Transport and membrane binding of the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) in Xenopus laevis oocytes

Summary We have examined transport and membrane binding of 6-diazo-5-oxo-l-norleucine (DON, a photoactive diazo-analogue of glutamine) and their relationships to glutamine transport in Xenopus laevis oocytes. DON uptake was stereospecific and saturable (V _max of 0.44 pmol/oocyte · min and a K _m of 0.065 mm). DON uptake was largely Na^u+ dependent (80% at 50 m DON) and inhibited (>75%) by glutamine and arginine (substrates of the System B^0,+ transporter) at 1 mm. Glutamine and DON show mutual competitive inhibition of Na⁺-dependent transport. Preincubation of oocytes in medium containing 0.1 mm DON for 24 or 48 hr depressed the V _max for System B^0,+ transport (as measured by Na⁺-dependent glutamine uptake), this effect was highly specific (neither d-DON nor the System B^0,+ substrates glutamine and d-alanine showed any independent effect) and required Na⁺ ions. Glutamine (1 mm in preincubation medium) protected transport from inhibition by DON. The possibility that specific inactivation of System B^0,+ by DON reflects attachment of DON to the transporter was tested by examining the binding of [¹⁴C]DON to Xenopus oocyte membranes. Oocytes incubated in 100 mm NaCl in the presence of [¹⁴C]DON for up to 48 hr showed 2.4-fold higher ¹⁴C-binding to membranes than oocytes incubated in choline chloride. Na⁺-dependent DON binding (31 ± 11 fmol/g membrane protein) was suppressed by external glutamine, arginine or alanine and was largely confined to a membrane protein fraction of 48–65 kDa (as assessed by SDS-polyacrylamide gel electrophoresis). The present studies indicate that DON and glutamine uptake in oocytes are both mediated by System B^0,+ and demonstrate that DON binding to a particular membrane protein fraction is associated with inactivation of the transporter, offering the prospect of using [¹⁴C]DON as a covalent label for the transport protein in order to facilitate its isolation and subsequent biochemical characterization.This work was supported by The Wellcome Trust, Action Research for the Crippled Child, Ajinomoto GmbH, Pfrimmer GmbH, the Rank Prize Funds, the Medical Research Council and the University of Dundee. We are grateful to Dr. C.I. Pogson (Wellcome Research Laboratories) and Drs. J.C. Ellory and B. Elford (University of Oxford) for gifts of [¹⁴C]DON.     

Influence of 6-diazo-5-oxo-L-norleucine (DON), a glutamine analogue, on cartilaginous differentiation in mouse limb buds in vitro.

The antibiotic, 6-diazo-5-oxo-L-norleucine (DON), an analogue of L-glutamine, causes limb malformations in several species, including mice. This report shows that DON also interferes with differentiation of cartilaginous rudiments of mouse limb buds grown as organ cultures for 3 to 8 days in medium containing the teratogen. DON (5 mug/ml) inhibits growth of the explants and interferes with production of normal matrix by chondrocytes. The cartilage of DON-treated cultures exhibits a striking lack of matrix, compared with that of control explants which contains abundant metachromatic matrix. Differentiation of osteoblasts, and secretion of osteoid around the scapula and humerus are enhanced by DON. The direct effects of DON on growth and chondrogenesis, which can be prevented by addition of L-glutamine (1 mg/ml) to the medium, can be attributed to the known interference of DON in L-glutamine-dependent steps in metabolism. The possible relationships between these effects of DON in vitro and the malformations produced in vivo, are discussed.     

Corneal development. V. Treatment of five-day-old embryos of domestic fowl with 6-diazo-5-oxo-L-norleucine (DON).

The embryogenesis of the corneal stroma of the domestic fowl was studied following the injection of the glutamine analog DON (6-diazo-5-oxo-l-norleucine) into the chorioallantoic veins of 5-day-old chick embryos. The 44 survivors were cilled between 1 hr and 13 days following injection. Their corneas were examined histologically and compared with those of untreated animals of similar age.All of the experimentally-treated corneas examined developed abnormally. The defects included: a temporary disappearance of portions of the endothelium; the deposition of disordered arrays of collagen fibers beneath the corneal epithelium, including a reversal in the direction of rotation of the axes of affected portions of the stromal lamellae; the appearance of stromal cysts; and the accumulation, beginning 6 days after injection, of pools of Gomori-silver-positive material within the epithelium.Abnormalities in corneal development following treatment with DON were compared with those previously obtained following administration of l-azetidine-2-carboxylic acid.The findings demonstrated that: (1) the characteristic, progressive rotation of fibril orientation which normally occurs in the outer lamellae of the avian, corneal, primary stroma is not a rigidly-determined configuration since its direction can be reversed consistently following treatment with DON; and (2) the primary stroma dictates the collagenous structure of the secondary stroma deposited within it.     

