Reduction of laccase type 1 copper by 3,4-dihydroxyphenylalanine and other catechol derivatives

3,4-Dihydroxyphenylalanine (DOPA) is not a preferred substrate of Rhus vernicifera laccase, as rate constants for the anaerobic reduction of the type 1 cupric atom by L-DOPA (6.3 X 10(1) M-1 s-1), D-DOPA (2.6 X 10(1) M-1 s-1), and L-DOPA methyl ester (2.6 X 10(1) M-1 s-1) are considerably smaller than k1 (catechol) (7 X 10(2) M-1 s-1) and rate constants characteristic of numerous other nonphysiological organic substrates (25 degrees C, pH 7.0, I = 0.5 M). The reactions of DOPA derivatives with laccase are unique, however, in that a two-term rate law pertains: kobsd = k0 + k1[phenol]; k0(L-DOPA) = 7 X 10(-2) s-1. The reactivities of other catechol derivatives (pyrogallol, gallic acid, and methyl gallate) with laccase type 1 copper were also examined.     

Role of fructose 2,6-bisphosphate in the regulation of glycolysis and gluconeogenesis in chicken liver

Glucagon and dibutyryl cyclic AMP inhibited glucose utilization and lowered fructose 2,6-bisphosphate levels of hepatocytes prepared from fed chickens. Partially purified preparations of chicken liver 6-phosphofructo-1-kinase and fructose 1,6-bisphosphatase were activated and inhibited by fructose 2,6-bisphosphate, respectively. The sensitivities of these enzymes and the changes observed in fructose 2,6-bisphosphate levels are consistent with an important role for this allosteric effector in hormonal regulation of carbohydrate metabolism in chicken liver. In contrast, oleate inhibition of glucose utilization by chicken hepatocytes occurred without change in fructose, 2,6-bisphosphate levels. Likewise, pyruvate inhibition of lactate gluconeogenesis in chicken hepatocytes cannot be explained by changes in fructose 2,6-bisphosphate levels. Exogenous glucose caused a marked increase in fructose 2,6-bisphosphate content of hepatocytes from fasted but not fed birds. Both glucagon and lactate prevented this glucose effect. Fasted chicken hepatocytes responded to lower glucose concentrations than fasted rat hepatocytes, perhaps reflecting the species difference in hexokinase isozymes.     

Synthesis and maturation of cross-reactive glycoprotein in fibroblasts deficient in arylsulfatase a activity

The biosynthesis of arylsulfatase A was studied in cultured fibroblasts by pulse-chase labeling with [2-³H]mannose; the enzyme was isolated by immunoprecipitation and denaturing polyacrylamide gel electrophoresis. In normal fibroblasts, and in fibroblasts from a patient with multiple sulfatase deficiency, the enzyme was synthesized as a glycoprotein of apparent molecular weight of 59,000; half of it was processed over a period of 4 days to M_r= 57,000. The precursor chain of M_r= 59,000 was secreted in the presence of 10 mM NH₄Cl. An immunoprecipitable glycoprotein of normal size was synthesized by fibroblasts from two unrelated patients with metachromatic leukodystrophy, but this material disappeared within twenty hours. In fibroblasts from an individual with pseudodeficiency of arylsulfatase A, the immunoprecipitable precursor glycoprotein was smaller (M_r= 56,000). The synthesis of cross-reactive proteins with altered properties supports the concept of allelic mutations as the genetic basis of metachromatic leukodystrophy and of arylsulfatase A pseudodeficiency.     

