Use of arginine-glycine-aspartic acid adhesion peptides coupled with a new collagen scaffold to engineer a myocardium-like tissue graft.

BACKGROUND: Cardiac tissue engineering might be useful in treatment of diseased myocardium or cardiac malformations. The creation of functional, biocompatible contractile tissues, however, remains challenging. We hypothesized that coupling of arginine-glycine-aspartic acid-serine (RGD+) adhesion peptides would improve cardiomyocyte viability and differentiation and contractile performance of collagen-cell scaffolds. METHODS: Clinically approved collagen scaffolds were functionalized with RGD+ cells and seeded with cardiomyocytes. Contractile performance, cardiomyocyte viability and differentiation were analyzed at days 1 and 8 and/or after culture for 1 month. RESULTS: The method used for the RGD+ cell-collagen scaffold coupling enabled the following features: high coupling yields and complete washout of excess reagent and by-products with no need for chromatography; spectroscopic quantification of RGD+ coupling; a spacer arm of 36 A, a length reported as optimal for RGD+-peptide presentation and favorable for integrin-receptor clustering and subsequent activation. Isotonic and isometric mechanical parameters, either spontaneous or electrostimulated, exhibited good performance in RGD+ constructs. Cell number and viability was increased in RGD+ scaffolds, and we saw good organization of cell contractile apparatus with occurrence of cross-striation. CONCLUSIONS: We report a novel method of engineering a highly effective collagen-cell scaffold based on RGD+ peptides cross-linked to a clinically approved collagen matrix. The main advantages were cell contractile performance, cardiomyocyte viability and differentiation.

The endogenous agonist quinolinic acid and the non endogenous homoquinolinic acid discriminate between NMDAR2 receptor subunits.

Quinolinic acid is an endogenous neurotoxin with NMDA receptor agonist properties. As such it may be the etiologic agent in many diseases. In this paper the NMDA receptor agonist properties of quinolinic acid, as well as those of homoquinolinic acid, a non endogenous analogue, were investigated in Xenopus oocytes injected with 12-day-old rat cortical mRNA or with recombinant NMDA receptors. In oocytes injected with cortical mRNA, quinolinic acid was a weak NMDA receptor agonist: millimolar concentrations were necessary to induce responses that were smaller than maximal responses induced by NMDA; homoquinolinic acid and NMDA had similar affinities but different efficacies: maximal responses induced by homoquinolinic acid were larger than maximal responses induced by NMDA. Cortical mRNA, as verified by RT-PCR and restriction analysis, contains various NMDA subunits. In order to investigate if the low affinity or efficacy of quinolinic acid could be explained by receptor composition, the pharmacological properties of the putative agonists were investigated in oocytes expressing binary combinations of recombinant NMDA receptors. Quinolinic acid did not activate receptors containing NR1 + NR2C but did activate receptors containing NR1 + NR2A and NR1 + NR2B even if only at millimolar concentrations; homoquinolinic acid activated all subunit combinations but was less efficient than NMDA only in the NR1 + NR2C subunit combination. The relative efficacies of quinolinic acid and homoquinolinic acid were evaluated by comparing the maximal responses induced by these agonists with those induced by NMDA and glutamate in the same oocytes. The rank order of potency was quinolinic acid < NMDA < homoquinolinic acid < or = glutamate for the NR1 + NR2A and NR1 + NR2B combinations whereas for NR1 + NR2C it was quinolinic acid << << homoquinolinic acid < NMDA < or = glutamate. The use of quinolinic acid and homoquinolinic acid may thus help to identify endogenous receptors containing the NR2C subunit.

Cat proenkephalin-A does not contain the opioid octapeptide.

The sequence of a large cDNA fragment of proenkephalin-A from the cat adrenal medulla was obtained using reverse transcription followed by polymerase chain reaction, and cloning. This cDNA encompasses the region normally containing all the opioid peptides, except the C-terminal heptapeptide. As with other species, cat proenkephalin-A contains four conserved copies of (Met5)-enkephalin, and one of (Leu5)-enkephalin, flanked by processing sites of paired basic amino acids. However, significant differences were found in the nucleotide and deduced amino acid sequences in the region of the octapeptide. In particular, the essential tyrosyl residue is substituted by a histidyl residue, making it unlikely that the cat equivalent would have opioid activity. Furthermore, the peptide is not flanked by paired basic residues, suggesting it is not processed.

Single cell RT-PCR proceeds without the risk of genomic DNA amplification.

