Extracellular matrix metabolism by chondrocytes 7. Evidence that L-glutamine is an essential amino acid for chondrocytes and other connective tissue cells

THe incorporation of [³H]glycine into acid-insoluble protein and of [³H]acetate into glysoaminoglycans by cultured chick chondrocytes was stimulated by the addition of L-glutamine to the incubation medium. The effect of exogenous L-glutamine on protein synthesis was studied further by examining changes in the sedimentation patterns on sucrose gardients of ribosomes isolated from chondrocytes incubated in presence and absence of L-glutamine. It was found that the absence of L-glutamine caused a disaggregation of poly-ribosomes that was reversed by the addition of this amino acid to the culture medium. No detectable glutamine synthetase activity could be measured in avian articular cartilage. These results indicate that L-glutamine is an essential amino acid for cartilage in that an extracellular supply of this amino acid is required for the maintenance of protein and glycosaminoglycan synthesis. A dependence on L-glutamine was also demonstrated for other avain connective tissues.     

Crosslinked elastic fibers are necessary for low energy loss in the ascending aorta

In the large arteries, it is believed that elastin provides the resistance to stretch at low pressure, while collagen provides the resistance to stretch at high pressure. It is also thought that elastin is responsible for the low energy loss observed with cyclic loading. These tenets are supported through experiments that alter component amounts through protease digestion, vessel remodeling, normal growth, or in different artery types. Genetic engineering provides the opportunity to revisit these tenets through the loss of expression of specific wall components. We used newborn mice lacking elastin (Eln^−/−) or two key proteins (lysyl oxidase, Lox^−/−, or fibulin-4, Fbln4^−/−) that are necessary for the assembly of mechanically-functional elastic fibers to investigate the contributions of elastic fibers to large artery mechanics. We determined component content and organization and quantified the nonlinear and viscoelastic mechanical behavior of Eln^−/−, Lox^−/−, and Fbln4^−/− ascending aorta and their respective controls. We confirmed that the lack of elastin, fibulin-4, or lysyl oxidase leads to absent or highly fragmented elastic fibers in the aortic wall and a 56–97% decrease in crosslinked elastin amounts. We found that the resistance to stretch at low pressure is decreased only in Eln^−/− aorta, confirming the role of elastin in the nonlinear mechanical behavior of the aortic wall. Dissipated energy with cyclic loading and unloading is increased 53–387% in Eln^−/−, Lox^−/−, and Fbln4^−/− aorta, indicating that not only elastin, but properly assembled and crosslinked elastic fibers, are necessary for low energy loss in the aorta.     

The Isolation and Characterization of Glycosylated Phosphoproteins from Herring Fish Bones

Past studies of bone extracellular matrix phosphoproteins such as osteopontin and bone sialoprotein have yielded important biological information regarding their role in calcification and the regulation of cellular activity. Most of these studies have been limited to proteins extracted from mammalian and avian vertebrates and nonvertebrates. The present work describes the isolation and purification of two major highly glycosylated and phosphorylated extracellular matrix proteins of 70 and 22 kDa from herring fish bones. The 70-kDa phosphoprotein has some characteristics of osteopontin with respect to amino acid composition and susceptibility to thrombin cleavage. Unlike osteopontin, however, it was found to contain high levels of sialic acid similar to bone sialoprotein. The 22-kDa protein has very different properties such as very high content of phosphoserine (∼270 Ser(P) residues/1000 amino acid residues), Ala, and Asx residues. The N-terminal amino acid sequence analysis of both the 70-kDa (NPIMA(M)ETTS(M)DSKVNPLL) and the 22-kDa (NQDMAMEASSDPEAA) fish phosphoproteins indicate that these unique amino acid sequences are unlike any published in protein databases. An enzyme-linked immunosorbent assay revealed that the 70-kDa phosphoprotein was present principally in bone and in calcified scales, whereas the 22-kDa phosphoprotein was detected only in bone. Immunohistological analysis revealed diffusely positive immunostaining for both the 70- and 22-kDa phosphoproteins throughout the matrix of the bone. Overall, this work adds additional support to the concept that the mechanism of biological calcification has common evolutionary and fundamental bases throughout vertebrate species.     

Three-dimensional perfused cell culture

Compelling evidence suggests the limitation and shortcomings of the current and well established cell culture method using multi-well plates, flasks and Petri dishes. These are particularly important when cell functions are sensitive to the local microenvironment, cell–cell and cell–extracellular matrix interactions. There is a clear need for advanced cell culture systems which mimic in vivo and more physiological conditions. This review summarises and analyses recent progress in three dimensional (3D) cell culture with perfusion as the next generation cell culture tools, while excluding engineered tissue culture where three dimensional scaffold has to be used for structural support and perfusion for overcoming mass transfer control. Apart from research activities in academic community, product development in industry is also included in this review.     

