Indoleamine 2,3-dioxygenase. Purification and some properties.

Indoleamine 2,3-dioxygenase was purified from rabbit small intestine to apparent homogeneity as judged by polyacrylamide gel electrophoresis and analytical ultracentrifugation. The native enzyme was a monomeric protein of a molecular weight of 41,000 +/- 1,000 with an s020,w value of 3.45 S. It had a relative abundance of hydrophobic amino acids such as valine, leucine, and isoleucine, and contained approximately 5% carbohydrate by weight. The estimated content of sugar residues per mol of enzyme was: galactose, 1.2; mannose, 2.6; N-acetylglucosamine, 5.2; and sialic acid, 0.8. One mole of enzyme had 0.8 mol of protoheme IX as a prosthetic group. However, copper was not detected in a significant amount and the ratio of copper to heme was less than 0.03. EPR spectra of the nitric oxide complex of the ferrous enzyme indicated that a nitrogen atom, possibly in an imidazole group, might be coordinated as the fifth ligand of the heme coenzyme. The anisotropic g values were gx = 2.08, gy = 1.98, and gz = 2.01. A single enzyme protein catalyzed the oxygenative ring cleavage of D- and L-tryptophan, D- and L-5-hydroxytryptophan, tryptamine, and serotonin. In addition, the purified enzyme had a peroxidase activity with guaiacol and potassium iodide as hydrogen donors, but not a catalase activity.     

beta-Adrenergic receptor desensitization stimulates glucose uptake in C6 rat glioma cells

When C₆ glioma cells were stimulated by β -adrenergic ligands, [³H]-deoxyglucose uptake by the cells decreased in the first 30 min, followed by its acceleration. The stimulation of deoxyglucose uptake was attributable to desensitization of β-adrenergic receptor-adenylate cyclase system. When the cells were treated with quinacrine or tetracaine, phospholipase inhibitors, the stimulation of deoxyglucose uptake by isoproterenol was diminished without changing the basal rate. On the other hand, when C₆ glioma cells were treated with melittin or phorbol ester, phospholipase A₂ activators, the deoxyglucose uptake increases even in the absence of isoproterenol. Since these compounds inhibit or enhance phospholipase A₂ as well as the desensitization of β -adrenergic receptors (Proc. Natl. Acad. Sci. USA 77, 1341–1345, 1980), these results suggest that turnovers of phospholipids in the vicinity of β -adrenergic receptors modify the glucose uptake of C₆ glioma cells.     

Calmodulin antagonists inhibit Ca2+ uptake of mitochondria of guinea pig peritoneal macrophages

The Ca²⁺ uptake of the mitochondria of guinea pig peritoneal macrophages was not stimulated by the addition of calmodulin. However, calmodulin antagonists, both phenotiazines and N-naphthalenesulfonamides, in low concentrations inhibited the Ca²⁺ uptake of the mitochondoria, as compared to the inhibition of the calmodulin-dependent stimulation of brain phosphodiesterase. These calmodulin antagonists appear to have severe side effects on active processes of the mitochondria and which are unrelated to the specific effect on calmodulin.     

Characterization of an abnormal fibrinogen Osaka V with the replacement of gamma-arginine 375 by glycine. The lack of high affinity calcium binding to D-domains and the lack of protective effect of calcium on fibrinolysis.

Prolonged thrombin time was completely corrected by the addition of millimolar concentrations of calcium in a new abnormal fibrinogen, Osaka V. Analysis of lysyl endopeptidase digests of A alpha-, B beta-, or gamma-chains by high performance liquid chromatography, and the following amino acid sequence analysis of relevant peptides revealed that about 50% of the gamma-chain has a replacement of gamma-arginine 375 by glycine. When fibrinogen was digested with plasmin in the presence of millimolar concentration of calcium, the amount of fragment D1 was about 50% of the normal control, and the rest was further cleaved to fragment D2, D3, or D62 with an apparent Mr of 62,000. Plasmic digestion of cross-linked fibrin in the presence of calcium resulted in the appearance of an abnormal fragment with an apparent Mr of 123,000 as well as fragments D2, D3, and D62, concomitant with the decrease of D dimer. The gamma-remnant of the abnormal fragment proved to be a cross-linked complex of the normal D1 gamma-remnant and residues 374-406/411 of the abnormal gamma-chain. The number of high affinity Ca(2+)-binding sites for the normal fibrinogen and fibrinogen Osaka V obtained by equilibrium dialysis was 2.88 (about 3) and 1.85, respectively, and that for the abnormal molecules was calculated as 0.9 (about 1) from their relative amounts in the samples, suggesting the lack of two Ca(2+)-binding sites in the D-domains. These data suggest that the normal structure of the COOH-terminal portion of the gamma-chain including residue 375 is required for the full expression of high affinity calcium binding to D-domains, the ability to be protected by calcium against plasmic digestion, and fibrin polymerization. During these studies, we found that the NH2-terminal amino acid of the gamma-remnant in fragments D or D dimer which were obtained after prolonged digestion with plasmin is gamma-Met89.     