We have previously described a method for detection of mRNAs expressed in single cells after patch-clamp recordings. The method, termed single cell RT-PCR, involves aspiration of the cell content, a reverse transcription (RT) step, and a polymerase chain reaction (PCR) using specific primers. Since the nucleus is frequently harvested together with the cytosol, genomic DNA may generate false positive results. Thus, we demonstrated that dilutions containing a few copies of plasmid could be detected by PCR in a range which, according to the Poisson law, suggests that the PCR method can amplify from the two genomic alleles. We performed single cell RT-PCR of intronless GluR2 or GluR5 fragments by comparing cerebellar cell types where these mRNAs are known to be present or absent. For each cell the nucleus was harvested together with the cytosol. Following RT-PCR with GluR5 primers, all Purkinje cells (n = 6) yielded the expected PCR product, whereas it was not generated from any of the granule cells (n = 5). In corresponding experiments with GluR2 primers, we obtained the GluR2 product from all Purkinje cells (n = 5), but not from any of the glial cells (n = 5). These results are in agreement with the known cellular expression of GluR2 and GluR5 mRNAs. We conclude that the single cell RT-PCR method does not amplify the genomic DNA when the nucleus is aspirated together with the cytosol. We suggest that genomic DNA amplification is avoided, because the genomic alleles are not exposed during the procedure.

Oligoclonal beta-galactoside-binding immunoglobulins antigenically related to 14 kDa lectin in human serum and cerebrospinal fluid: purification and characterization.

1. An antiserum raised against a 14 kDa beta-galactoside specific lectin from human brain (HBL14) was used to probe blots from samples of serum and cerebrospinal fluid. The only HBL14-immunoreactive material detected was heavy and light chains of a beta-galactoside-binding IgG fraction (lectin-like IgG). 2. Lectin-like IgG, as well as IgG Fab fragments, compete with HBL14 for binding either to anti-HBL14 antibody or to a lactosyl polyacrylamide-based copolymer. 3. Purification of lectin-like IgG was obtained by affinity chromatography on immobilized rabbit anti-lectin immunoglobulins. The carbohydrate-binding specificity of the purified molecules was restricted to beta-Gal-containing structures and close to the HBL14 one.

Subunit composition at the single-cell level explains functional properties of a glutamate-gated channel.

The diversity of known glutamate-gated channels has been markedly increased by the discovery of multiple subunits and their spliced and edited variants. These subunits can potentially form different oligomeric complexes with diverging properties. A crucial question is therefore to determine the actual subunit composition of naturally occurring glutamate receptors. We have coupled patch-clamp recordings and reverse transcription followed by PCR amplification to correlate the presence of mRNAs for each subunit and the functional properties of native glutamate receptors at the single-cell level. In a homogeneous population of functionally identified hippocampal neurons (type II) in culture bearing a glutamate receptor of the AMPA subtype with a high calcium permeability, we found that, among the multiple subunits, only two, the flop forms of GluR1 and GluR4, were expressed. In particular, GluR2 was never detected. This composition explains the uncommon properties of AMPA receptors in type II neurons.

Molecular biology of excitatory amino acid receptors: subtypes and subunits.

Glutamate receptors coupled to ion channels have been named according to their selective agonist: N-methyl-D-Aspartate (NMDA), kainate, quisqualate and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA). The pharmacology of the NMDA receptor is clearly different from that of the kainate, quisqualate and AMPA receptors, thus differentiating two types: NMDA and non-NMDA receptors. Molecular cloning and expression of non-NMDA receptor subunits have now established that the different neuronal responses to kainate, quisqualate and AMPA are mediated by at least two subtypes of ligand-gated channels: one responding to the three ligands, the other responding to kainate and quisqualate but not to AMPA.

Electron microscopic localization of photoaffinity-labelled delta opioid receptors in the neostriatum of the rat.

The distribution of delta opioid receptors, selectively labelled in vitro with the photoaffinity probe monoiodo azido-DTLET ([D-Thr2,pN3Phe4, Leu5]enkephaly-Thr6), was analyzed by light and electron microscopic radioautography in sections from rat neostriatum. Preliminary experiments indicated that up to 65% of specific 125I-azido-DTLET binding to rat striatal sections was still detectable following prefixation of the brain with 0.5% glutaraldehyde. These experiments also showed that up to 20-30% of the specifically bound radioactivity was covalently linked following ultraviolet irradiation and was thereby retained in tissue during subsequent postfixation and dehydration steps. Accordingly, the topographic distribution of the covalently attached azido-DTLET molecules was similar to that seen in fresh frozen sections and characteristic of that previously described for delta sites. Light and electron microscopic examination of the label in prefixed, striatal sections irradiated with ultraviolet light revealed that a significant proportion of specifically bound 125I-azido-DTLET molecules was intraneuronal. Specifically, 16% of the labelled binding sites were found in dendrites, 12% in perikarya and 4% in axon terminals. These results suggest that an important proportion of delta opioid binding sites labelled in the neostriatum correspond to receptors that are undergoing synthesis, transport and/or recycling. They also imply that a major fraction of delta sites are associated with intrastriatal neurons, as opposed to afferent axons. Approximately 44% of the labelled binding sites were associated with neuronal plasma membranes. Although most of these were found at the level of axodendritic (20%) and dendrodendritic (7%) appositions, comparison of the labelling incidence of these two compartments with their frequency of occurrence in tissue suggested that delta sites are fairly widely dispersed along neuronal plasma membranes. Only a small proportion (smaller than that of mu or kappa sites labelled in the same region) was associated with synaptic specializations. These results support the concept that delta receptors correspond to molecular entities that are distinct from mu and kappa sites and suggest that delta ligands act primarily nonjunctionally on the plasma membrane of striatal neurons.

