Dg Function Research Articles

Glycyrrhizinate attenuates exosome induced endothelial cell proliferation and permeability through p38 pathway

Purpose: To investigate the mechanism of action involved in the treatment of severe burn injury with glycyrrhizinate (DG). Methods: Exosomes were purified from sera of patients with burn injury using an ultra-high-speed centrifuge, and verified by western blot. Cell proliferation and permeability were assessed using cell counting kit (CCK)-8, transepithelial-transendothelial electrical resistance (TEER), and Fluorescein Isothiocyanate (FITC) dextran assays, while immunoblotting was used for assay of the levels of p38, occluding, and zonula occludens 1 (ZO-1). Results: Serum-derived exosomes (serum-exo) significantly suppressed cell proliferation while causing hyperpermeability in HUVECs (p < 0.001). Furthermore, DG alleviated the hyperpermeability and inhibition of cell proliferation caused by serum-exo (p < 0.001). In addition, the upregulation of p-P38 induced by serum-exo decreased upon DG treatment. Interestingly, the effect of DG was blocked by anisomycin, a specific p38 activator, indicating that p38 signaling pathway may contribute to the function of DG. Conclusion: Glycyrrhizinate attenuates serum-exo-induced cell proliferation and permeability alteration via p38 signaling pathway, thereby making it a potential agent for the management of severe burn injury.

Probabilistic seismic assessment of the pounding risk based on the local demands of a multistory RC frame structure

The aim of this study is to incorporate the local inelastic demands of a multistory reinforced concrete (RC) frame structure for the first time in the probabilistic seismic assessment of the pounding risk. Two distinct types of structural pounding are examined: a) the floor-to-floor structural pounding - Type A, and b) the inter-story (floor-to-column) structural pounding - Type B. Three different initial gap distances dg between the adjacent structures are considered. The seismic performance of the RC frame structure without the pounding effect is also estimated. The probabilistic evaluation of the pounding effect is performed through fragility curves in terms of global and local engineering demand parameters (EDPs) as a function of the peak ground acceleration (PGA). The first part of this research is focused on the development of global and local probabilistic seismic demand models (PSDMs) against pounding risk. For this purpose, linear and bilinear regression models have been used. Afterwards, the fragility assessment of the RC frame against pounding is performed (a) as a function of the separation gap distance dg, (b) based on the global seismic performances, and (c) based on the local seismic performances. A compounded fragility-based solution on evaluating the pounding risk at different performance levels is also presented and new performance levels thresholds are introduced. Results indicate that the local performances of the columns of the RC structure are crucial demand parameters for the probabilistic assessment of the pounding risk. The type of the structural pounding (Type A or Type B) that is occurred between the adjacent structures significantly alters the results of the probabilistic assessment when the local performances are considered. The compounded evaluation of the pounding risk clearly indicates that the RC frame structure exceeds the examined performance levels at a lower value of PGA when subjected to pounding in comparison to the corresponding cases without pounding. Finally, a decision method is presented as an approach to estimate the minimum separation gap distance dg,min between adjacent structures when global and local performances are incorporated in the fragility assessment process.

Dorsal and ventral mossy cells differ in their axonal projections throughout the dentate gyrus of the mouse hippocampus.

Glutamatergic hilar mossy cells (MCs) have axons that terminate both near and far from their cell body but stay within the DG, making synapses primarily in the molecular layer. The long‐range axons are considered the primary projection, and extend throughout the DG ipsilateral to the soma, and project to the contralateral DG. The specificity of MC axons for the inner molecular layer (IML) has been considered to be a key characteristic of the DG. In the present study, we made the surprising finding that dorsal MC axons are an exception to this rule. We used two mouse lines that allow for Cre‐dependent viral labeling of MCs and their axons: dopamine receptor D2 (Drd2‐Cre) and calcitonin receptor‐like receptor (Crlr‐Cre). A single viral injection into the dorsal DG to label dorsal MCs resulted in labeling of MC axons in both the IML and middle molecular layer (MML). Interestingly, this broad termination of dorsal MC axons occurred throughout the septotemporal DG. In contrast, long‐range axons of ventral MCs terminated in the IML, consistent with the literature. Taken together, these results suggest that dorsal and ventral MCs differ significantly in their axonal projections. Since MC projections in the ML are thought to terminate primarily on GCs, the results suggest a dorsal–ventral difference in MC activation of GCs. The surprising difference in dorsal and ventral MC projections should therefore be considered when evaluating dorsal–ventral differences in DG function.