Inactivation of microbial glutamin-(asparagin-)ase by azaserine and 6-diazo-5-oxo-L-norleucine

The effect of substrate analogues on glutamin-(asparagin-)ase from Pseudomonas aurantiaca-548 has been studied. The enzyme was demonstrated to be highly sensitive to the the action of 6-diazo-5-oxo-L-norleucine and azaserine. L-isomers of glutamine, aspartate, glutamate and several other substrate analogues with free alpha-amino groups protected the enzyme against the inhibitory DON effect. Thus, thorough preliminary selection of appropriate inhibitors, their dosage and treatment duration is needed for the recommendation of combined enzyme-inhibitor application in anti-tumour chemotherapy.     

6-diazo-5-oxo-L-norleucine and azaserine as affinity inhibitors of glutamin(asparagin)ase

Incubation of homogeneous glutamin(asparagin)ase from Pseudomonas aurantiaca with 6-diazo-5-oxo-L-norleucine (DON) and azaserine leads to an almost complete inactivation of the enzyme. The inactivation process in both cases involves the step of reversible binding of the enzyme with the inhibitor into a complex and subsequent modification of the enzyme within this complex. The data on saturation of the enzyme by low concentrations of inhibitors, the protective effect of substrate and its analogs as well as of the competitive inhibitor and product of the enzymatic reaction, L-aspartate, suggest that the modification of functional groups takes place in the enzyme active site. The presence of essential threonine hydroxyl groups in/or near the enzyme active site is surmised.     

The effects of 6-diazo-5-oxo-L-norleucine, a glutamine analogue, on the structure of the major cartilage proteoglycan synthesized by cultured chondrocytes

Incubation of embryonic chick chondrocytes with 6-diazo-5-oxo-L-norleucine (DON), a glutamine analogue, led to a dose-dependent inhibition of [35S]sulfate incorporation into proteoglycan. In the absence of exogenous L-glutamine, a maximal inhibition of 50-60% was achieved with DON concentrations greater than or equal to 1 microgram/ml (6 microM); the ED50 was approximately 0.2 microM. This inhibitory effect could be partially restored by the addition of 100-fold molar excess of either exogenous L-glutamine or M-glucosamine. The quantitative changes were due neither to inhibition of protein core synthesis nor to undersulfation of glycosaminoglycan chains. Rather, the proteoglycan synthesized in the presence of DON contained substantially fewer (approximately 50% of control) and smaller (10-15% of control, on the average) chondroitin sulfate chains as well as a paucity of keratan sulfate chains. The result of these structural changes was a proteoglycan with significantly lower molecular weight, buoyant density, and anionic charge. In spite of these modifications, the altered proteoglycan synthesized in the presence of DON was secreted normally and retained the ability to interact with exogenous hyaluronic acid and link proteins. The results of our experiments also indicate that DON substantially diminished the pool of hexosamine precursors required for glycosaminoglycan synthesis. We conclude that this decrease was responsible for the molecular alterations described above; and these, in turn, can account for the morphological changes previously seen in cartilage matrix synthesized in the presence of DON.     

Inhibition of morphogenetic cell interactions by 6-diazo-5-oxo-norleucine (DON).

Inasmuch as cell adhesion and spreading are fundamental properties involved in a variety of vital processes, the dependence of the expression of spontaneous macrophage-mediated cytotoxicity on these attributes was investigated using unique means by which substratum adhesiveness of effector and target cells could be either markedly diminished or varied within a large range. Spontaneous cytotoxicity by activated macrophages against various tumor targets growing in adherent or in suspension culture was unaltered, irrespective of whether interaction took place on plastic, on Teflon, or on plastic coated with varying concentrations of the polymer, poly(HEMA). The findings indicate that whatever the nature is of spontaneous macrophage-mediated target cell killing, it is quite independent of cell adhesion and cell shape considerations.     

