The transplantation of encapsulated islets of Langerhans is one approach to treat type 1 diabetes without the need of lifelong immunosuppression. Capillaries have been used for macroencapsulation because they have a favorable surface-to-volume ratio and because they can be refilled. It is unclear at present whether the outer surface of such capillaries should be smooth to prevent, or rough to promote, cell adhesions. In this study we tested a new capillary made of modified polysulfone (MWCO: 50 kDa) with a rough, open-porous outer surface for islet transplantation. Compared with free-floating islets, encapsulation of freshly isolated rat islets affected neither the kinetics nor the efficiency of glucose-induced insulin release in perifusion experiments. Free-floating islets maintained insulin secretion during cell culture but encapsulated islets gradually lost their glucose responsiveness and released VEGF. This indicated hypoxia in the capillary lumen. Transplantation of encapsulated rat islets into diabetic rats significantly reduced blood glucose concentrations from the first week of implantation. This hypoglycaemic effect persisted until explantation 4 weeks later. Transplantation of encapsulated porcine islets into diabetic rats reduced blood glucose concentrations depending on the islet purity. With semipurified islets a transient reduction of blood glucose concentrations was observed (2, 8, 18, 18 days) whereas with highly purified islets a sustained normoglycaemia was achieved (more than 28 days). Explanted capillaries containing rat islets were covered with blood vessels. Vascularization was also observed on capillaries containing porcine islets that were explanted from normoglycaemic rats. In contrast, on capillaries containing porcine islets that were explanted from hyperglycemic rats a fibrous capsule and lymphocyte accumulations were observed. No vascularization on the surface of transplanted capillaries was observed in the absence of islets. In conclusion, encapsulated islets can release VEGF, which appears to be an important signal for the vascularization of the capillary material. The rough, open-porous outer surface of the polysulfone capillary provides a site well suited for vascular tissue formation and may allow a prolonged islet function after transplantation.
A purification procedure for guanylate kinase from pig brain has been developed consisting of ammonium sulfate precipitation and heptane extraction of the crude extract, hydrophobic-interaction chromatography, affinity chromatography and chromatofocussing. From 1.75 kg pig brain, 1.2 mg enzyme was isolated with a yield of 18% and a purity of about 90%. For sequence determination, the protein was cleaved with trypsin, cyanogen bromide and endoproteinase Glu-C. Some of the isolated peptides were subcleaved with chymotrypsin, thermolysin or trifluoroacetic acid. The blocked N-terminus was analyzed by mass spectrometry and by amino acid analysis of a tryptic peptide, while the C-terminus was found in a tryptic and a chymotryptic peptide and confirmed by a carboxypeptidase Y digestion. The sequence contains 197 amino acids with a M, of 21 831, one tryptophan and one cysteine residue. It has been compared to those of the homologous enzymes of yeast and Escherichiu coli, as well as to proteins from sequence data banks that show similarities. The sequence is discussed in the light of the known spatial structure of yeast guanylate kinase.Guanylate kinase (ATP:GMP-phospho-transferase) is a monomeric enzyme catalyzing the reaction MgATP + [2,19, 201. For the E. coli enzyme, the DNA sequence has been elucidated [21]. Guanylate kinase is essential for recycling GMP and, indirectly, also cGMP. A potent inhibitor of the enzyme is the antitumor drug 6-thioguanine [17]. The antiviral drugs Acyclovir [ l l ] and 9-(1,3-dihydroxy-2-propoxymethy1)guanine monophosphate [12] need the phosphorylation and, therefore, activation through guanylate kinase for inhibiting the replication of Herpes simplex virus. Therefore, it was of interest to isolate and sequence a guanylate kinase from a mammalian origin. Pig brain was selected because of the ready availability of the tissue and its relatively high guanylate kinase content [l].
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