Profiling of the muscle-specific dystroglycan interactome reveals the role of Hippo signaling in muscular dystrophy and age-dependent muscle atrophy

BackgroundDystroglycanopathies are a group of inherited disorders characterized by vast clinical and genetic heterogeneity and caused by abnormal functioning of the ECM receptor dystroglycan (Dg). Remarkably, among many cases of diagnosed dystroglycanopathies, only a small fraction can be linked directly to mutations in Dg or its regulatory enzymes, implying the involvement of other, not-yet-characterized, Dg-regulating factors. To advance disease diagnostics and develop new treatment strategies, new approaches to find dystroglycanopathy-related factors should be considered. The Dg complex is highly evolutionarily conserved; therefore, model genetic organisms provide excellent systems to address this challenge. In particular, Drosophila is amenable to experiments not feasible in any other system, allowing original insights about the functional interactors of the Dg complex.MethodsTo identify new players contributing to dystroglycanopathies, we used Drosophila as a genetic muscular dystrophy model. Using mass spectrometry, we searched for muscle-specific Dg interactors. Next, in silico analyses allowed us to determine their association with diseases and pathological conditions in humans. Using immunohistochemical, biochemical, and genetic interaction approaches followed by the detailed analysis of the muscle tissue architecture, we verified Dg interaction with some of the discovered factors. Analyses of mouse muscles and myocytes were used to test if interactions are conserved in vertebrates.ResultsThe muscle-specific Dg complexome revealed novel components that influence the efficiency of Dg function in the muscles. We identified the closest human homologs for Dg-interacting partners, determined their significant enrichment in disease-associations, and verified some of the newly identified Dg interactions. We found that Dg associates with two components of the mechanosignaling Hippo pathway: the WW domain-containing proteins Kibra and Yorkie. Importantly, this conserved interaction manages adult muscle size and integrity.ConclusionsThe results presented in this study provide a new list of muscle-specific Dg interactors, further analysis of which could aid not only in the diagnosis of muscular dystrophies, but also in the development of new therapeutics. To regulate muscle fitness during aging and disease, Dg associates with Kibra and Yorkie and acts as a transmembrane Hippo signaling receptor that transmits extracellular information to intracellular signaling cascades, regulating muscle gene expression.

A Stress-Dilatancy Relationship for Rockfill Incorporating Particle Breakage and Intermediate Principal-Stress Ratio

Rockfill material is widely used for construction of high rockfill dam, which undergoes significant particle breakage and three-dimensional stress path induced by the water pressure, earthquake, etc. The dilatancy equation, defined as the function of the dilatancy dg and the stress ratio η, is regarded as the foundation of elastoplastic constitutive model. Therefore, a dilatancy equation considering the influence of particle breakage and intermediate principal stress ratio b was proposed in this paper. A series of test data from rockfill materials including conventional triaxial test, constant-b test and plane strain test were used to verified the applicability of the equation. It was found that the proposed dilatancy equation considering two important factors (i.e., particle breakage and b-value) agree well with the rearranged test data of the rockfill material in terms of dg-η. While the other dilatancy equations showed significant poorer predictions when ignoring one or two of the factors. That is, the proposed dilatancy equation can well describe the dilatancy behavior of rockfill material when considering the combined influences of the particle breakage and b-value.

Corneal kindled C57BL/6 mice exhibit saturated dentate gyrus long-term potentiation and associated memory deficits in the absence of overt neuron loss

