Fibroblasts from wounds of different stages of repair vary in their ability to contract a collagen gel in response to growth factors

Wound contraction is one function of granulation tissue which is critical to repair. This study compares the ability of fibroblast-like cells derived from granulation tissue of various ages to contract a tissue equivalent, or a collagen gel, and examines the influence of growth factors implicated in wound repair on collagen gel contraction by these different cell populations. Cells from older granulation tissue (21 and 28 days) have an enhanced ability to contract a tissue equivalent when compared to cells from younger granulation tissue (7 and 14 days) or normal rat skin fibroblasts. Transforming growth factor-beta 1 (TGF-beta 1) enhanced contractility most in those cells which had a greater basal contractile ability. While basic fibroblast growth factor (bFGF) alone had moderately stimulatory effects at low doses (0.1-1.0 ng/ml), higher doses (greater than or equal to 10 ng/ml) inhibited basal contraction. Pretreatment with bFGF followed by exposure to TGF-beta 1, with or without the continued presence of bFGF, delayed gel contraction by cells from skin and early granulation tissue, but bFGF enhanced TGF-beta 1 activity in highly contractile cells. Transforming growth factor-alpha moderately enhanced contraction by cells from older granulation tissue. While both TGF-beta 1 and bFGF enhanced wound repair, their differential effects on the fibroblast-like cell derived from granulation tissue of different ages suggest that phenotypic differences exist between these cell populations. In addition, our results predict significant interactions between polypeptide cytokines at the site of repair.     

Variation in binding of Bacillus sphaericus toxin and wheat germ agglutinin to larval midgut cells of six species of mosquitoes

Bacillus sphaericus toxin labeled with fluorescein isothiocyanate was readily ingested by Culex pipiens, Aedes aegypti, Anopheles stephensi, Anopheles gambiae, Anopheles quadrimaculatus, and Anopheles albimanus larvae. Fluorescent toxin bound to the luminal cell surface in discrete regions of the posterior midgut and gastric caecum in C. pipiens. In Anopheles spp., toxin bound in a variable pattern to these structures and central and anterior midgut as well. The toxin did not bind to midgut cells of A. aegypti. The toxin was internalized in bright fluorescent vesicles in C. pipiens, but was not internalized in Anopheles spp. and appeared to be weakly bound in these larvae, leaking rapidly from the gut surface. The lectin, wheat germ agglutinin, which interferes with binding of the B. sphaericus toxin, bound to the posterior midgut and gastric caecum of all species, but was not internalized. These results suggest that the sugar moiety of the receptor is not solely responsible for specificity of this toxin, and that binding to Culex spp. midgut cells may be highly specific and of high affinity, whereas binding to Anopheles spp. cells may be nonspecific and/or of low affinity.     

Molecular evolution of enzyme structure: Construction of a hybrid hamster/Escherichia coli aspartate transcarbamoylase

Summary Aspartate transcarbamoylase (ATCase, EC 2.1.3.2) is the first unique enzyme common to de novo pyrimidine biosynthesis and is involved in a variety of structural patterns in different organisms. InEscherichia coli, ATCase is a functionally independent, oligomeric enzyme; in hamster, it is part of a trifunctional protein complex, designated CAD, that includes the preceding and subsequent enzymes of the biosynthetic pathway (carbamoyl phosphate synthetase and dihydroorotase). The complete complementary DNA (cDNA) nucleotide sequence of the ATCase-encoding portion of the hamster CAD gene is reported here. A comparison of the deduced amino acid sequences of the hamster andE. coli catalytic peptides revealed an overall 44% amino acid similarity, substantial conservation of predicted secondary structure, and complete conservation of all the amino acids implicated in the active site of theE. coli enzyme. These observations led to the construction of a functional hybrid ATCase formed by intragenic fusion based on the known tertiary structure of the bacterial enzyme. In this fusion, the amino terminal half (the “polar domain”) of the fusion protein was provided by a hamster ATCase cDNA subclone, and the carboxyl terminal portion (the “equatorial domain”) was derived from a clonedpyrBI operon ofE. coli K-12. The recombinant plasmid bearing the hybrid ATCase was shown to satisfy growth requirements of transformedE. coli pyrB ⁻ cells. The functionality of thisE. coli-hamster hybrid enzyme confirms conservation of essential structure-function relationships between evolutionarily distant and structurally divergent ATCases.     

