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.     

THE ROOTING OF LIQUID-GROWN ASPEN CALLUS

Liquid-grown callus was used to study the nutritional requirements for rooting of a triploid form of normally diploid quaking aspen (Populus tremuloides Michx.). In modified Wolter's liquid medium, tan rough-surfaced spheres of callus grew rapidly when supplied with a high concentration of 2,4-D (0.5 mg/liter), but light-yellow uniformly smooth and firm spheres grew more slowly with a low level of 2,4-D (0.04 mg/liter) plus kinetin. When tissue was grown uncut in liquid for 1, 2, or 3 months, then subcultured to a low-2,4-D agar medium, rooting increased primarily with the age of the source tissue rather than the initial explant size. The surface of the youngest tissue source was almost smooth, but free columnar proliferations extended out from the surface for two or three cells in 2-month-old tissue and for three to six cells in the 3-month-old tissue. The relationship of increased rooting with increased surface cell-proliferation of older tissue was not determined anatomically.     

Cleavage of proteoglycan aggregate by leucocyte elastase.

The partial degradation of proteoglycan aggregate by human leucocyte elastase yielded products that banded with Mr 190,000, 140,000, 88,000, and 71,000 when analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis. Analysis of these bands revealed that the 190,000- and 140,000-Da bands contained chondroitin and keratan sulfate stubs and had N-terminal amino acid sequences corresponding to a sequence starting at residue 398 of the core protein of rat or human aggrecan. With increased time of digestion, the staining intensities of the 190,000-, 140,000-, and 88,000-Da bands decreased relative to the 71,000-Da band. Analysis of the 88,000- and 71,000-Da bands showed that they contained peptides substituted only with keratan sulfate stubs and that each band contained two peptides with different N-terminal sequences. One of these corresponded to a sequence that started at residue 398 of rat or human aggrecan and the other to the N-terminal sequence of bovine aggrecan. Under conditions of complete digestion, bands of 71,000 and 56,000 Da which contained only keratan sulfate stubs were observed on SDS-polyacrylamide electrophoresis. The 71,000-Da band was shown to have a single sequence similar to that starting at residue 398 of human and rat aggrecan and thus represents the globular domain 2 (G2) of the core protein of aggrecan. The 56,000-Da band was shown to have a sequence similar to that of the N-terminal sequence of bovine aggrecan indicating that this peptide corresponds to the globular domain 1 (G1) of the molecule. These results suggest that leucocyte elastase cleaves the core protein of aggrecan between valine 397 and isoleucine 398, which are located in the interglobular domain linking the G1 and G2 domains of the core protein of aggrecan. Further digestion of the proteoglycan aggregate with elastase resulted in the cleavage of the core protein within the chondroitin sulfate attachment domains.     

The ATP-dependent interaction of eukaryotic initiation factors with mRNA

The interaction of three protein synthesis initiation factors, eukaryotic initiation factor (eIF)-4A, -4B, and -4F, with mRNA has been examined. Three assays specifically designed to evaluate this interaction are RNA-dependent ATP hydrolysis, retention of mRNAs on nitrocellulose filters, and cross-linking to periodate-oxidized mRNAs. The ATPase activity of eIF-4A is only activated by RNA which is lacking in secondary structure, and the minimal size of an oligonucleotide capable of effecting an optimal activation is 12-18 bases. In the presence of ATP, eIF-4A is capable of binding mRNA. Consistent with the ATPase activity, this binding shows a definite preference for single-stranded RNA. In the absence of ATP, eIF-4F is the only factor to bind capped mRNAs, and this binding, unlike that of eIF-4A, is sensitive to m7GDP inhibition. The activities of both eIF-4A and eIF-4F are stimulated by eIF-4B, which seems to have no specific independent activity in our assays. Evidence from the cross-linking studies indicates that in the absence of ATP, only the 24,000-dalton polypeptide of eIF-4F binds to the 5' cap region of the mRNA. From the data presented in conjunction with the current literature, a suggested sequence of factor binding to mRNA is: eIF-4F is the first initiation factor to bind mRNA ind an ATP-independent fashion; eIF-4B then binds to eIF-4F, if in fact it was not already bound prior to mRNA binding; and finally, eIF-4A binds to the eIF-4F X eIF-4B X mRNA complex and functions in an ATP-dependent manner to allow unwinding of the mRNA.     

Glycosylation reduces the bioactivity of calcitonin

The biological significance of peptide hormone glycosylation is uncertain. To examine the effect of Asn-linked glycosylation on calcitonin's bioactivity we purified glycosylated calcitonin from a transplantable rat medullary thyroid carcinoma. Glycosylated calcitonin constituted 2.3% of the total extracted immunoreactive calcitonin. The structure of this peptide differed from nonglycosylated calcitonin only by the oligosaccharide modification of asparagine 3. Affinity of glycosylated calcitonin for lentil lectin indicated that the oligosaccharide was a complex processed form. In a standard in vivo bioassay glycosylated calcitonin had a markedly reduced hypocalcemic activity compared to nonglycosylated calcitonin, an effect most likely due to the presence of the oligosaccharide.     

Dissociation of double-stranded polynucleotide helical structures by eukaryotic initiation factors, as revealed by a novel assay

A new technique has been applied to the study of the RNA secondary structure unwinding activity of the eukaryotic initiation factors (eIFs) 4F, 4A, and 4B. Secondary structures were generated at the 5' ends of reovirus and globin mRNA molecules by hybridization with 32P-labeled cDNA molecules 15 nucleotide residues long. The dissociation of the labeled cDNAs from the mRNAs was assayed by a gel filtration chromatography procedure which separates the free cDNAs from mRNAs and mRNA/cDNA hybrids. When the three factors were tested alone, only eIF-4F stimulated dissociation of hybrids. The combination of eIF-4A plus eIF-4B also exhibited a strong hybrid dissociating activity, which was markedly temperature dependent. Under optimum conditions, up to 90% of the hybrid structures are disrupted in 60 min. These results demonstrate for the first time that stable double-stranded regions can be melted and dissociated by eIFs. They also characterize more precisely the first step in the structure unwinding reaction.     

Coronary reperfusion in dogs inhibits endothelium-dependent relaxation: role of superoxide radicals

Previous studies indicate that release of superoxide radicals during coronary reperfusion following occlusion may relate to the loss of endothelium-dependent coronary arterial relaxation. We examined coronary arterial ring relaxation in dogs subjected to temporary circumflex (Cx) coronary artery occlusion and treated with saline or the superoxide radical scavenger superoxide dismutase (SOD). In dogs treated with saline, Cx coronary ring relaxation in response to leukotriene D4 (LTD4) and acetylcholine (ACh) was attenuated (p less than 0.01), but coronary relaxation in response to nitroglycerin was preserved, suggesting loss of endothelium-dependent relaxation following coronary reperfusion. In contrast, Cx coronary relaxation in response to LTD4 and ACh was preserved in the SOD-treated dogs (p less than 0.01 compared to saline-treated dogs). To further examine the role of superoxide radicals in the loss of endothelium-dependent relaxation, normal nonischemic canine coronary artery and rat aortic rings were exposed to a superoxide radical generating system of xanthine and xanthine oxidase in vitro. Xanthine plus xanthine oxidase treatment caused a significant (p less than 0.01) decrease in the relaxant effects of ACh. Pretreatment of rat aortic rings with SOD protected against the loss of ACh-induced relaxation. These observations suggest that release of superoxide radicals during reperfusion is the basis of loss of endothelium-dependent coronary arterial relaxation. Treatment with superoxide radical scavengers prior to coronary reperfusion protects against this loss.