Memory deficits have a significant impact on the quality of life of patients with epilepsy and currently no effective treatments exist to mitigate this comorbidity. While these cognitive comorbidities can be associated with varying degrees of hippocampal cell death and hippocampal sclerosis, more subtle changes in hippocampal physiology independent of cell loss may underlie memory dysfunction in many epilepsy patients. Accordingly, animal models of epilepsy or epileptic processes exhibiting memory deficits in the absence of cell loss could facilitate novel therapy discovery. Mouse corneal kindling is a cost-effective and non-invasive model of focal to bilateral tonic-clonic seizures that may exhibit memory deficits in the absence of cell loss. Therefore, we tested the hypothesis that corneal kindled C57BL/6 mice exhibit spatial pattern processing and memory deficits in a task reliant on DG function and that these impairments would be concurrent with physiological remodeling of the DG as opposed to overt neuron loss. Following corneal kindling, C57BL/6 mice exhibited deficits in a DG-associated spatial memory test – the metric task. Compatible with this finding, we also discovered saturated, and subsequently impaired, LTP of excitatory synaptic transmission at the perforant path to DGC synapse. This saturation of LTP was consistent with evidence suggesting that perforant path to DGC synapses in kindled mice had previously experienced LTP-like changes to their synaptic weights: increased postsynaptic depolarizations in response to equivalent presynaptic input and significantly larger amplitude AMPA receptor mediated spontaneous EPSCs. Additionally, there was evidence for kindling-induced changes in the intrinsic excitability of DGCs: reduced threshold to population spikes under extracellular recording conditions and significantly increased membrane resistances observed in DGCs. Importantly, quantitative immunohistochemical analysis revealed hippocampal astrogliosis in the absence of overt neuron loss. These changes in spatial pattern processing and memory deficits in corneal kindled mice represent a novel model of seizure-induced cognitive dysfunction associated with pathophysiological remodeling of excitatory synaptic transmission and granule cell excitability in the absence of overt cell loss.

An adhesome comprising laminin, dystroglycan and myosin IIA is required during notochord development in Xenopus laevis

Dystroglycan (Dg) is a transmembrane receptor for laminin that must be expressed at the right time and place in order to be involved in notochord morphogenesis. The function of Dg was examined in Xenopus laevis embryos by knockdown of Dg and overexpression and replacement of the endogenous Dg with a mutated form of the protein. This analysis revealed that Dg is required for correct laminin assembly, for cell polarization during mediolateral intercalation and for proper differentiation of vacuoles. Using mutations in the cytoplasmic domain, we identified two sites that are involved in cell polarization and are required for mediolateral cell intercalation, and a site that is required for vacuolation. Furthermore, using a proteomic analysis, the cytoskeletal non-muscle myosin IIA has been identified for the first time as a molecular link between the Dg-cytoplasmic domain and cortical actin. The data allowed us to identify the adhesome laminin-Dg-myosin IIA as being required to maintain the cortical actin cytoskeleton network during vacuolation, which is crucial to maintain the shape of notochordal cells.

An adhesome comprising laminin, dystroglycan and myosin IIA is required during notochord development in Xenopus laevis.

Dystroglycan (Dg) is a transmembrane receptor for laminin that must be expressed at the right time and place in order to be involved in notochord morphogenesis. The function of Dg was examined in Xenopus laevis embryos by knockdown of Dg and overexpression and replacement of the endogenous Dg with a mutated form of the protein. This analysis revealed that Dg is required for correct laminin assembly, for cell polarization during mediolateral intercalation and for proper differentiation of vacuoles. Using mutations in the cytoplasmic domain, we identified two sites that are involved in cell polarization and are required for mediolateral cell intercalation, and a site that is required for vacuolation. Furthermore, using a proteomic analysis, the cytoskeletal non-muscle myosin IIA has been identified for the first time as a molecular link between the Dg-cytoplasmic domain and cortical actin. The data allowed us to identify the adhesome laminin-Dg-myosin IIA as being required to maintain the cortical actin cytoskeleton network during vacuolation, which is crucial to maintain the shape of notochordal cells.

Long-term exercise is needed to enhance synaptic plasticity in the hippocampus

Exercise can have many benefits for the body, but it also benefits the brain by increasing neurogenesis, synaptic plasticity, and performance on learning and memory tasks. The period of exercise needed to realize the structural and functional benefits for the brain have not been well delineated, and previous studies have used periods of exercise exposure that range from as little as 3 d to up to 6 mo. In this study, we systematically evaluated the effects of differential running periods (3, 7, 14, 28, and 56 d) on both structural (cell proliferation and maturation) and functional (in vivo LTP) changes in the dentate gyrus of adult male Sprague-Dawley rats. We found that voluntary access to a running wheel for both short- and long-term periods can increase cell proliferation in the adult DG; however, increases in neurogenesis required longer term exposure to exercise. Increases in immature neurons were not observed until animals had been running for a minimum of 14 d. Similarly, short-term periods of wheel running did not facilitate LTP in the DG of adult animals, and reliable increases in LTP were only observed with 56 d of running. These results provide us with a greater understanding of the time course of wheel running access needed to enhance DG function. Furthermore, the results indicate that the new neurons produced in response to exercise in rats do not contribute significantly to synaptic plasticity until they mature.

