Composition and function of macroencapsulated human embryonic stem cell-derived implants: comparison with clinical human islet cell grafts.

β-Cells generated from large-scale sources can overcome current shortages in clinical islet cell grafts provided that they adequately respond to metabolic variations. Pancreatic (non)endocrine cells can develop from human embryonic stem (huES) cells following in vitro derivation to pancreatic endoderm (PE) that is subsequently implanted in immune-incompetent mice for further differentiation. Encapsulation of PE increases the proportion of endocrine cells in subcutaneous implants, with enrichment in β-cells when they are placed in TheraCyte-macrodevices and predominantly α-cells when they are alginate-microencapsulated.

Human mesotrypsin exhibits restricted S1' subsite specificity with a strong preference for small polar side chains.

Mesotrypsin, an inhibitor-resistant human trypsin isoform, does not activate or degrade pancreatic protease zymogens at a significant rate. These observations led to the proposal that mesotrypsin is a defective digestive protease on protein substrates. Surprisingly, the studies reported here with alpha1-antitrypsin (alpha1AT) revealed that, even though mesotrypsin was completely resistant to this serpin-type inhibitor, it selectively cleaved the Lys10-Thr11 peptide bond at the N-terminus.

A degradation-sensitive anionic trypsinogen (PRSS2) variant protects against chronic pancreatitis.

Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease.

Expression of human cationic trypsinogen with an authentic N terminus using intein-mediated splicing in aminopeptidase P deficient Escherichia coli.

High-level expression of human trypsinogens as inclusion bodies in Escherichia coli requires deletion of the secretory signal sequence and placement of an initiator methionine at the N terminus. Trypsinogen preparations obtained this way contain a mixture of abnormal N termini, as a result of processing by cytoplasmic aminopeptidases. Here, we describe an expression system that produces recombinant human cationic trypsinogen with a native, intact N terminus, using intein-mediated protein splicing and an aminopeptidase P (pepP) deficient E.

Inactivity of recombinant ELA2B provides a new example of evolutionary elastase silencing in humans.

Background: The archetypal mammalian elastase (ELA1) is not expressed in the human pancreas, because evolutionary mutations suppressed transcription of the ELA1 gene.

Aims: In this study, we tested the theory that the unique duplication of the ELA2 gene in humans might compensate for the loss of ELA1.

Methods: Recombinant ELA2A and ELA2B were expressed in Escherichia coli, and their activity was tested on Glt-Ala-Ala-Pro-Leu-p-nitroanilide, DQ elastin and bovine milk protein.

Multiple subphases of DNA repair and poly (ADP-ribose) synthesis in Chinese hamster ovary (CHO-K1) cells.

The two types of DNA synthesis as well as poly(ADP-ribose) biosynthesis were measured simultaneously in synchronized intact populations of CHO cells throughout the duration of S phase. Naturally occurring DNA fragmentation was detected by random primed oligonucleotide synthesis (ROPS assay). Fractions of synchronous cell populations were obtained by counterflow centrifugal elutriation.

Subphases of DNA replication in Drosophila cells.

Exponentially growing Drosophila S2 cells in suspension culture were synchronized at low- and high-resolution centrifugal elutriation, and DNA synthesis was measured by [(3)H]-thymidine incorporation throughout the S phase. At low resolution, one repair peak at the G(1)/G(0) border and two replication peaks known as early and late S subphases were observed. At high resolution, six chronologic compartments were distinguished.

Effect of cadmium on the relationship between replicative and repair DNA synthesis in synchronized CHO cells.

Repair and replicative DNA synthesis were measured at different stages of the cell cycle in control and cadmium-treated Chinese hamster ovary (CHO-K1) cells. Cells were synchronized by counterflow centrifugal elutriation. Elutriation resulted in five repair and four replication subphases.