Inactivation of rat renal phosphate-dependent glutaminase with 6-diazo-5-oxo-L-norleucine. Evidence for interaction at the glutamine binding site.

Inactivation of rat renal phosphate-dependent glutaminase by 6-diazo-5-oxo-L-norleucine occurs only under conditions where the enzyme is catalytically active. The glutaminase activity and the rate of inactivation by the diazoketone exhibit very similar phosphate concentration-dependent activation profiles. Because of this phosphate dependency, it was not possible to differentiate an apparent protection by glutamine from the strong inhibition of inactivation caused by glutamate. The ability of glutamate to protect the glutaminase against inactivation is reversed by increasing concentrations of phosphate.The observed characteristics of inactivation by 6-diazo-5-oxo-L-norleucine differ considerably from those reported for the inactivation by L-2-amino-4-oxo-5-chloropentanoic acid. In addition, the presence of o-carbamoyl-L-serine was found to stimulate inactivation by 6-diazo-5-oxo-L-norleucine, but to protect the glutaminase against inactivation by the chloroketone. Preinactivation of the glutaminase by the diazoketone only slightly reduced the stoichiometry of binding of [5-14C]chloroketone. These observations suggest that 6-diazo-5-oxo-L-norleucine and L-2-amino-4-oxo-5-chloropentanoic acid interact with different sites on the glutaminase which are specific for binding glutamine and glutamate, respectively.     

In vitro cartilage formation of composites of synovium-derived mesenchymal stem cells with collagen gel

Graft implantation is one of the more popular procedures for repairing cartilage defects; however, sacrifices of the donor site have been an issue. Mesenchymal stem cells (MSCs) are a fascinating source for regenerative medicine because they can be harvested in a less invasive manner and are easily isolated and expanded, with multipotentiality including chondrogenesis. MSCs can be isolated from various adult mesenchymal tissues including synovium. Here, we attempted to form cartilage from the composites of synovium-derived MSCs with collagen gel in vitro. After 21 days of culture, the composites had increased their cartilage matrix, as demonstrated by toluidine blue staining and immunohistochemistry for type II collagen. The composites consisting of 5×10⁷ and 10⁸ cells/ml in gel were richer in proteoglycans than those consisting of lower cell densities. After 1 day, MSCs/gel composites contracted and the diameter decreased by 30%; however, they were stable thereafter. Round cells with short processes producing collagen fibrils showing a similar morphology to that of chondrocytes were seen in the composites by transmission electron microscopy. During composite culture, chondroitin sulfate and mRNA expression for cartilage-related genes increased, demonstrating cartilage maturation. Using an optimized method, we obtained cartilage discs with a diameter of 7 mm and a thickness of 500 μm. Our procedure should thus make it possible to produce a large cartilage matrix in vitro. The tissue engineering of autologous cartilage from the composites of synovium-derived MSCs with collagen gel in vitro for transplantation may be a future alternative to graft implantation for patients with cartilage defects. This study is supported in part by grants from the Japanese Society for the Promotion of Science (16591478), the Japanese Orthopaedics and Traumatology Foundation, and the Nakatomi Foundation to I.S., and the Japanese Society for the Promotion of Science (16591477), the Japanese Sports Medicine Foundation, the Japanese Latest Osteoarthritis Society, and the Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone in Tokyo Medical and Dental University to T.M.     

Avian corneal innervation: Inhibition of nerve ring formation by 6-diazo-5-oxo-l-norleucine

Normal innervation of embryonic avian cornea is achieved in two distinct phases. During phase I, nerves extend from the ventrotemporal region both dorsally and ventrally around the cornea, but not into it, ultimately encircling the 10th-day cornea. Phase II commences as nerves extend radially from the ring into the corneal stroma and from there into the epithelium. The effect of the glutamine analog, 6-diazo-5-oxo-l-norleucine (DON), on this normal sequence of events has been examined. In ovo administration of 5 μg DON on the 5th day of development inhibits the incorporation of [³⁵S]sulfate in sulfated glycosaminoglycans in both the cornea and control tissues and inhibits the completion of phase I. Phase II of corneal innervation appears to be affected only indirectly and extension of nerves into the cornea does occur. However, the number of nerves entering the DON-treated cornea is dramatically reduced. Administration of DON on the 7th or 9th days of development does not affect corneal innervation, but does demonstrate a clear effect on [³⁵S]sulfate incorporation in sulfated glycosaminoglycans by the cornea and control tissues. These data suggest that nerve ring completion is not a prerequisite for extension of nerves into the cornea and suggest an integral role for glycosaminoglycans in facilitating phase I, but not phase II, of corneal innervation.     

