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Novel affinity-purified antibodies against human SGLT1 (hSGLT1) and SGLT2 (hSGLT2) were used to localize hSGLT2 in human kidney and hSGLT1 in human kidney, small intestine, liver, lung, and heart. The renal locations of both transporters largely resembled those in rats and mice; hSGLT2 and SGLT1 were localized to the brush border membrane (BBM) of proximal tubule S1/S2 and S3 segments, respectively. Different to rodents, the renal expression of hSGLT1 was absent in thick ascending limb of Henle (TALH) and macula densa, and the expression of both hSGLTs was sex-independent. In small intestinal enterocytes, hSGLT1 was localized to the BBM and subapical vesicles. Performing double labeling with glucagon-like peptide 1 (GLP-1) or glucose-dependent insulinotropic peptide (GIP), hSGLT1 was localized to GLP-1-secreting L cells and GIP-secreting K cells as has been shown in mice. In liver, hSGLT1 was localized to biliary duct cells as has been shown in rats. In lung, hSGLT1 was localized to alveolar epithelial type 2 cells and to bronchiolar Clara cells. Expression of hSGLT1 in Clara cells was verified by double labeling with the Clara cell secretory protein CC10. Double labeling of human heart with aquaporin 1 immunolocalized the hSGLT1 protein in heart capillaries rather than in previously assumed myocyte sarcolemma. The newly identified locations of hSGLT1 implicate several extra renal functions of this transporter, such as fluid absorption in the lung, energy supply to Clara cells, regulation of enteroendocrine cells secretion, and release of glucose from heart capillaries. These functions may be blocked by reversible SGLT1 inhibitors which are under development.

To clarify the physiological role of Na(+)-D-glucose cotransporter SGLT1 in small intestine and kidney, Sglt1(-/-) mice were generated and characterized phenotypically. After gavage of d-glucose, small intestinal glucose absorption across the brush-border membrane (BBM) via SGLT1 and GLUT2 were analyzed. Glucose-induced secretion of insulinotropic hormone (GIP) and glucagon-like peptide 1 (GLP-1) in wild-type and Sglt1(-/-) mice were compared. The impact of SGLT1 on renal glucose handling was investigated by micropuncture studies. It was observed that Sglt1(-/-) mice developed a glucose-galactose malabsorption syndrome but thrive normally when fed a glucose-galactose-free diet. In wild-type mice, passage of D-glucose across the intestinal BBM was predominantly mediated by SGLT1, independent the glucose load. High glucose concentrations increased the amounts of SGLT1 and GLUT2 in the BBM, and SGLT1 was required for upregulation of GLUT2. SGLT1 was located in luminal membranes of cells immunopositive for GIP and GLP-1, and Sglt1(-/-) mice exhibited reduced glucose-triggered GIP and GLP-1 levels. In the kidney, SGLT1 reabsorbed ∼3% of the filtered glucose under normoglycemic conditions. The data indicate that SGLT1 is 1) pivotal for intestinal mass absorption of d-glucose, 2) triggers the glucose-induced secretion of GIP and GLP-1, and 3) triggers the upregulation of GLUT2.

