C nuclear magnetic resonance study of acetate incorporation into malate during ca-uptake by isolated leaf tissues

¹³C Nuclear magnetic resonance spectroscopy of leaflets of Gleditsia triacanthos and Albizia julibrisin was used to determine the fate of acetate taken up during the absorption of calcium from ¹³C-labeled Ca-acetate solution. Small amounts of acetate accumulated temporarily in the leaf tissues, but the bulk of acetate was incorporated into malate. The initial rate of malate synthesis was very low, but increased rapidly during acetate treatment and reached its maximum after 8 hours; the enzymes involved in malate synthesis thus appear to be substrate induced. Use of acetate-2-¹³C yielded malate labeled in C-3, indicating that vacuolar malate accumulating during Ca-uptake might be synthesized via malate synthase from acetate and glyoxalate. However, a source of glyoxalate condensing with acetate during malate synthesis could not be identified. Glycolate produced in photorespiration is an unlikely source, because glycolate-2-¹³C was absorbed and metabolized by the leaf tissues into products of the glycolate pathway, but was not a major precursor in malate synthesis. Malate synthesis via the glyoxalate cycle is also unlikely, because no evidence for the recycling of a ¹³C-labeled 4-carbon organic acid was found. Malate synthesis in the leaflets of Gleditsia and Albizia thus appears to involve the inducible condensation of acetate with a 2-carbon compound of unidentified nature and origin.     

Cobinding of bilirubin and sulfonamide and of two bilirubin molecules to human serum albumin: a site model

Differential light absorption spectra of the bilirubin-albumin 1:1 complex, obtained on addition of 20 different sulfonamides, differ with respect to shape and amplitude. This finding seems to indicate that the sulfonamide molecule is bound in direct touch with the bilirubin. The light absorption spectrum of bilirubin-albumin 1:1 undergoes changes on cobinding of a fatty acid anion, laurate, and on variation of pH, previously explained by a change of dihedral angle between the two chromophores of the bilirubin molecule. In bilirubin-albumin 2:1, binding of laurate and variation of pH cause little change of the spectrum. This is best explained by binding of the two bilirubin molecules in close proximity, preventing conformational changes in the complex. From measurements of fluorescence of the lone tryptophan group in albumin and quenching on binding of bilirubin, we calculated the distance of 22 A from tryptophan to the first bound bilirubin molecule, and of 18 A to the second. Mutual quenching of the bilirubin fluorescence from two bound bilirubin molecules seemed to indicate that the two are bound closely together. A model of bilirubin-albumin with a binding site capable of accommodating one bilirubin and one sulfonamide molecule, or two molecules of bilirubin, is compatible with our findings.     

Acyl-CoA-binding protein from cow. Binding characteristics and cellular and tissue distribution.

Hydroxyl radicals (OH.) in free solution react with scavengers at rates predictable from their known second-order rate constants. However, when OH. radicals are produced in biological systems by metal-ion-dependent Fenton-type reactions scavengers do not always appear to conform to these established rate constants. The detector molecules deoxyribose and benzoate were used to study damage by OH. involving a hydrogen-abstraction reaction and an aromatic hydroxylation. In the presence of EDTA the rate constant for the reaction of scavengers with OH. was generally higher than in the absence of EDTA. This radiomimetic effect of EDTA can be explained by the removal of iron from the detector molecule, where it brings about a site-specific reaction, by EDTA allowing more OH. radicals to escape into free solution to react with added scavengers. The deoxyribose assay, although chemically complex, in the presence of EDTA appears to give a simple and cheap method of obtaining rate constants for OH. reactions that compare well with those obtained by using pulse radiolysis.     

Neural network detects errors in the assignment of mRNA splice sites.

The use of databanks in genetic research assumes reliability of the information they contain. Currently, error-detection in the manually or electronically entered data contained in the nucleotide sequence databanks at EMBL, Heidelberg and GenBank at Los Alamos is limited. We have used a subset of sequences from these databanks to train neural networks to recognize pre-mRNA splicing signals in human genes. During the training on 33 human genes from the EMBL databank seven genes appeared to disturb the learning process. Subsequent investigation revealed discrepancies from the original published papers, for three genes. In four genes, we found wrongly assigned splicing frames of introns. We believe this to be a reflection of the fact that splicing frames cannot always be unambiguously assigned on the basis of experimental data. Thus incorrect assignment appear both due to mere typographical misprints as well as erroneous interpretation of experiments. Training on 241 human sequences from GenBank revealed nine new errors. We propose that such errors could be detected by computer algorithms designed to check the consistency of data prior to their incorporation in databanks.     