ATP-Evoked Ca2+ Mobilisation and Prostanoid Release from Astrocytes: P2-Purinergic Receptors Linked to Phosphoinositide Hydrolysis

Astrocyte cultures prelabelled with either [3H]inositol or 45Ca2+ were exposed to ATP and its hydrolysis products. ATP and ADP, but not AMP and adenosine, produced increases in the accumulation of intracellular 3H-labelled inositol phosphates (IP), efflux of 45Ca2+, and release of thromboxane A2 (TXA2). Whereas ATP-stimulated 3H-IP accumulation was unaffected, its ability to promote TXA2 release was markedly reduced by mepacrine, an inhibitor of phospholipase A2 (PLA2). ATP-evoked 3H-IP production was also spared following treatment with the cyclooxygenase inhibitor, indomethacin. We conclude that ATP-induced phosphoinositide (PPI) breakdown and 45 Ca2+ mobilisation occurred in parallel with, if not preceded, the release of TXA2. Following depletion of intracellular Ca2+ with a brief preexposure to ATP in the absence of extracellular Ca2+, the release of TXA2 in response to a subsequent ATP challenge was greatly reduced when compared with control. These results suggest that mobilisation of cytosolic Ca2+ may be the stimulus for PLA2 activation and, thus, TXA2 release. Stimulation of alpha 1-adrenoceptors also caused PPI breakdown and 45 Ca2+ efflux but not TXA2 release. The effects of ATP and noradrenaline (NA) on 3H-IP accumulation were additive, but their combined ability to increase 45Ca2+ efflux was not. Interestingly, in the presence of NA, ATP-stimulated TXA2 release was reduced. Our data provide evidence that functional P2-purinergic receptors are present on astrocytes and that ATP is the first physiologically relevant stimulus found to initiate prostanoid release from these cells.     

Reactions of benzylamines with methylamine dehydrogenase. Evidence for a carbanionic reaction intermediate and reaction mechanism similar to eukaryotic quinoproteins.

It had been previously reported that aromatic amines were not substrates for the bacterial quinoprotein methylamine dehydrogenase. In this study, benzylamine-dependent activity was also not observed in the steady-state assay of this enzyme with the artificial electron acceptor phenazine ethosulfate (PES). Benzylamines did, however, stoichiometrically reduce the protein-bound tryptophan tryptophylquinone (TTQ) prosthetic group and acted as reversible competitive inhibitors of methylamine oxidation when the enzyme was assayed with PES. When methylamine dehydrogenase activity was monitored using a steady-state assay which employed its physiological electron acceptor amicyanin instead of PES, very low but detectable benzylamine-dependent activity was observed. The reactions of a series of para-substituted benzylamines with methylamine dehydrogenase were examined. A Hammett plot of the log of Ki values for the competitive inhibition by these amines against sigma p exhibited a negative slope. Rapid kinetic measurements allowed the determination of values of k3 and Ks for the reduction of TTQ by each of these amines. A Hammett plot of log k3 versus sigma p exhibited a positive slope, which suggests that the oxidation of these amines by methylamine dehydrogenase proceeds through a carbanionic reaction intermediate. A negative slope was observed for the correlation between log Ks and sigma p. Plots of log k3 and log Ks against substituent constants which reflected either resonance or field/inductive parameters for each para substituent indicated that the magnitude of k3 was primarily influenced by field/inductive effects while Ks was primarily influenced by resonance effects. No correlation was observed between either k3 or Ks and the relative hydrophobicity of the para-substituted benzylamines or steric parameters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temporal and ontogenetic aspects of protein induction in foliage of the tomato, Lycopersicon esculentum