To localise antigens by immunocytochemistry (IC), the samples of tissues or cells are usually denatured by fixation, and either frozen and cryosectioned, or embedded in paraffin before sectioning. p-Formaldehyde (PFA; formalin) is a common fixative, which preserves antigenicity of proteins, but damages the tissue/cell morphology and \"masks\" the antibody binding sites (epitopes). In order to \"unmask\" epitopes, some kind of antigen retrieval (AR) is used. The aim of this study was: a) to find an optimal AR method in cryosections of in vivo PFA-fixed kidneys for organic anion transporters (Oat) that reside in the basolateral (Oat1, Oat3) and brush-border membrane (Oat2, Oat5) of the rat renal proximal tubules, and b) using optimal method, to compare IC staining of Oats in kidneys that had been PFA-fixed in vivo or in vitro. IC staining in untreated cryosections was compared with that following detergent treatment or microwave heating in citrate buffer of pH 3, pH 6, or pH 8, with or without alcohol pre-treatment. The preferred AR method for Oat1, Oat2, and Oat5 was heating of cryosections at pH 6, and for Oat3 heating at pH 3, without alcohol pre-treatment. Compared with tissue fixed in vivo, tissue fixed in vitro exhibited damaged tubule morphology, similar staining intensity of Oat1 and Oat3, and higher staining intensity of Oat2 and Oat5. We conclude that for optimal IC presentation, each Oat in the rat kidney has to be treated individually, with different fixation and AR approach. Za imunocitokemijsku (IC) lokalizaciju antigena uzorci tkiva ili stanica obično se denaturiraju fiksacijom, a potom se odmah smrznu i naresci režu kriostatom ili se uklope u parafin prije rezanja narezaka. Parafin se kasnije odstrani alkoholima. p-Formaldehid (PFA; formalin) čest je fiksativ, koji čuva antigeničnost proteina, ali oštećuje morfologiju stanica i \"maskira\" vezna mjesta za protutijela (epitope). Za \"demaskiranje\" epitopa potrebno je primijeniti neku od metoda otkrivanja (regeneracije) antigena (RA). Namjera ove studije jest: a) odrediti optimalnu metodu RA u kriostatskim narescima PFA-fiksiranih bubrega in vivo za prijenosnike organskih aniona (Oat) smještene u bazolateralnoj (Oat1, Oat3) i četkastoj (Oat2, Oat5) membrani proksimalnih kanalića u bubrezima štakora i b) rabeći optimalnu metodu, usporediti IC bojenje Oat-a u bubrezima koji su bili PFA-fiksirani in vivo ili in vitro. IC bojenje u neobrađenim kriostatskim narescima uspoređeno je s bojenjem nakon obrade detergentom ili mikrovalnim kuhanjem u citratnom puferu pH 3, pH 6 ili pH 8, s prethodnom obradom alkoholima ili bez nje. Optimalni RA-uvjet za Oat1, Oat2 i Oat5 bio je kuhanje narezaka pri pH 6, a za Oat3 pri pH 3, bez prethodne obrade alkoholima. U usporedbi s tkivom fiksiranim in vivo, tkivo fiksirano in vitro imalo je: oštećenu morfologiju kanalića, sličnu jačinu bojenja Oat1 i Oat3, a jače bojenje Oat2 i Oat5. Zaključujemo da svaki Oat u bubregu štakora treba obraditi pojedinačno, s različitom fiksacijom i metodom RA, kako bi se našli optimalni uvjeti za njegovo IC prikazivanje.

To clarify the physiological role of [Na.sup.+]-D-glucose cotransporter SGLT1 in small intestine and kidney, [Sglt1.sup.-/-] mice were generated and characterized phenotypically. After gavage of D-glucose, small intestinal glucose absorption across the brush-border membrane (BBM) via SGLT1 and GLUT2 were analyzed. Glucose-induced secretion of insulinotropic hormone (GIP) and glucagon-like peptide 1 (GLP-1) in wild-type and [Sglt1.sup.-/-] mice were compared. The impact of SGLT1 on renal glucose handling was investigated by micropuncture studies. It was observed that [Sglt1.sup.-/-] mice developed a glucose-galactose malabsorption syndrome but thrive normally when fed a glucose-galactose-free diet. In wild-type mice, passage of D-glucose across the intestinal BBM was predominantly mediated by SGLT1, independent the glucose load. High glucose concentrations increased the amounts of SGLT1 and GLUT2 in the BBM, and SGLT1 was required for upregulation of GLUT2. SGLT1 was located in luminal membranes of cells immunopositive for GIP and GLP-1, and [Sglt1.sup.-/-] mice exhibited reduced glucose-triggered GIP and GLP-1 levels. In the kidney, SGLT1 reabsorbed ~3% of the filtered glucose under normoglycemic conditions. The data indicate that SGLT1 is 1) pivotal for intestinal mass absorption of D-glucose, 2) triggers the glucose-induced secretion of GIP and GLP-1, and 3) triggers the upregulation of GLUT2. Diabetes 61:187-196, 2012