Pulse sequences generated by a degenerate analog neuron model

The response characteristics of an electronic neuron model proposed by the authors are investigated. Periodic stimulating pulse sequences with a fixed frequency are applied to the analog neuron model and the response pulse sequences are studied. In the degenerate case, the state transition of the neuron model during one period of the stimulating pulse sequence is described by a first order piecewise linear difference equation with a jump. It is shown that the periodic response pulse sequences of the neuron model belong to a special class of pulse sequences generated by a simple algorithm, and that the relation between the pulse width (or amplitude) of the stimulating pulse and the firing rate of the neuron model takes the form of an extended Cantor function.     

Biosynthesis of triterpenoids from amino acids in Pisum sativum. The distribution of the radioactivity in squalene biosynthesized from radioisotopically labelled l-leucine and l-valine

Radioisotopically labelled l-leucine and l-valine were fed to Pisum sativum and incorporated into squalene and β-amyrin. Chemical degradation of the radioactive squalene revealed an equal distribution of the radioactivity in the isopentenyl pyrophosphate(IPP)-derived and the 3,3-dimethylallyl pyrophosphate(DMAPP)-derived moieties of the squalene molecule, unlike the unbalanced distribution in favour of the DMAPP-derived moiety of a monoterpenoid molecule biosynthesized from these amino acids by higher plants.     

An electron microscopic study on the mating tube formation in the heterobasidiomycete Tremella mesenterica

Ultrastructure of the mating tube formed in yeast haplont of the heterobasidiomycete Tremella mesenterica was studied by electron microscopy. Cell wall of the mating tube emerged as evagination of the inner layers, rupturing outer layers of the mother cell wall. Comparison with budding cells suggested that the tube emergence place at bud scar and the process of tube emergence was the same as that of bud emergence. Electron transparent vesicles of 0.1 m diameter were scattered in the cytoplasm of the mating tube. Nucleus-associated organelle was located at one side of the nuclear envelope which extended towards the mating tube. A few microtubules were detected in the mating tube, but their association with a nucleus was not clear. The cytoplasmic structure of the mating tube was discussed in comparison with that of hyphae of the filamentous fungi.     

Image-based parameter inference for epithelial mechanics

Measuring mechanical parameters in tissues, such as the elastic modulus of cell-cell junctions, is essential to decipher the mechanical control of morphogenesis. However, their in vivo measurement is technically challenging. Here, we formulated an image-based statistical approach to estimate the mechanical parameters of epithelial cells. Candidate mechanical models are constructed based on force-cell shape correlations obtained from image data. Substitution of the model functions into force-balance equations at the cell vertex leads to an equation with respect to the parameters of the model, by which one can estimate the parameter values using a least-squares method. A test using synthetic data confirmed the accuracy of parameter estimation and model selection. By applying this method to Drosophila epithelial tissues, we found that the magnitude and orientation of feedback between the junction tension and shrinkage, which are determined by the spring constant of the junction, were correlated with the elevation of tension and myosin-II on shrinking junctions during cell rearrangement. Further, this method clarified how alterations in tissue polarity and stretching affect the anisotropy in tension parameters. Thus, our method provides a novel approach to uncovering the mechanisms governing epithelial morphogenesis.     

Tumor cells enhance their own CD44 cleavage and motility by generating hyaluronan fragments

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that interacts with cell-surface receptors, including CD44. Although HA usually exists as a high molecular mass polymer, HA of a much lower molecular mass that shows a variety of biological activities can be detected under certain pathological conditions, particularly in tumors. We previously reported that low molecular weight HAs (LMW-HAs) of a certain size range induce the proteolytic cleavage of CD44 from the surface of tumor cells and promote tumor cell migration in a CD44-dependent manner. Here, we show that MIA PaCa-2, a human pancreatic carcinoma cell line, secreted hyaluronidases abundantly and generated readily detectable levels of LMW-HAs ranging from approximately 10- to 40-mers. This occurred in the absence of any exogenous stimulation. The tumor-derived HA oligosaccharides were able to enhance CD44 cleavage and tumor cell motility. Inhibition of the CD44-HA interaction resulted in the complete abrogation of these cellular events. These results are consistent with the concept that tumor cells generate HA oligosaccha-rides that bind to tumor cell CD44 through the expression of their own constitutive hyaluronidases. This enhances their own CD44 cleavage and cell motility, which would subsequently promote tumor progression. Such an autocrine/paracrine-like process may represent a novel activation mechanism that would facilitate and promote the malignant potential of tumor cells.