three soluble proteins (rat serum albumin, ribonuclease T1, and azurin), and N-acetyltryptophanamide (NATA) was investigated by time-resolved measurements extended over 65 ns. A long lifetime component with a characteristic time of 25 ns and an amplitude below 1% was found for outer membrane protein A, lactose permease, the peptide in lipid membranes, and azurin in water, but not for rat serum albumin, ribonuclease T1, and NATA in water. When outer membrane protein A was dissolved and unfolded in guanidinum hydrochloride, the long lifetime component disappeared. Hence, a hydrophobic environment seems to be a necessary requirement for the long lifetime component to be present. However, NATA dissolved in butanol does not exhibit the long lifetime component, while the peptide dissolved in the same solvent under conditions which preserve its helical structure does show the long lifetime. Thus, a regular secondary structure for the polypeptide chain to which the tryptophan residue belongs seems to be a second necessary requirement for the long lifetime component to be present. The long lifetime component may therefore be seen in the context of protein substates.

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)

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.

Gradient for D-glucose and linoleic acid uptake along the crypt-villus axis of rabbit jejunal brush border membrane vesicles.

Glucose uptake into jejunal brush border membrane (BBM) varies along the crypt-villus axis (CVA). In the present study, the question was addressed whether uptake of the essential long-chain fatty acid linoleic acid also varies along the CVA. Using agitation techniques, five jejunal enterocyte fractions were sequentially isolated from female New Zealand white rabbits. A sixth and final fraction of lower-villus/crypt cells was obtained by the scraping of the remaining jejunal mucosa. Cell fraction along the CVA was proven histologically, by noting decreasing alkaline phosphatase activities in sequentially isolated fractions, and by demonstrating [3H-methyl]thymidine uptake mainly in the final fraction of the lower villus/crypt cells. BBM vesicles were prepared from the upper, mid- and lower-villus/crypt enterocyte fractions, using differential centrifugation and divalent ion precipitation. D-Glucose uptake into each fraction showed an Na(+)-gradient dependent time-course "overshoot" with linear uptake to 15 s and a subsequent decline to a steady-state plateau. Varying D-glucose concentrations from 50-1000 microM demonstrated saturation kinetics of uptake, with maximal transport rates (Vmax) and Michaelis affinity constants (Km) varying between fractions; the Km and Vmax were both lowest in the upper-villus fraction. A linear relationship existed between linoleic acid concentration (25-200 microM) and uptake in each fraction. Linoleic acid uptake was equivalent in all fractions when expressed per mg protein, but when expressed in terms of the estimated minimal BBM, vesicle surface area uptake was greater in the upper- than in the lower-villus/crypt fractions. Thus, BBM vesicle uptake of both linoleic acid and glucose vary along the crypt-villus axis of the rabbit jejunum.