ABSTRACT
FiberWire is a popular suture in flexor tendon repair that allows for early mobilization, but its poor knot-holding properties have raised concerns over the potential effects on tendon healing and strength. We examined how the number of knot throws affects the 2 mm gap force, ultimate tensile strength, and mode of failure in a four-strand cruciate locked tendon repair in porcine flexor tendons in order to elucidate the optimal number of suture throws. There was no effect on the 2 mm gap force with increasing knot throws, but there was a significant increase in ultimate tensile strength. A minimum of six-knot throws prevents unravelling, whereas five out of 10 of repairs unravelled with less than six throws.
Subject(s)
Suture Techniques , Sutures , Tendons/surgery , Analysis of Variance , Animals , Biomechanical Phenomena , Equipment Failure Analysis , Forelimb , Materials Testing , Swine , Tensile StrengthABSTRACT
Fluor-PACAP, a fluorescent derivative of PACAP-27, has been confirmed to share a high affinity for PAC1 receptors transfected into NIH/3T3 cells and to have comparable pharmacological characteristics to the unconjugated, native form. Through competitive binding with 125I-PACAP-27, the two ligands exhibited similar dose- dependent inhibition. Additional examination of the efficacy of activating adenylyl cyclase revealed that both ligands analogously stimulated the production of cyclic AMP. Furthermore, PAC1 internalization visualized by our Fluor-PACAP, is compareable to that performed with the radioligand, 125I-PACAP-27, with maximal internalization achieved within thirty minutes. Thus, Fluor-PACAP exhibits intracellular signaling abilities homologous to the native ligand.
Subject(s)
Neuropeptides/chemistry , 3T3 Cells , Adenylyl Cyclases/metabolism , Animals , Binding, Competitive , Cyclic AMP/metabolism , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Fluorescent Dyes/pharmacology , Hydrazines/pharmacology , Immunohistochemistry , Ligands , Mice , Microscopy, Confocal , Neuropeptides/pharmacology , Pituitary Adenylate Cyclase-Activating Polypeptide , Protein Binding , Signal Transduction , Time Factors , TransfectionABSTRACT
The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 (PAC1) receptor is a G protein-coupled receptor and class II receptor member. The receptor domains critical for signaling are unknown. To explore the role of the C terminus, truncations of 63 residues (Tr406), 53 residues (Tr416), 49 residues (Tr420), 44 residues (Tr424), and 37 residues (Tr433) were constructed and expressed in NIH/3T3 cells, and immunofluorescence, radioligand binding, adenylyl cyclase (AC) and phospholipase C (PLC) assays were performed. (125)I-PACAP-27 binding (K(d) = 0.6-1.5 nm) for the Tr406 and Tr433 were similar to wild type Hop and Null splice variants (K(d) = approximately 1.1 nm). Although internalization of ligand for both the Tr406 and Tr433 mutants was reduced to 50-60% at 60 min compared with 76-87% for WT, loss of G protein coupling did not account for differences in internalization. Despite similar binding properties Tr406 and Tr416 mutants showed no AC or PLC response. Addition of 14 amino acids distal to HopTr406 resulted in normal AC and PLC responses. Site-directed mutagenesis indicated that Arg(416) and Ser(417) are essential for G protein activation. The proximal C terminus mediates signal transduction, and the distal is involved with internalization. Two residues within the C terminus, Arg(416) and Ser(417) conserved among class II receptors are the likely sites for G protein coupling.
Subject(s)
Down-Regulation , Endocytosis , Receptors, Pituitary Hormone/chemistry , Receptors, Pituitary Hormone/metabolism , Signal Transduction , 3T3 Cells , Adenylyl Cyclases/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding, Competitive , Cyclic AMP/metabolism , Fluorescent Antibody Technique , Half-Life , Humans , Inositol Phosphates/metabolism , Iodine Radioisotopes , Mice , Molecular Sequence Data , Mutation , Pituitary Gland/enzymology , Pituitary Gland/metabolism , Protein Binding , Protein Structure, Tertiary , Protein Transport , Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide , Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide , Receptors, Pituitary Hormone/genetics , Sequence Alignment , TransfectionABSTRACT
We describe a program for the analysis of RNA secondary structure. There are two new features in this program. (i) To get vector speeds on a vector pipeline machine (such as Cray X-MP/24) we have vectorized the secondary structure dynamic algorithm. (ii) The statistical significance of a locally 'optimal' secondary structure is assessed by a Monte Carlo method. The results can be depicted graphically including profiles of the stability of local secondary structures and the distribution of the potentially significant secondary structures in the RNA molecules. Interesting regions where both the potentially significant secondary structures and 'open' structures (single-stranded coils) occur can be identified by the plots mentioned above. Furthermore, the speed of the vectorized code allows repeated Monte Carlo simulations with different overlapping window sizes. Thus, the optimal size of the significant secondary structure occurring in the interesting region can be assessed by repeating the Monte Carlo simulation. The power of the program is demonstrated in the analysis of local secondary structures of human T-cell lymphotrophic virus type III (HIV).
