Prediction of impending type 1 diabetes through automated dual-label measurement of proinsulin:C-peptide ratio

Background : The hyperglycemic clamp test, the gold standard of beta cell function, predicts impending type 1 diabetes in islet autoantibody-positive individuals, but the latter may benefit from less invasive function tests such as the proinsulin: C-peptide ratio (PI:C). The present study aims to optimize precision of PI:C measurements by automating a dual-label trefoil-type time-resolved fluorescence immunoassay (TT-TRFIA), and to compare its diagnostic performance for predicting type 1 diabetes with that of clamp-derived C-peptide release. Methods : Between-day imprecision (n = 20) and split-sample analysis (n = 95) were used to compare TT-TRFIA (Auto Delfia, Perkin-Elmer) with separate methods for proinsulin (in-house TRFIA) and C-peptide (Elecsys, Roche). High-risk multiple autoantibody-positive firstdegree relatives (n = 49; age 5-39) were tested for fasting PI:C, HOMA2-IR and hyperglycemic clamp and followed for 20-57 months (interquartile range). Results : TT-TRFIA values for proinsulin, C-peptide and PI:C correlated significantly (r(2) = 0.96-0.99; P<0.001) with results obtained with separate methods. TT-TRFIA achieved better between-day % CV for PI:C at three different levels (4.5-7.1 vs 6.7-9.5 for separate methods). In high-risk relatives fasting PI:C was significantly and inversely correlated ( r(s) = -0.596; P<0.001) with first-phase C-peptide release during clamp ( also with second phase release, only available for age 12-39 years; n = 31), but only after normalization for HOMA2-IR. In ROC- and Cox regression analysis, HOMA2-IR-corrected PI:C predicted 2-year progression to diabetes equally well as clamp-derived C-peptide release. Conclusions : The reproducibility of PI:C benefits from the automated simultaneous determination of both hormones. HOMA2-IR-corrected PI:C may serve as a minimally invasive alternative to the more tedious hyperglycemic clamp test

The involvement of actin, calcium channels and exocytosis proteins in somato-dendritic oxytocin and vasopressin release

Hypothalamic magnocellular neurons release vasopressin and oxytocin not only from their axon terminals into the blood, but also from their somata and dendrites into the extracellular space of the brain, and this can be regulated independently. Differential release of neurotransmitters from different compartments of a single neuron requires subtle regulatory mechanisms. Somato-dendritic, but not axon terminal release can be modulated by changes in intracellular calcium concentration [(Ca(2+))] by release of calcium from intracellular stores, resulting in priming of dendritic pools for activity-dependent release. This review focuses on our current understanding of the mechanisms of priming and the roles of actin remodeling, voltage-operated calcium channels (VOCCs) and SNARE proteins in the regulation somato-dendritic and axon terminal peptide release

Thermodynamic and kinetic insights into stop codon recognition by release factor 1.

Stop codon recognition is a crucial event during translation termination and is performed by class I release factors (RF1 and RF2 in bacterial cells). Recent crystal structures showed that stop codon recognition is achieved mainly through a network of hydrogen bonds and stacking interactions between the stop codon and conserved residues in domain II of RF1/RF2. Additionally, previous studies suggested that recognition of stop codons is coupled to proper positioning of RF1 on the ribosome, which is essential for triggering peptide release. In this study we mutated four conserved residues in Escherichia coli RF1 (Gln185, Arg186, Thr190, and Thr198) that are proposed to be critical for discriminating stop codons from sense codons. Our thermodynamic and kinetic analysis of these RF1 mutants showed that the mutations inhibited the binding of RF1 to the ribosome. However, the mutations in RF1 did not affect the rate of peptide release, showing that imperfect recognition of the stop codon does not affect the proper positioning of RF1 on the ribosome

Novel Molecules for Intra-Oral Delivery of Antimicrobials to Prevent and Treat Oral Infectious Diseases

New molecules were designed for efficient intra-oral delivery of antimicrobials to prevent and treat oral infection. The salivary statherin fragment, which has high affinity for the tooth enamel, was used as a carrier peptide. This was linked through the side chain of the N-terminal residue to the C-terminus of a defensin-like 12-residue peptide to generate two bifunctional hybrid molecules, one with an ester linkage and the other with an anhydride bond between the carrier and the antimicrobial components. They were examined for their affinity to a HAP (hydroxyapatite) surface. The extent of the antimicrobial release in human whole saliva was determined using 13C-NMR spectroscopy. The candidacidal activity of the molecules was determined as a function of the antimicrobial release from the carrier peptide in human saliva. The hybrid-adsorbed HAP surface was examined against Candida albicans and Aggregatibacter actinomycetemcomitans using the fluorescence technique. The bifunctional molecules were tested on human erythrocytes, GECs (gingival epithelial cells) and GFCs (gingival fibroblast cells) for cytotoxicity. They were found to possess high affinity for the HAP mineral. In human whole saliva, a sustained antimicrobial release over a period of more than 40–60 h, and candidacidal activity consistent with the extent of hybrid dissociation were observed. Moreover, the bifunctional peptide-bound HAP surface was found to exhibit antimicrobial activity when suspended in clarified human saliva. The hybrid peptides did not show any toxic influence on human erythrocytes, GECs and GFCs. These novel hybrids could be safely used to deliver therapeutic agents intra-orally for the treatment and prevention of oral infectious diseases

Development of affinity-based delivery of NGF from a chondroitin sulfate biomaterial.