Dystroglycan is involved in skin morphogenesis downstream of the Notch signaling pathway.

Dystroglycan (Dg) is a transmembrane protein involved both in the assembly and maintenance of basement membrane structures essential for tissue morphogenesis, and the transmission of signals across the plasma membrane. We used a morpholino knockdown approach to investigate the function of Dg during Xenopus laevis skin morphogenesis. The loss of Dg disrupts epidermal differentiation by affecting the intercalation of multiciliated cells, deposition of laminin, and organization of fibronectin in the extracellular matrix (ECM). Depletion of Dg also affects cell-cell adhesion, as shown by the reduction of E-cadherin expression at the intercellular contacts, without affecting the distribution of β(1) integrins. This was associated with a decrease of cell proliferation, a disruption of multiciliated-cell intercalation, and the down-regulation of the transcription factor P63, a marker of differentiated epidermis. In addition, we demonstrated that inhibition or activation of the Notch pathway prevents and promotes transcription of X-dg. Our study showed for the first time in vivo that Dg, in addition to organizing laminin in the ECM, also acts as a key signaling component in the Notch pathway.

Functional Glycosylation of Dystroglycan Is Crucial for Thymocyte Development in the Mouse

BackgroundAlpha-dystroglycan (α-DG) is a cell surface receptor providing a molecular link between the extracellular matrix (ECM) and the actin-based cytoskeleton. During its biosynthesis, α-DG undergoes specific and unusual O-glycosylation crucial for its function as a high-affinity cellular receptor for ECM proteins.Methodology/Principal FindingsWe report that expression of functionally glycosylated α-DG during thymic development is tightly regulated in developing T cells and largely confined to CD4−CD8− double negative (DN) thymocytes. Ablation of DG in T cells had no effect on proliferation, migration or effector function but did reduce the size of the thymus due to a significant loss in absolute numbers of thymocytes. While numbers of DN thymocytes appeared normal, a marked reduction in CD4+CD8+ double positive (DP) thymocytes occurred. In the periphery mature naïve T cells deficient in DG showed both normal proliferation in response to allogeneic cells and normal migration, effector and memory T cell function when tested in acute infection of mice with either lymphocytic choriomeningitis virus (LCMV) or influenza virus.Conclusions/SignificanceOur study demonstrates that DG function is modulated by glycosylation during T cell development in vivo and that DG is essential for normal development and differentiation of T cells.

The conserved WW-domain binding sites in Dystroglycan C-terminus are essential but partially redundant for Dystroglycan function

BackgroundDystroglycan (Dg) is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC) which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C-terminal PPXY motif has been established as a binding site for Dystrophin (Dys) WW-domain. However, our previous studies indicate that both Dystroglycan PPXY motives, WWbsI and WWbsII can bind Dystrophin protein in vitro.ResultsWe now find that both WW binding sites are important for maintaining full Dg function in the establishment of oocyte polarity in Drosophila. If either WW binding site is mutated, the Dg protein can still be active. However, simultaneous mutations in both WW binding sites abolish the Dg activities in both overexpression and loss-of-function oocyte polarity assays in vivo. Additionally, sequence comparisons of WW binding sites in 12 species of Drosophila, as well as in humans, reveal a high level of conservation. This preservation throughout evolution supports the idea that both WW binding sites are functionally required.ConclusionBased on the obtained results we propose that the presence of the two WW binding sites in Dystroglycan secures the essential interaction between Dg and Dys and might further provide additional regulation for the cytoskeletal interactions of this complex.