The stability of the collagen phenotype during stimulated collagen,glycosaminoglycan, and DNA synthesis by articular cartilage organ cultures

Rabbit articular cartilage slices were grown in organ culture for 9 weeks. Eightfold increases in the synthesis of both glycosaminoglycan and collagen were observed at 1 and 3 weeks, respectively. These levels of synthesis gradually declined in parallel to fourfold at 9 weeks. DNA synthesis was stimulated more than 30-fold at 3 weeks and then declined to sevenfold at 9 weeks. In contrast, the content of glycosaminoglycans and collagen per milligram of original wet slices did not vary significantly, while the number of cells increased 1.7-fold by the end of the study. The collagen phenotype of these cultures was determined by sodium dodecyl sulfate electrophoresis of recently synthesized, [³H]proline-labeled intact collagen chains and CNBr peptides. Throughout the study the major collagen synthesized was type II, ranging from 95 to 68% of the collagen synthesized at 0 and 5 weeks, respectively. Increases in the proportions of X₂Y and type III collagen were first observed at 3 weeks in culture. The synthesis of type I collagen was detected only after 5 weeks in culture and never represented more than 11% of the total collagen synthesized. The synthesis of type I trimer could not be verified at any time. This study demonstrates that in vitro organ culture of articular cartilage slices allows chondrocytes to maintain the normal chondrocyte collagen phenotype of predominantly type II synthesis while stimulating their proliferation and matrix synthesis.     

Immunological effects of collagen and collagen peptide from blue shark cartilage on 6T-CEM cells

The physicochemical and in vitro mechanism of immunologic tolerance of pepsin-soluble collagen and its peptide, CII-P, from blue shark cartilage were studied. Protein patterns showed three identical (α₁)₃ chains, suggesting that it was a type-II collagen (CII). CII-P had high antioxidant activity and low carbohydrate content. Collagens had better biocompatibility with decreased the viability of 6T-CEM cell compared to control cells (without collagen). Immunological indices such as FAS/APO-1, cytokine, and caspase levels were higher in CII-treated 6T-CEM cells. Collagen bound to 6T-CEM cell receptors in a dose-dependent manner, and an optimum effect was observed with 10 μg/mL collagen. The high carbohydrate content of CII could activate the FAS receptor, which led to increased apoptotic gene expression in 6T-CEM cells. Breakdown of 6T-CEM cell nuclei through the induction of apoptosis by CII was confirmed by fluorescence microscopy. Collagen molecular weight and glycosylation patterns were crucial factors for immunologic tolerance and 6T-CEM cellular apoptosis.     

In vitro toxicity induced by deoxynivalenol (DON) on human and rat granulomonocytic progenitors

Deoxynivalenol (DON) is a trichothecene mycotoxin produced by various species of fungi. Trichothecenes are known as major contaminants of cereals and cereal-containing foods. DON has been detected in agricultural products worldwide and persists in products after processing. In humans as well as in animals, DON has been shown to induce both alimentary and hematological toxicities. Granulo-monocytic progenitors (CFU-GM) from human umbilical cord blood from rat bone marrow were cultured in the presence of DON (from 10-6 to 10-8 mol/L) for 14 days. DON rapidly inhibits human and rat CFU-GM in a concentration-dependent manner between 10-6 and 2.5 × 10-7 mol/L. IC50 values on days 7, 10, and 14 were, respectively, 3 × 10-8, 2.9 × 10-8, 3.9 × 10-8 mol/L for human CFU-GM and 2.6 × 10-7, 1.5 × 10-7, and 1.6 × 10-7 mol/L for rat CFU-GM. The present study defines the cytotoxic and inhibitory DON concentrations for rat and human CFU-GM and provides a system for further investigation of cellular DON targets and elucidation of the mechanism of trichothecene hematotoxicity. Moreover, we propose one of the trichothecenes tested in our studies as a reference molecule for in vitro studies, since one mycotoxin seems to be the most potent myelotoxic inhibitor of CFU-GM detected to date.