Novel affinity-purified antibodies against human SGLT1 (hSGLT1) and SGLT2 (hSGLT2) were used to localize hSGLT2 in human kidney and hSGLT1 in human kidney, small intestine, liver, lung, and heart. The renal locations of both transporters largely resembled those in rats and mice; hSGLT2 and SGLT1 were localized to the brush border membrane (BBM) of proximal tubule S1/S2 and S3 segments, respectively. Different to rodents, the renal expression of hSGLT1 was absent in thick ascending limb of Henle (TALH) and macula densa, and the expression of both hSGLTs was sex-independent. In small intestinal enterocytes, hSGLT1 was localized to the BBM and subapical vesicles. Performing double labeling with glucagon-like peptide 1 (GLP-1) or glucose-dependent insulinotropic peptide (GIP), hSGLT1 was localized to GLP-1-secreting L cells and GIP-secreting K cells as has been shown in mice. In liver, hSGLT1 was localized to biliary duct cells as has been shown in rats. In lung, hSGLT1 was localized to alveolar epithelial type 2 cells and to bronchiolar Clara cells. Expression of hSGLT1 in Clara cells was verified by double labeling with the Clara cell secretory protein CC10. Double labeling of human heart with aquaporin 1 immunolocalized the hSGLT1 protein in heart capillaries rather than in previously assumed myocyte sarcolemma. The newly identified locations of hSGLT1 implicate several extra renal functions of this transporter, such as fluid absorption in the lung, energy supply to Clara cells, regulation of enteroendocrine cells secretion, and release of glucose from heart capillaries. These functions may be blocked by reversible SGLT1 inhibitors which are under development.

Za imunocitokemijsku (IC) lokalizaciju antigena uzorci tkiva ili stanica obično se denaturiraju fi ksacijom, a potom se odmah smrznu i naresci režu kriostatom ili se uklope u parafi n prije rezanja narezaka. Parafi n se kasnije odstrani alkoholima. p-Formaldehid (PFA; formalin) čest je fi ksativ, koji čuva antigeničnost proteina, ali oštećuje morfologiju stanica i “maskira” vezna mjesta za protutijela (epitope). Za “demaskiranje” epitopa potrebno je primijeniti neku od metoda otkrivanja (regeneracije) antigena (RA). Namjera ove studije jest: a) odrediti optimalnu metodu RA u kriostatskim narescima PFA-fi ksiranih bubrega in vivo za prijenosnike organskih aniona (Oat) smještene u bazolateralnoj (Oat1, Oat3) i četkastoj (Oat2, Oat5) membrani proksimalnih kanalića u bubrezima štakora i b) rabeći optimalnu metodu, usporediti IC bojenje Oat-a u bubrezima koji su bili PFA-fi ksirani in vivo ili in vitro. IC bojenje u neobrađenim kriostatskim narescima uspoređeno je s bojenjem nakon obrade detergentom ili mikrovalnim kuhanjem u citratnom puferu pH 3, pH 6 ili pH 8, s prethodnom obradom alkoholima ili bez nje. Optimalni RA-uvjet za Oat1, Oat2 i Oat5 bio je kuhanje narezaka pri pH 6, a za Oat3 pri pH 3, bez prethodne obrade alkoholima. U usporedbi s tkivom fi ksiranim in vivo, tkivo fi ksirano in vitro imalo je: oštećenu morfologiju kanalića, sličnu jačinu bojenja Oat1 i Oat3, a jače bojenje Oat2 i Oat5. Zaključujemo da svaki Oat u bubregu štakora treba obraditi pojedinačno, s različitom fi ksacijom i metodom RA, kako bi se našli optimalni uvjeti za njegovo IC prikazivanje.

The named “green chemistry” has been receiving increasing prominence due to its environmentally friendly characteristics. The use of enzymes as catalysts in processes of synthesis to replace the traditional use of chemical catalysts present as main advantage the fact of following the principles of the green chemistry. However, processes of enzymatic nature generally provide lower yields when compared to the conventional chemical processes. Therefore, in the last years, the ultrasound has been extensively used in enzymatic processes, such as the production of esters with desirable characteristics for the pharmaceutical, cosmetics, and food industry, for the hydrolysis and glycerolysis of vegetable oils, production of biodiesel, etc. Several works found in the open literature suggest that the energy released by the ultrasound during the cavitation phenomena can be used to enhance mass transfer (substrate/enzyme), hence increasing the rate of products formation, and also contributing to enhance the enzyme catalytic activity. Furthermore, the ultrasound is considered a “green” technology due to its high efficiency, low instrumental requirement and significant reduction of the processing time in comparison to other techniques. The main goal of this review was to summarize studies available to date regarding the application of ultrasound in enzyme-catalyzed esterification, hydrolysis, glycerolysis and transesterification reactions.