In vivo analyzes of dystroglycan function during somitogenesis in Xenopus laevis

Dystroglycan (Dg) is a cell adhesion receptor for laminin that has been reported to play a role in skeletal muscle cell stability, cytoskeletal organization, cell polarity, and signaling. Here we show that Dg is expressed at both the notochord/somite and the intersomitic boundaries, where laminin and fibronectin are accumulated during somitogenesis. Inhibition of Dg function with morpholino antisense oligonucleotides or a dominant negative mutant results in the normal segmentation of the presomitic mesoderm but affects the number, the size, and the integrity of somites. Depletion of Dg disrupts proliferation and alignment of myoblasts without affecting XMyoD and XMRF4 expression. It also leads to defects in laminin deposition at the intersomitic junctions, whereas expression of integrin beta1 subunits and fibronectin assembly occur normally. Our results show that Dg is critical for both proliferation and elongation of somitic cells and that the Dg-cytoplasmic domain is required for the laminin assembly at the intersomitic boundaries. Developmental Dynamics 238:1332-1345, 2009. (c) 2008 Wiley-Liss, Inc.

Muscle Dystroglycan Organizes the Postsynapse and Regulates Presynaptic Neurotransmitter Release at the Drosophila Neuromuscular Junction

BackgroundThe Dystrophin-glycoprotein complex (DGC) comprises dystrophin, dystroglycan, sarcoglycan, dystrobrevin and syntrophin subunits. In muscle fibers, it is thought to provide an essential mechanical link between the intracellular cytoskeleton and the extracellular matrix and to protect the sarcolemma during muscle contraction. Mutations affecting the DGC cause muscular dystrophies. Most members of the DGC are also concentrated at the neuromuscular junction (NMJ), where their deficiency is often associated with NMJ structural defects. Hence, synaptic dysfunction may also intervene in the pathology of dystrophic muscles. Dystroglycan is a central component of the DGC because it establishes a link between the extracellular matrix and Dystrophin. In this study, we focused on the synaptic role of Dystroglycan (Dg) in Drosophila.Methodology/Principal FindingsWe show that Dg was concentrated postsynaptically at the glutamatergic NMJ, where, like in vertebrates, it controls the concentration of synaptic Laminin and Dystrophin homologues. We also found that synaptic Dg controlled the amount of postsynaptic 4.1 protein Coracle and alpha-Spectrin, as well as the relative subunit composition of glutamate receptors. In addition, both Dystrophin and Coracle were required for normal Dg concentration at the synapse. In electrophysiological recordings, loss of postsynaptic Dg did not affect postsynaptic response, but, surprisingly, led to a decrease in glutamate release from the presynaptic site.Conclusion/SignificanceAltogether, our study illustrates a conservation of DGC composition and interactions between Drosophila and vertebrates at the synapse, highlights new proteins associated with this complex and suggests an unsuspected trans-synaptic function of Dg.

Dystroglycan and protein O-mannosyltransferases 1 and 2 are required to maintain integrity of Drosophila larval muscles.

In vertebrates, mutations in Protein O-mannosyltransferase1 (POMT1) or POMT2 are associated with muscular dystrophy due to a requirement for O-linked mannose glycans on the Dystroglycan (Dg) protein. In this study we examine larval body wall muscles of Drosophila mutant for Dg, or RNA interference knockdown for Dg and find defects in muscle attachment, altered muscle contraction, and a change in muscle membrane resistance. To determine if POMTs are required for Dg function in Drosophila, we examine larvae mutant for genes encoding POMT1 or POMT2. Larvae mutant for either POMT, or doubly mutant for both, show muscle attachment and muscle contraction phenotypes identical to those associated with reduced Dg function, consistent with a requirement for O-linked mannose on Drosophila Dg. Together these data establish a central role for Dg in maintaining integrity in Drosophila larval muscles and demonstrate the importance of glycosylation to Dg function in Drosophila. This study opens the possibility of using Drosophila to investigate muscular dystrophy.