Chondroitin sulfate is a major component of the extracellular matrix in both the central and peripheral nervous systems. Chondroitin sulfate is upregulated at injury, thus methods to promote neurite extension through chondroitin sulfate-rich matrices and synthetic scaffolds are needed. We describe the use of both chondroitin sulfate and a novel chondroitin sulfate-binding peptide to control the release of nerve growth factor. Interestingly, the novel chondroitin sulfate-binding peptide enhances the controlled release properties of the chondroitin sulfate gels. While introduction of chondroitin sulfate into a scaffold inhibits primary cortical outgrowth, the combination of chondroitin sulfate, chondroitin sulfate-binding peptide and nerve growth factor promotes primary cortical neurite outgrowth in chondroitin sulfate gels

Optimization of macromolecular prodrugs of the antitumor antibiotic adriamycin

In our earlier work [10] on aminoribosyl-bound prodrugs of adriamycin (ADR) using poly(α-l-glutamic acid) (PGA) grafted in high yield (90–100 mol.%) with various peptide spacers as a plasma-soluble macromolecular carrier we observed rather low cytotoxic activities in L1210 leukemia and B16 melanoma in vitro assays. These results may be tentatively explained by a decreased susceptibility of the spacer-bound adriamycin moiety to hydrolysis by lysosomal enzymes due to the high spacer load. This hypothesis was tested by the study of two conjugates prepared by a different route. Peptide conjugates of adriamycin (Gly-Gly-Leu—ADR and Gly-Gly-Gly-Leu—ADR) were synthesized using the trityl N-protecting group and were coupled to PGA in 4.5 mol.% load according to the method described earlier [11]. However, these conjugates were almost totally devoid of cell growth-inhibiting activity in L1210 and B16 in vitro tests. The data suggest that either the uptake of the polymeric prodrugs into the cell by pinocytosis is highly dependent on spacer load or molecular weight, or that lysosomal digestion is too slow for efficient release of ADR. Possibly, enzymatic degradation of PGA which is known to occur only between pH 4 and 6 is rate-limiting for release of the drug. Current studies include the enzymatic degradation of PGA—peptide spacer—drug systems using p-nitroaniline as a model drug and papain as the enzyme. By variation of the length and load of spacer it can be estimated under which conditions the release of drug (using UV spectrometry) is faster than degradation of the polymer (as determined by viscometry). In addition, the uptake of PGA and derivatives with a fluorescent label into tumor cells is studied using laser flow cytometry and laser microscopy

Atomic mutagenesis of stop codon nucleotides reveals the chemical prerequisites for release factor-mediated peptide release.

Termination of protein synthesis is triggered by the recognition of a stop codon at the ribosomal A site and is mediated by class I release factors (RFs). Whereas in bacteria, RF1 and RF2 promote termination at UAA/UAG and UAA/UGA stop codons, respectively, eukaryotes only depend on one RF (eRF1) to initiate peptide release at all three stop codons. Based on several structural as well as biochemical studies, interactions between mRNA, tRNA, and rRNA have been proposed to be required for stop codon recognition. In this study, the influence of these interactions was investigated by using chemically modified stop codons. Single functional groups within stop codon nucleotides were substituted to weaken or completely eliminate specific interactions between the respective mRNA and RFs. Our findings provide detailed insight into the recognition mode of bacterial and eukaryotic RFs, thereby revealing the chemical groups of nucleotides that define the identity of stop codons and provide the means to discriminate against noncognate stop codons or UGG sense codons

Alzheimer’s disease-associated peptide Aβ₄₂ mobilizes ER Ca²⁺ via InsP₃R-dependent and -independent mechanisms

Dysregulation of Ca2+ homeostasis is considered to contribute to the toxic action of the Alzheimer’s Disease (AD) associated Amyloid β-peptide (Aβ). Ca2+ fluxes across the plasma membrane and release from intracellular stores have both been reported to underlie the Ca2+ fluxes induced by Aβ42. Here, we investigated the contribution of Ca2+ release from the endoplasmic reticulum (ER) to the effects of Aβ42 upon Ca2+ homeostasis and the mechanism by which Aβ42 elicited these effects. Consistent with previous reports, application of soluble oligomeric forms of Aβ42 exhibited Ca2+ mobilizing properties. The Aβ42-stimulated Ca2+ signals persisted in the absence of extracellular Ca2+ indicating a significant contribution of Ca2+ release from the ER Ca2+ store to the generation of these signals. Moreover, inositol 1,4,5-trisphosphate (InsP3) signaling contributed to Aβ42-stimulated Ca2+ release. The Ca2+ mobilizing effect of Aβ42 was also observed when applied to permeabilized cells deficient in InsP3 receptors revealing an additional direct effect of internalized Aβ42 upon the ER, and a mechanism for induction of toxicity by intracellular Aβ42