Objective To investigate the mechanisms underlying the satiety-promoting effects of a novel protease inhibitors concentrate derived from potato (PPIC). Methods Acute and prolonged effects of oral PPIC administration (100 mg/kg per day) on food intake, body weight, and gastric emptying were evaluated in healthy rats. Parameters of body weight, food intake, plasma glucose, insulin, and cholecystokinin (CCK) were measured. Duodenal proteolytic activity and CCK expression were determined in tissue extracts. Intestinal STC-1 cell culture model was used to investigate the direct effect of PPIC on CCK transcript level and secretion. Results Acute oral administration of PPIC reduced immediate food intake during the first two hours following the treatment, delayed gastric emptying, and decreased proteolytic activity in the duodenum. Repeated oral ingestion of PPIC reduced weight gain in male rats and significantly elevated the plasma CCK levels. Although duodenal mucosal CCK mRNA levels increased in response to PPIC administration, the concentrate failed to elevate CCK expression or release in STC-1 cells. The 14-day ascending dose range study (33 to 266 mg/kg PPIC per day) showed no adverse side effects associated with PPIC administration. Conclusion These findings provided evidence that PPIC is effective in reducing food intake and body weight gain in healthy rats when administered orally by increasing circulating CCK levels through a trypsin-dependent mechanism.

PTF1 to the top, side and apical faces of the COM crystal. RESULTSPTF1 alone always adsorbed predominantly to the COM apical surfaces, while HSA bound principally to the side faces under inorganic conditions, but to the apical faces in urine. In the presence of each other, both proteins competed for adsorption to the apical faces, with attachment of PTF1 dominating over that of HSA. Modelling showed that urinary PTF1 had equal theoretical bonding potential for all three COM surfaces. CONCLUSIONS(i) Anisotropic inclusion of HSA and PTF1 into urinary and inorganic COM crystals results from their preferential binding to specific COM faces; (ii) the binding preference of HSA differs under inorganic and urinary conditions; (iii) preferential binding of PTF1 to the apical faces of COM is more complex than can be explained by interactions between Gla groups and surface atomic arrays; (iv) future studies of interactions between urinary proteins and stone mineral crystal surfaces should be performed in urine. KEYWORDScalcium oxalate, urolithiasis, intracrystalline protein, prothrombin fragment 1, human serum albumin, biomineralization OBJECTIVETo compare the intracrystalline distributions of prothrombin fragment 1 (PTF1) and human serum albumin (HSA) within inorganic and urinary calcium oxalate (CaOx) monohydrate (COM) crystals and to determine whether binding of PTF1 can be explained by interactions between particular γ -carboxyglutamic (Gla) residues and atomic arrays on individual faces of the COM crystal. MATERIALS AND METHODSCOM crystals were precipitated from inorganic solutions and ultrafiltered urine containing fluorescent HSA or PTF1 at different relative concentrations and examined by fluorescence microscopy. Accelrys Materials Studio and Discovery Studio were used to model the binding of

Our aim was to examine the attachment to, and incorporation of intact, highly phosphorylated osteopontin (OPN) into inorganic (i) and urinary (u) calcium oxalate monohydrate (COM) and dihydrate (COD) crystals. uCOM and uCOD crystals were precipitated from ultrafiltered (UF) urine containing human milk OPN (mOPN) labelled with Alexa Fluor 647 fluorescent dye at concentrations of 0.1-5.0 mg/L. iCOM and iCOD crystals were generated in aqueous solutions at concentrations of 0.01-0.5 mg/L. Crystals were demineralised with EDTA and the resulting extracts analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and western blotting, or examined by fluorescent confocal microscopy and field emission scanning electron microscopy before and after washing to remove proteins bound reversibly to the crystal surfaces. Binding of mOPN to pre-formed iCOM crystals was also studied in phosphate-buffered saline (PBS) and ultrafiltered (UF) urine. mOPN attached to the {100} faces and to the {010} sides of the {100}/{010} edges of iCOM crystals was removed by washing, indicating that it was not incorporated into the mineral bulk. In both PBS and urine, mOPN was attached to the {021} faces of pre-formed iCOM crystals as well as to the {100}/{010} edges, but was concentrated at the intersection points of the {100} and {121} faces at the crystal tips. Attachment in UF urine appeared to be greater than in PBS and stronger at higher calcium concentrations than lower calcium concentrations. In uCOM crystals, the distribution of fluorescence and patterns of erosion after washing suggested attachment of mOPN to the four end faces, followed by interment within the mineral phase. Fluorescence distributions of mOPN associated with both iCOD and uCOD crystals were consistent with uniform binding of the protein to all equivalent {101} faces and concentration along the intersections between them. Persistence of fluorescence after washing indicated that most mOPN was incarcerated within the mineral phase. We concluded that attachment of mOPN to calcium oxalate crystals is face-specific and depends upon the anatomical and genetic source of the protein and whether the crystals are (1) COM or COD; (2) pre-formed or precipitated from solution, and (3) precipitated from urine or aqueous solutions. Our findings emphasise the need for caution when drawing conclusions about possible roles of OPN or other proteins in urolithiasis from experimental data obtained under inorganic conditions.

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