AMPA receptor subunits expressed by single Purkinje cells.

Several subunits of the glutamate receptor of the AMPA subtype have been cloned recently. These subunits, named GluR1, GluR2, GluR3, and GluR4, exist as two splicing variants (flip and flop). We have determined the subset of AMPA receptor subunits expressed by single cerebellar Purkinje cells in culture. This was achieved by combining whole-cell patch-clamp recordings and a molecular analysis, based on the polymerase chain reaction, of the messenger RNAs harvested into the patch pipette at the end of each recording. We found that each single cell expresses the messenger RNAs encoding the following five subunits: the flip and flop versions of GluR1 and GluR2 as well as GluR3flip, GluR2 being the most abundant. In addition, GluR3flop and GluR4flip were scarcely expressed in half of these neurons, and GluR4flop was never detected.

In the GluR1 glutamate receptor subunit a glutamine to histidine point mutation suppresses inward rectification but not calcium permeability.

Recent papers have described glutamine to arginine point mutations of the cloned AMPA/Kainate receptor subunits that alter current-voltage relationship and suppress Ca2+ permeability, thus linking these two characteristics. We describe a glutamine to histidine mutation at the same position, which alters current-voltage relationship but retains Ca2+ permeability, thus dissociating the two properties.

A chimeric glutamate receptor subunit: discrete changes modify the properties of the channel.

GluR1 and GluR2 are two highly homologous subunits of the glutamate AMPA receptor but with different functional properties. In ligand gated channels the transmembrane domain II is thought to form the wall of the ionic pore and determine the electrical properties. A chimeric AMPA receptor subunit was constructed by replacing the region comprising transmembrane domains I and II in GluR1 by the corresponding region of GluR2. Alone or forming an heteromer with GluR1, the resulting chimera has the properties of GluR2. Sequence comparison suggests that an arginine at position 600 in the chimera instead of a glutamine in GluR1 is responsible for these properties.

Photoaffinity labeling of opioid delta receptors with an iodinated azido-ligand: [125I][D-Thr2,pN3Phe4,Leu5]enkephalyl-Thr6.

The photoaffinity ligand of the delta opioid receptor Tyr-D-Thr-Gly-pN3Phe-Leu-Thr (azido-DTLET) was iodinated and purified by high performance liquid chromatography. Monoiodo-azido-DTLET displayed a high affinity (KD = 15 nM) and is selective (Kl mu/Kl delta = 9.8) for rat brain delta opioid receptors (for comparison, the corresponding values for tritiated azido-DTLET are KD = 1.66 nM and Kl mu/Kl delta = 27). On rat brain sections, the anatomical distribution of [125I]azido-DTLET binding sites revealed by autoradiography corresponds to that of delta receptors. On rat brain membrane homogenates and NG108-15 hybrid cells, UV irradiation of the receptor-ligand complex results in the irreversible binding to membrane proteins of 14% of the bound radioactivity Gel electrophoresis of [125I]azido-DTLET-labeled proteins followed by autoradiography shows a different pattern in rat brain and NG108-15 cells. In rat brain, labeling of two of these proteins, with molecular weights of 44,000 and 34,000, was inhibited by 30 nmol/liter of nonradioactive DTLET, a delta-selective ligand but not by the same concentration of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, a mu-selective ligand. In NG108-15 cells, this 44-kDa protein was not visualized; the main band was at 33 kDa and disappeared in the presence of levorphanol.

Photoaffinity labeling of a 33 kDa protein subunit of the delta-opioid receptor in neuroblastoma and hybrid cell lines.

Tritiated DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr) binds with high affinity, specificity and saturability to neuroblastoma N18TG2 and hybrid neuroblastoma x glioma NG108-15 and NG108-5 intact cells. The delta-opioid receptor density in cells cultured in chemically defined medium was increased about 2 times compared to that in cells cultured in 10% fetal calf serum. A major and a minor protein species covalently and specifically bound to [125I]azido-DTLET (Tyr-D-Thr-Gly-pN3Phe-Leu-Thr), photoactivatable ligand, migrated on SDS-gel electrophoresis with Mr values near 33,000 and 58,000, respectively.