Improved application of the diphenylamine reaction for the determination of DNA

An improved procedure is described for the separation and quantitation of DNA from biological samples. Following papain digestion, DNA was separated from other cell material with CdCl₂ precipitation onto a glass-fiber filter and quantitated easily by the modified diphenylamine reaction.     

Pathological mechanism of chondrocytes and the surrounding environment during osteoarthritis of temporomandibular joint

Temporomandibular joint (TMJ) osteoarthritis is a common chronic degenerative disease of the TMJ. In order to explore its aetiology and pathological mechanism, many animal models and cell models have been constructed to simulate the pathological process of TMJ osteoarthritis. The main pathological features of TMJ osteoarthritis include chondrocyte death, extracellular matrix (ECM) degradation and subchondral bone remodelling. Chondrocyte apoptosis accelerates the destruction of cartilage. However, autophagy has a protective effect on condylar chondrocytes. Degradation of ECM not only changes the properties of cartilage but also affects the phenotype of chondrocytes. The loss of subchondral bone in the early stages of TMJ osteoarthritis plays an aetiological role in the onset of osteoarthritis. In recent years, increasing evidence has suggested that chondrocyte hypertrophy and endochondral angiogenesis promote TMJ osteoarthritis. Hypertrophic chondrocytes secrete many factors that promote cartilage degeneration. These chondrocytes can further differentiate into osteoblasts and osteocytes and accelerate cartilage ossification. Intrachondral angiogenesis and neoneurogenesis are considered to be important triggers of arthralgia in TMJ osteoarthritis. Many molecular signalling pathways in endochondral osteogenesis are responsible for TMJ osteoarthritis. These latest discoveries in TMJ osteoarthritis have further enhanced the understanding of this disease and contributed to the development of molecular therapies. This paper summarizes recent cognition on the pathogenesis of TMJ osteoarthritis, focusing on the role of chondrocyte hypertrophy degeneration and cartilage angiogenesis.     

Proliferation and differentiation of primary bovine myoblasts using Chlorella vulgaris extract for sustainable production of cultured meat

Recently, cultured meat obtained from livestock-derived cells is being considered as a sustainable food source that reduces the use of natural resources. This study aimed to show that nutrients extracted from Chlorella vulgaris were beneficial in the culture of primary bovine myoblasts (PBMs), a major cell source for cultured meat production. Nutrients (glucose, amino acids, and vitamins) present in the animal-cell culture media were effectively recovered from C. vulgaris using acid hydrolysis treatment. On culture in nutrient-free inorganic salt solution, cell death was induced in most PBMs after 6 days of cultivation. However, the addition of C. vulgaris extract (CVE) significantly improved PBM viability, which was comparable to the viability in conventional culture medium (Dulbecco's modified Eagle's medium). Furthermore, by adding horse serum to induce differentiation, the formation of myotubes was confirmed when CVE were used. Together, the results showed that CVE could be used as an alternative to the conventional culture medium for PBMs. These findings will not only lower the environmental risks associated with the establishment of this eco-friendly cell culture system, but also highlight microalgae as a potent nutrient source that can replace conventional grain-dependent nutrient sources.     

Tracing the skeletal progenitor transition during postnatal bone formation

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Effect of TaiCatoxin (TCX) on the electrophysiological,mechanical and biochemical characteristics of spontaneously beating ventricular cardiomyocytes

TaiCatoxin (TCX), a complex toxin isolated from Taipan snake venom, is believed to have a specific blocking activity on voltage-dependent cardiac calcium channels. The aim of this study was to investigate the effects of TCX on a broad range of heart muscle cell functions, i.e. electrophysiology, contractility, automaticity and the related biochemical modifications. Myocyte-enriched cultures were prepared from newborn rat heart ventricles. The transmembrane potentials were recorded with glass microelectrodes. The contractions were monitored photometrically. TCX decreased the action potential amplitudes, mainly by lowering the plateau. The action potential duration and the contraction parameters were decreased. Although TCX has a minor overall negative chronotropic effect, it evoked transient but severe arrhythmias and prolonged changes in the intercellular electrical coupling. Moreover, the action of TCX appeared to be dose-dependent. These effects are consistent with a specific blockade of the L-type, voltage-dependent calcium channels, but effects of other components of the toxin complex cannot be excluded. TCX also exhibits phospholipase A₂ activity leading to the release of lysophospholipids and FFA (acyl CoA and acyl carnitine), which have detrimental effects on cellular integrity and function.     

Effect of epidermal growth factor on cultured adult rat hepatocytes

When adult rat hepatocytes were cultured in plastic Petri dishes in a medium containing insulin and glucagon, supplementation with epidermal growth factor (EGF) had a pronounced effect on their viability, morphology, and biochemical integrity. Transmission and scanning electron microscopic studies showed that after 1 week cells denied EGF accumulated numerous non-electron-dense bodies and filamentous whorls, had irregular nuclei, and exhibited atypical cell surfaces. In contrast, cells grown for 2-3 weeks in the presence of EGF had well-preserved cellular organelles and remained as an epithelial-like monolayer. After 3 weeks EGF-exposed cultures were still inducible for liver-specific tyrosine aminotransferase, and both rat albumin and rat transferrin were recoverable from the culture medium. Virtually no viable cells were present at 3 weeks in EGF-deprived cultures.     

Effects of vanadate on 6-phosphofructo 2-kinase activity and fructose 2,6-bisphosphate levels in cultured rat hepatocytes

The presence of vanadate in primary cultures of rat hepatocytes produced a significant increase in the concentration of fructose 2,6-bisphosphate and in the activity of 6-phosphofructo 2-kinase. Compared with insulin, vanadate had a more potent action on the metabolite increase, but a similar effect on the 6-phosphofructo 2-kinase activity. Both the insulin- and the vanadate-dependent enhancements of 6-phosphofructo 2-kinase were inhibited by cycloheximide which specifically blocks protein synthesis on the translational level, suggesting that the increase of the enzyme activity was due to induction rather than to a change in the catalytic activity.     

Cystic fibrosis: Demonstration of an abnormality in mucopolysaccharides in cultured lymphoid lines

Cultured lymphoid cells of both homozygotes and heterozygotes for cystic fibrosis could be distinguished from those of normals by (1) growth pattern, gross clumping, and (2) a relative increase in dermatan sulfate, with a normal total mucopolysaccharide content. Lines derived from the genetic mucopolysaccharidoses also had these characteristics, but their total mucopolysaccharide content was markedly increased. These observations support the hypothesis that the cellular disturbance in cystic fibrosis resides in those cellular regions whose functions would be altered by mucopolysaccharide composition.This research was made possible through a grant from The National Foundation-March of Dimes and, in part, supported by The Cystic Fibrosis Children's Fund and The National Cystic Fibrosis Research Foundation.Career Scientist of the Health Research Council of New York City (I-797).