A Putative Src Homology 3 Domain Binding Motif but Not the C-terminal Dystrophin WW Domain Binding Motif Is Required for Dystroglycan Function in Cellular Polarity in Drosophila

The conserved dystroglycan-dystrophin (Dg.Dys) complex connects the extracellular matrix to the cytoskeleton. In humans as well as Drosophila, perturbation of this complex results in muscular dystrophies and brain malformations and in some cases cellular polarity defects. However, the regulation of the Dg.Dys complex is poorly understood in any cell type. We now find that in loss-of-function and overexpression studies more than half (34 residues) of the Dg proline-rich conserved C-terminal regions can be truncated without significantly compromising its function in regulating cellular polarity in Drosophila. Notably, the truncation eliminates the WW domain binding motif at the very C terminus of the protein thought to mediate interactions with dystrophin, suggesting that a second, internal WW binding motif can also mediate this interaction. We confirm this hypothesis by using a sensitive fluorescence polarization assay to show that both WW domain binding sites of Dg bind to Dys in humans (K(d) = 7.6 and 81 microM, respectively) and Drosophila (K(d) = 16 and 46 microM, respectively). In contrast to the large deletion mentioned above, a single proline to an alanine point mutation within a predicted Src homology 3 domain (SH3) binding site abolishes Dg function in cellular polarity. This suggests that an SH3-containing protein, which has yet to be identified, functionally interacts with Dg.

Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy

Perturbation in the Dystroglycan (Dg)-Dystrophin (Dys) complex results in muscular dystrophies and brain abnormalities in human. Here we report that Drosophila is an excellent genetically tractable model to study muscular dystrophies and neuronal abnormalities caused by defects in this complex. Using a fluorescence polarization assay, we show a high conservation in Dg-Dys interaction between human and Drosophila. Genetic and RNAi-induced perturbations of Dg and Dys in Drosophila cause cell polarity and muscular dystrophy phenotypes: decreased mobility, age-dependent muscle degeneration and defective photoreceptor path-finding. Dg and Dys are required in targeting glial cells and neurons for correct neuronal migration. Importantly, we now report that Dg interacts with insulin receptor and Nck/Dock SH2/SH3-adaptor molecule in photoreceptor path-finding. This is the first demonstration of a genetic interaction between Dg and InR.

Alpha-Dystroglycan is a laminin receptor involved in extracellular matrix assembly on myotubes and muscle cell viability.

alpha-Dystroglycan (alpha-DG) is a laminin-binding protein and member of a glycoprotein complex associated with dystrophin that has been implicated in the etiology of several muscular dystrophies. To study the function of DG, C2 myoblasts were transfected stably with an antisense DG expression construct. Myotubes from two resulting clones (11F and 11E) had at least a 40-50% and 80-90% reduction, respectively, in alpha-DG but normal or near normal levels of alpha-sarcoglycan, integrin beta1 subunit, acetylcholine receptors (AChRs), and muscle-specific kinase (MuSK) when compared with parental C2 cells or three clones (11A, 9B, and 10C) which went through the same transfection and selection procedures but expressed normal levels of alpha-DG. Antisense DG-expressing myoblasts proliferate at the same rate as parental C2 cells and differentiate into myotubes, however, a gradual loss of cells was observed in these cultures. This loss correlates with increased apoptosis as indicated by greater numbers of nuclei with condensed chromatin and more nuclei labeled by the TUNEL method. Moreover, there was no sign of increased membrane permeability to Trypan blue as would be expected with necrosis. Unlike parental C2 myotubes, 11F and 11E myotubes had very little laminin (LN) on their surfaces; LN instead tended to accumulate on the substratum between myotubes. Exogenous LN bound to C2 myotubes and was redistributed into plaques along with alpha-DG on their surfaces but far fewer LN/alpha-DG plaques were seen after LN addition to 11F or 11E myotubes. These results suggest that alpha-DG is a functional LN receptor in situ which is required for deposition of LN on the cell and, further, implicate alpha-DG in the maintenance of myotube viability.

Synthesis and structure assignments of amide protected nucleosides and their use as phosphoramidites in deoxyoligonucleotide synthesis.

The syntheses of several amide protected deoxyguanosine- as well as thymidine nucleosides are described. These compounds were synthesized according to the Mitsunobu reaction and Michael addition. In contradiction to previous studies we have discovered that the Michael addition gives only products derived from N-alkylation. The occurrence of N- or O-alkylation was assigned by means of two dimensional 1H, 1 3 C-COLOC-NMR spectroscopy. Further, we have found that the Mitsunobu reaction used for the protection of the amide function of dG is limited to alcohols without acidic hydrogen atoms. Amide protected phosphormidites (15, 16) were used for the preparation of deoxyoligonucleotides with a large number of guanine and thymine bases using two different coupling times. We have shown that there is no experimentally detectable difference in the quality of the products if the starting monomer is amide protected or not.