Inhibition of zipper-interacting protein kinase function in smooth muscle by a myosin light chain kinase pseudosubstrate peptide

As a regulator of smooth muscle contractility, zipper-interacting protein kinase (ZIPK) appears to phosphorylate the regulatory myosin light chain (RLC20), directly or indirectly, at Ser19 and Thr18 in a Ca²⁺-independent manner. The calmodulin-binding and autoinhibitory domain of myosin light chain kinase (MLCK) shares similarity to a sequence found in ZIPK. This similarity in sequence prompted an investigation of the SM1 peptide, which is derived from the autoinhibitory region of MLCK, as a potential inhibitor of ZIPK. In vitro studies showed that SM1 is a competitive inhibitor of a constitutively active 32-kDa form of ZIPK with an apparent K_i value of 3.4 µM. Experiments confirmed that the SM1 peptide is also active against full-length ZIPK. In addition, ZIPK autophosphorylation was reduced by SM1. ZIPK activity is independent of calmodulin; however, calmodulin suppressed the in vitro inhibitory potential of SM1, likely as a result of nonspecific binding of the peptide to calmodulin. Treatment of ileal smooth muscle with exogenous ZIPK was accompanied by an increase in RLC20 diphosphorylation, distinguishing between ZIPK [and integrin-linked kinase (ILK)] and MLCK actions. Administration of SM1 suppressed steady-state muscle tension developed by the addition of exogenous ZIPK to Triton-skinned rat ileal muscle strips with or without calmodulin depletion by trifluoperazine. The decrease in contractile force was associated with decreases in both RLC20 mono- and diphosphorylation. In summary, we present the SM1 peptide as a novel inhibitor of ZIPK. We also conclude that the SM1 peptide, which has no effect on ILK, can be used to distinguish between ZIPK and ILK effects in smooth muscle tissues. inhibitory peptide; calcium sensitization     

Histochemical studies on the amphibian opercularis muscle (Amphibia)

Summary Histochemical studies of the opercularis muscle of the bullfrog (Rana catesbeiana) and the tiger salamander (Ambystoma tigrinum) provide evidence that the opercularis muscle of anurans is a specialized, tonic portion of the levator scapulae superior muscle. Staining results for myosin adenosine triphosphatase (ATPase) and succinate dehydrogenase (SDH), combined with measurements of muscle fiber diameters, demonstrate that the opercularis/levator scapulae superior muscle mass of both the tiger salamander and bullfrog consists of an anterior tonic portion, a middle fast oxidative-glycolytic (FOG) twitch portion, and a posterior fast-glycolytic (FG) twitch portion. In R. catesbeiana the tonic fibers represent 57.3% of the fiber total and (because they have relatively narrow diameters) about 29% of the cross-sectional area of the muscle mass, and form that part of the muscle (=opercularis muscle) that inserts on the operculum. In Ambystoma the tonic fibers represent only 8.8% of the fiber total and represent about 4% of the cross-sectional area. In the tiger salamander, the entire levator scapulae superior muscle inserts on the operculum and therefore represents the opercularis muscle. The bullfrog differs from the tiger salamander, therefore, in that the anterior tonic part of the opercularis/levator scapulae superior complex is greatly enlarged and the insertion on the operculum is limited to these tonic fibers. No evidence of a columellar muscle was found in R. catesbeiana. Previous reports of one in this species and in other anurans may be based on the tripartite nature of the opercularis/levator scapulae superior muscle mass. The middle FOG portion of the muscle may have been considered a muscle distinct from the anterior tonic portion (=opercularis muscle) and the posterior FG portion.     

Myxozoan parasitism in waterfowl

Myxozoans are spore-forming, metazoan parasites common in cold-blooded aquatic vertebrates, especially fishes, with alternate life cycle stages developing in invertebrates. We report nine cases of infection in free-flying native and captive exotic ducks (Anseriformes: Anatidae) from locations across the United States and describe the first myxozoan in birds, Myxidium anatidum n. sp. We found developmental stages and mature spores in the bile ducts of a Pekin duck (domesticated Anas platyrhynchos). Spores are lens-shaped in sutural view, slightly sigmoidal in valvular view, with two polar capsules, and each valve cell has 14-16 longitudinal surface ridges. Spore dimensions are 23.1 microm x 10.8 microm x 11.2 microm. Phylogenetic analysis of the ssrRNA gene revealed closest affinity with Myxidium species described from chelonids (tortoises). Our novel finding broadens the definition of the Myxozoa to include birds as hosts and has implications for understanding myxozoan evolution, and mechanisms of geographical and host range extension. The number of infection records indicates this is not an incidental occurrence, and the detection of such widely dispersed cases suggests more myxozoans in birds will be encountered with increased surveillance of these hosts for pathogens.     

In vivo gene expression: combining hydrodynamics-based transfection and electrotransfer

Clinical development of gene therapy is hampered by the lack of an efficient and safe method for in vivo gene transfer. New methodologies for plasmid transfer are being developed. Recently, significant expression of a reporter gene was achieved in liver cells by coupling intravenous injection and stimulation of the tissue with electric pulses. This combination of hydrodynamics-based transfection and electrotransfer could provide the basis for many promising clinical applications.     

A Retained Secretory Signal Peptide Mediates High Density Lipoprotein (HDL) Assembly and Function of Haptoglobin-related Protein

Haptoglobin-related protein (Hpr) is a component of a minor subspecies of high density lipoproteins (HDL) that function in innate immunity. Here we show that assembly of Hpr into HDL is mediated by its retained N-terminal signal peptide, an unusual feature for a secreted protein and the major difference between Hpr and the soluble acute phase protein haptoglobin (Hp). The 18-amino acid signal peptide is necessary for binding to HDL and interacts directly with the hydrocarbon region of lipids. Utilizing model liposomes, we show that the rate of assembly and steady-state distribution of Hpr in lipid particles is mediated by the physical property of lipid fluidity. Dye release assays reveal that Hpr interacts more rapidly with fluid liposomes. Conversely, steady-state binding assays indicate that more rigid lipid compositions stabilize Hpr association. Lipid association also plays a role in facilitating hemoglobin binding by Hpr. Our data may offer an explanation for the distinct distribution of Hpr among HDL subspecies. Rather than protein-protein interactions mediating localization, direct interaction with phospholipids and sensitivity to lipid fluidity may be sufficient for localization of Hpr and may represent a mechanism of HDL subspeciation.     

The Interplay between Chondrocyte Redifferentiation Pellet Size and Oxygen Concentration

Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1×10⁵–5×10⁵ chondrocytes are aggregated, resulting in “macro” pellets having diameters ranging from 1–2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1–2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2×10⁵ human chondrocytes into micropellets of 166 cells each, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360×360 µm microwells cast into polydimethylsiloxane (PDMS), that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG) production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O₂). Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues. Our results indicate that micropellet amalgamation efficiency is inversely related to the time cultured as discreet microtissues. In summary, we describe a micropellet production platform that represents an efficient tool for studying chondrocyte redifferentiation and demonstrate that the micropellets could be assembled into larger tissues, potentially useful in cartilage defect repair.