No abstract

Tissue engineering scaffolds are commonly formed using proteins extracted from animal tissues, such as bovine hide. Risks associated with the use of these materials include hypersensitivity and pathogenic contamination. Human-derived proteins lower the risk of hypersensitivity, but possess the risk of disease transmission. Methods engineering recombinant human proteins using plant material provide an alternate source of these materials without the risk of disease transmission or concerns regarding variability. To investigate the utility of plant-derived human collagen (PDHC) in the development of engineered skin (ES), PDHC and bovine hide collagen were formed into tissue engineering scaffolds using electrospinning or freeze-drying. Both raw materials were easily formed into two common scaffold types, electrospun nonwoven scaffolds and lyophilized sponges, with similar architectures. The processing time, however, was significantly lower with PDHC. PDHC scaffolds supported primary human cell attachment and proliferation at an equivalent or higher level than the bovine material. Interleukin-1 beta production was significantly lower when activated THP-1 macrophages where exposed to PDHC electrospun scaffolds compared to bovine collagen. Both materials promoted proper maturation and differentiation of ES. These data suggest that PDHC may provide a novel source of raw material for tissue engineering with low risk of allergic response or disease transmission.

Rapidly growing tomato (Lycopersicon esculentum) cell suspension cultures contain transiently high levels of cell surface, salt-elutable, monomeric precursors to the covalently cross-linked extensin network of the primary cell wall. Thus, we purified a highly soluble monomeric extensin substrate from rapidly growing cells, and devised a soluble in vitro cross-linking assay based on Superose-6 fast protein liquid chromatography separation, which resolved extensin monomers from the newly formed oligomers within 25 minutes. Salt elution of slowly growing (early stationary phase) cells yielded little or no extensin monomers but did give a highly active enzymic preparation that specifically cross-linked extensin monomers in the presence of hydrogen peroxide, judging from: (a) a decrease in the extensin monomer peak on fast protein liquid chromatography gel filtration, (b) appearance of oligomeric peaks, and (c) direct electron microscopical observation of the cross-linked oligomers. The cross-linking reaction had a broad pH optimum between 5.5 and 6.5. An approach to substrate saturation of the enzyme required extensin monomer concentrations of 20 to 40 milligrams per milliliter. Preincubation with catalase completely inhibited the cross-linking reaction, which was highly dependent on hydrogen peroxide and optimal at 15 to 50 micromolar. We therefore identified the cross-linking activity as extensin peroxidase.the identification of IDT2 in cell wall hydrolysates (15), and hence a new protein cross-linking amino acid.Demonstration of IDT as a short intramolecular cross-link in two extensin tryptic peptides (13) made the suggested involvement of peroxidase in extensin cross-linkage (9, 29) more plausible and led to the discovery of a monomeric extensin pool in muro readily eluted from intact cells of rapidly growing suspension cultures (40). Pool turnover kinetics were consistent with the status of these monomers (P1 and P2) as soluble precursors to an insoluble network (40). Moreover, peptide mapping and amino acid sequencing identified the hexapeptide Val-Lys-ProTyr-His-Pro of P1 as a putative cross-link domain, occurring about five times in the monomer (41).Our extensin precursor preparations routinely yield substrate (mg) amounts, which have enabled us to develop a quantitative assay for in vitro cross-linking. We describe here methods for the isolation and assay of extensin precursor cross-linking activity. This involved CaCl2 elution of extensin cross-linking activity from intact cells and assay of the enzymic activity as the conversion rate ofextensin monomers to oligomers, measured by FPLC on Superose-6. We confirmed cross-linking by direct TEM visualization of the oligomeric products.The essential enzymic characterization of the cross-linking activity included: pH optimum, substrate saturation, inhibitors, and dependence on exogenous hydrogen peroxide. We therefore propose the trivial name 'extensin peroxidase' for the isolated cross-linking activity, which we have previously described briefly in progr...

Ovine pregnancy-specific protein B (oPSPB) was isolated from sheep placentas. Antiserum to oPSPB was developed in rabbits. A quantitative RIA was developed and used to assay the serum concentrations of oPSPB during and after pregnancy in ewes bearing single or twin fetuses. Suffolk and Panama ewes, kept with rams equipped with a marking harness, were checked daily for breeding marks as an indication of estrus and bled daily between 10 and 30 d after marking. Ovine PSPB became detectable at 19.7 +/- .14 (mean +/- SE) d after breeding and increased steadily to d 30. Panama oPSPB concentration increased at a greater rate than that of Suffolks (breed x day interaction, P < .01). Ten ewes were bled twice weekly 3 wk before their expected date of lambing and weekly for 7 wk postpartum. Serum concentrations differed considerably between prepartum ewes, but concentrations remained stable within the period of 20 d prepartum. Following parturition, oPSPB concentrations dropped rapidly. In nine ewes, oPSPB was last detectable at 12.78 +/- 2.26 (mean +/- SE) d postpartum. In the 10th ewe, oPSPB was .65 ng/mL at the last sample on d 46 postpartum. To determine the effect of fetal number on oPSPB concentrations, ewes in which estrus was synchronized were bled at d 18, 25, 38, 60, 90, and 120 after breeding. Ewes were killed at d 60, 90, 120, and 148 and fetal number determined. There was a significant (P < .01) difference in the log of oPSPB concentrations according to number of fetuses, day postbreeding, and the day x fetal number interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Background and Purpose Translation of non‐clinical markers of delayed ventricular repolarization to clinical prolongation of the QT interval corrected for heart rate (QTc) (a biomarker for torsades de pointes proarrhythmia) remains an issue in drug discovery and regulatory evaluations. We retrospectively analysed 150 drug applications in a US Food and Drug Administration database to determine the utility of established non‐clinical in vitro IKr current human ether‐à‐go‐go‐related gene (hERG), action potential duration (APD) and in vivo (QTc) repolarization assays to detect and predict clinical QTc prolongation. Experimental Approach The predictive performance of three non‐clinical assays was compared with clinical thorough QT study outcomes based on free clinical plasma drug concentrations using sensitivity and specificity, receiver operating characteristic (ROC) curves, positive (PPVs) and negative predictive values (NPVs) and likelihood ratios (LRs). Key Results Non‐clinical assays demonstrated robust specificity (high true negative rate) but poor sensitivity (low true positive rate) for clinical QTc prolongation at low‐intermediate (1×–30×) clinical exposure multiples. The QTc assay provided the most robust PPVs and NPVs (ability to predict clinical QTc prolongation). ROC curves (overall test accuracy) and LRs (ability to influence post‐test probabilities) demonstrated overall marginal performance for hERG and QTc assays (best at 30× exposures), while the APD assay demonstrated minimal value. Conclusions and Implications The predictive value of hERG, APD and QTc assays varies, with drug concentrations strongly affecting translational performance. While useful in guiding preclinical candidates without clinical QT prolongation, hERG and QTc repolarization assays provide greater value compared with the APD assay.