The effect of phenformin and other adenosine triphosphate (ATP)-lowering agents on insulin binding to IM-9 human cultured lymphocytes

In the present study, we investigated the mechanism by which the antidiabetic drug phenformin increases insulin binding to its receptors in IM-9 human cultured lymphocytes. After a 24-hr preincubation, phenformin induced a twofold increase in specific 125I-insulin binding, and removal of phenformin was followed 6 hr later by a return in binding to control levels. This effect of phenformin on insulin binding was not a consequence of either inhibition of cell growth, changes in cellular cyclic adenosine monophosphate (AMP) levels, or changes in guanosine triphosphate (GTP) content. Since phenformin is known to inhibit various aspects of cellular energy metabolism, the relationship between 125I-insulin binding and energy metabolism in IM-9 cells was investigated. The phenformin-induced increase in insulin binding to IM-9 cells was related to a time- and dose-dependent decrease in ATP levels. Other agents that lowered ATP levels, including antimycin, dinitrophenol, and 2-deoxyglucose, also raised insulin binding. These studies indicated, therefore, that phenformin enhances insulin binding to receptors on IM-9 cells and that this effect on insulin receptors may be related to alterations in metabolic functions that are reflected by a lowering of ATP levels.     

Synthesis of peptides from amino acids and ATP with lysine-rich proteinoid

Summary Lysine-rich proteinoids in aqueous solution catalyze the formation of peptides from free amino acids and ATP. This catalytic activity is not found in acidic proteinoids, even though the latter contain some basic amino acid. The pH optimum for the synthesis is about 11, but is appreciable below 8 and above 13. Temperature data indicate an optimum at 20°C or above, with little increase in rate to 60°C. Pyrophosphate can be used instead of ATP, with lesser yields resulting. The ATP-aided syntheses of peptides in aqueous solution occur with several types of proteinous amino acid.Proofs should be sent to S.W. Fox, Institute for Molecular and Cellular Evolution, University of Miami, 521 Anastasia Avenue, Coral Gables, FL 33134     

Pulmonary neuroendocrine cells sense succinate to stimulate myoepithelial cell contraction

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Mechanical,histological, and functional properties remain inferior in conservatively treated Achilles tendons in rodents: Long term evaluation

Conservative treatment (non-operative) of Achilles tendon ruptures is suggested to produce equivalent capacity for return to function; however, long term results and the role of return to activity (RTA) for this treatment paradigm remain unclear. Therefore, the objective of this study was to evaluate the long term response of conservatively treated Achilles tendons in rodents with varied RTA. Sprague Dawley rats (n = 32) received unilateral blunt transection of the Achilles tendon followed by randomization into groups that returned to activity after 1-week (RTA1) or 3-weeks (RTA3) of limb casting in plantarflexion, before being euthanized at 16-weeks post-injury. Uninjured age-matched control animals were used as a control group (n = 10). Limb function, passive joint mechanics, tendon properties (mechanical, histological), and muscle properties (histological, immunohistochemical) were evaluated. Results showed that although hindlimb ground reaction forces and range of motion returned to baseline levels by 16-weeks post-injury regardless of RTA, ankle joint stiffness remained altered. RTA1 and RTA3 groups both exhibited no differences in fatigue properties; however, the secant modulus, hysteresis, and laxity were inferior compared to uninjured age-matched control tendons. Despite these changes, tendons 16-weeks post-injury achieved secant stiffness levels of uninjured tendons. RTA1 and RTA3 groups had no differences in histological properties, but had higher cell numbers compared to control tendons. No changes in gastrocnemius fiber size or type in the superficial or deep regions were detected, except for type 2x fiber fraction. Together, this work highlights RTA-dependent deficits in limb function and tissue-level properties in long-term Achilles tendon and muscle healing.     

Impacts of maturation on the micromechanics of the meniscus extracellular matrix

To elucidate how maturation impacts the structure and mechanics of meniscus extracellular matrix (ECM) at the length scale of collagen fibrils and fibers, we tested the micromechanical properties of fetal and adult bovine menisci via atomic force microscopy (AFM)-nanoindentation. For circumferential fibers, we detected significant increase in the effective indentation modulus, E_ind, with age. Such impact is in agreement with the increase in collagen fibril diameter and alignment during maturation, and is more pronounced in the outer zone, where collagen fibrils are more aligned and packed. Meanwhile, maturation also markedly increases the E_ind of radial tie fibers, but not those of intact surface or superficial layer. These results provide new insights into the effect of maturation on the assembly of meniscus ECM, and enable the design of new meniscus repair strategies by modulating local ECM structure and mechanical behaviors.