Saint Lucia revisited

The eastern Caribbean island of Saint Lucia is now famous in parasitological history as the setting for a major programme of schistosomiasis control'. Perhaps less well-known are the island's effective control of many intestinal parasites, and elimination of malaria, such that the current patterns of mortality and other demographic indicators now resemble those of industrialized countries. More recently, the island has become the focus for another community-based health programme as the Caribbean region again comes to grips with Aedes aegypti and its recently imported relative, Aedes albopictus, important vectors of yellow fever and dengue viruses (see Box 1).     

Matrix plasminogen activator inhibitor. Modulation of the extracellular proteolytic environment

We have previously demonstrated that plasminogen activator inhibitor (PAI-1) is associated with the extracellular matrix of cultured bovine smooth muscle cells (Knudsen, B.S., Harpel, P.C., Nachman, R.L. (1987) J. Clin. Invest. 80, 1082-1089). In this report we describe the physiologic role of PAI-1 during the interaction of the tissue plasminogen activator (t-PA) secreting Bowes human melanoma cell line with endothelial extracellular matrices. In addition we have characterized the t-PA.PAI complexes formed during this interaction in the presence and absence of plasminogen. In the absence of plasminogen, a 104-kDa complex between Bowes t-PA and PAI-1 appears in the supernatant. In the presence of plasminogen, PAI initially prevents plasmin formation on the matrix and protects the matrix from degradation by plasmin. The 104-kDa t-PA.PAI complex is degraded into a 68 and a 47-kDa complex by small amounts of plasmin generated from secreted Bowes t-PA and plasminogen. Analysis of these complexes revealed that t-PA is rapidly cleaved by plasmin within the complex whereas complexed PAI-1 is not further degraded. Matrix-associated PAI-1 may play an important role in the protection of extracellular matrices from remodeling and degradation by cellular t-PA and plasminogen.     

Alterations in cell surface galactosyltransferase activity during limb chondrogenesis in brachypod mutant mouse embryos

The autosomal mutation brachypod (bpH/bpH) in the mouse affects the development of precartilage mesenchymal condensation in the limb-bud. We have previously shown that this defect is localized to the expression of terminal N-acetylglucosamine (GlcNAc) glycoproteins in the plasma membrane (Elmer and Wright, '83). The present study is focused on cell surface galactosyltransferase (GalTase), an ectoenzyme that transfers galactose to its GlcNAc substrate. Purified plasma membrane preparations derived from wild-type (+/+), heterozygote (+/bpH) and brachypod (bpH/bpH) embryonic mouse limb cells were assayed for GalTase activity during in vitro and in utero chondrogenesis using High-Performance Liquid Chromatography (HPLC). On embryonic day E12, prior to overt expression of the mutant gene, no significant difference in GalTase activity was observed. By the third day in culture, all major chondrogenic elements of the autopod were present in +/+ and +/bpH embryos, whereas the mutant autopods were markedly deficient in staining and appeared consistently shorter. The accumulation of alcianophilic cartilage matrix in the wild-type was accompanied by a 29% increase in GalTase activity, which reflected the net change (29%) observed during development from days E12 to E13 in utero. The GalTase activity for the in utero E13 mutant (13%) was significantly different from control. In culture, day E12 mutant autopods actually decreased in their GalTase level by 3 days so that the activity was reduced to only 57% of the wild-type. Though GalTase activity in the heterozygote showed an intermediate expression, optical image analysis did not reveal consistent differences in cartilage development when compared to +/+, arguing against a gene-dosage effect at the gross anatomical level. These data indicate that an increase in plasma membrane GalTase activity is a natural developmental event that occurs during limb-bud chondrogenesis and a decrease in GalTase activity contributes to the dysmorphogenesis in brachypod limb-buds.