Damage to foliage of the tomato, Lycopersicon esculentum, causes the induction of proteinase inhibitors and of the oxidative enzymes polyphenol oxidase, peroxidase, and lipoxygenase. The time courses of induction of these proteins by feeding of two caterpillar species (Manduca sexta and Helicoverpa zea) were studied in a series of experiments. In another series of experiments, the effects of plant age on the inducibility of these proteins were studied. In the time course experiments, induction of proteinase inhibitors and oxidative enzymes in the damaged leaflet was rapid, with higher protein activities evident in damaged leaflets within 12–24 h of damage, depending on the enzyme and the species of insect used to damage the plant. Systemic induction of proteinase inhibitors was also rapid, but systemic induction of polyphenol oxidase was delayed relative to systemic induction of proteinase inhibitors, possibly because high constitutive polyphenol oxidase activities obscured expression of systemic induction at earlier time points. Lipoxygenase and peroxidase were not induced systemically. Induction of all proteins persisted for at least 21 days. In the phenology experiments, inducibility of all proteins decreased in magnitude and was less consistent as plants aged. The results of these experiments exemplify the numerous constraints on induction in tomato plants. Knowledge of these physiological constraints is important to an understanding of the ecological role and causal basis of induced resistance.     

Fatty acid double bond orientation alters interaction with L-cell fibroblasts

Relatively little is known of fatty acid specificity in cellular fatty acid uptake. In this study L-cells, a fibroblastic cell line with very low levels of endogenous cytosolic fatty acid binding protein, were used to examine the role of cis and trans unsaturation on fatty acid uptake. The fluorescent fatty acids, trans-parinaric acid and cis-parinaric acid, were used as analogs of straight-chain saturated, and kinked-chain unsaturated fatty acids, respectively, in order to evaluate the fatty acid specificity of the uptake system. Parinaric acid is poorly metabolizable; greater than 97% was unesterified while ³H-oleic acid was almost totally metabolized after 30 min uptake. Cis- and trans-parinaric acid uptake was saturable and dependent on the concentration of fatty acid. However, the initial rate and maximal amount of trans-parinaric acid taken up by the L-cells was greater than for cis-parinaric acid under the same conditions. The affinity of L-cell uptake for trans-parinaric acid (K_m = 0.12 uM) was 35-fold higher than that for cis-parinaric acid (K_m = 4.17 uM) . Based on competition studies with oleic and stearic acids, it was concluded that the cis- and trans-parinaric acid were taken up by the same L-cell fatty acid uptake system. The results suggest that the L-cell fatty acid uptake system has selectivity for straight chain rather than kinked chain unsaturated fatty acids.Abbreviations Cis-parinaric acid 9Z, 11E, 13E, 15Z-octatetraenoic acid - trans-parinaric acid 9E, I IE, 13E, 15E-octatetraenoic acid - EGTA ethylene glycol-bis(beta-amlno-ethyl ether) N,N,N,N-tetratacetic acid - BSA bovine serum albumin - PBS phosphate buffered saline     

Structure and analysis of phospholipase D gene from Ricinus communis L

Phospholipase D (PLD; EC 3.1.4.4) has been proposed to play a pivotal role in various cellular processes, but molecular understanding of this enzyme is rather limited. This report describes the nucleotide sequence, structure, and genomic organization of a PLD gene from castor bean (Ricinus communis L. cv. Hale). The PLD gene was isolated from a castor bean genomic library using the PLD cDNA as a hybridization probe. Sequence comparison with the PLD cDNA revealed that the PLD gene consisted of four exons and three introns, one of which interrupts the 5-untranslated region. Southern blot analysis indicated that the cloned PLD gene was present as a single-copy gene, and yet there were other PLD or PLD-related sequences in the castor bean genome.