The influence of contact guidance on chemotaxis of human neutrophil leukocytes

Chemotaxis of human neutrophil leukocytes moving on or in aligned 3D fibrin gels is more efficient if the cells are moving along the axis of fibre alignment than if they have to cross the fibres. This was shown by using two assays, one in which the cells were responding to a distant (600 micrometers) gradient source diffusing from a filter paper impregnated with formyl-Met-Leu-Phe and incorporated into the gel, the other in which the cells were responding to nearby (20--30 micrometers) Candida albicans spores in serum. In the former assay, impairment of chemotaxis across the axis of fibre alignment was highly significant. In the latter, cells showed efficient chemotaxis to the spores, but took more irregular paths when crossing the aligned fibres than when running along them. Neutrophils show contact guidance in aligned collagen or fibrin gels (Wilkinson et al., Exp cell res 140 (1982) 55) [1], thus the cells were subjected simultaneously to two directional cues in these experiments, one the chemotactic gradient and the other a contact guidance field. These cues may reinforce or interfere with each other depending on their relative orientation. Since many tissues in vivo show alignment or more complex forms of patterning, tissue architecture is likely to be an important determinant of the efficiency of cellular mobilization in inflamed or infected sites.     

Mitogenic response to epidermal growth factor (EGF) modulated by platelet-derived growth factor in cultured fibroblasts

The mitotic effects of epidermal growth factor (EGF) were investigated in two cultured fibroblast lines, BALB/c-3T3 and C3H 10T1/2 cells. EGF (30 ng/ml) added to quiescent 3T3 cells in medium containing either platelet-poor plasma or 10(-5) M insulin caused only minimal increases in the percentage of cells stimulated to initiate DNA synthesis. In contrast, EGF acted synergistically with either insulin or plasma to stimulate DNA synthesis in quiescent cultures of 10T1/2 cells, although the maximum effects of EGF were measured at concentrations several-fold greater than those found in either serum or plasma. In either 3T3 or 10T1/2 cells a transient preexposure to platelet-derived growth factor (PDGF) caused over a 10-fold increase in the sensitivity to the mitogenic effects of EGF. It is therefore possible that a primary action of PDGF is to increase the sensitivity of fibroblasts to EGF, independent of whether EGF alone is found to be mitogenic.     

Formation of vinculin plaques precedes other cytoskeletal changes during retinoic acid-induced teratocarcinoma cell differentiation

Immunofluorescence and immunoblotting techniques were used to study the presence and distribution of vimentin and keratin type intermediate filaments, actin, and vinculin (130 kD protein) during retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma (EC) cells. The undifferentiated F9 cells regularly expressed vimentin, usually concentrated close to the nucleus, but not keratin. Actin appeared as short intracellular filaments and as spikes at the edges of the colonies, together with some diffuse cytoplasmic staining. F9 cells also showed a weak, diffuse cytoplasmic vinculin-specific fluorescence in addition to occasional small focal vinculin patches at the edges of the cell colonies. RA treatment led into a series of changes in the cytoskeletal organization of F9 cells. These changes were initiated by the appearance of distinct vinculin plaques and followed by formation of actin stress fibers and by profound changes in the organization of vimentin in the flattening cells. RA treatment finally led to the appearance and co-expression of keratin fibrils in many of the vimentin-containing F9 cells. This sequence of changes suggests that the vinculin-containing adhesion plaques may be important in the mechanism of RA-induced differentiation of EC cells.     

Maintenance and reversibility of active albumin secretion by adult rat hepatocytes co-cultured with another liver epithelial cell type

When adult rat hepatocytes were co-cultured with another liver epithelial cell type in a medium supplemented or not with fetal calf serum (FCS), it was found that 1. They survived for more than 2 months 2. Albumin secretion levels remained high over the whole culture period 3. Decreased secretion might be reversed 4. This protein secretion activity appeared to be dependent upon both the presence of cell-cell contacts and the production of an extracellular material. The results demonstrate for the first time long-term stabilization and reversibility of a specific function (albumin secretion) at high levels by adult hepatocytes cultured in serum-free medium and suggest that both the presence of other liver cell type(s) and the production of an extracellular matrix are needed for the maintenance of specific functions in cultured hepatocytes.     

Association of ganglioside-protein conjugates into cell and Sendai virus. Requirement for the HN subunit in viral fusion

We have prepared several electron and light microscopic labels of epidermal growth factor (EGF) to analyse the morphologic features of its binding and internalization by cultured cells. These include a ferritin conjugate of EGF, a covalent conjugate of EGF and horseradish peroxidase (EGF-HRP), a colloidal gold marker system using EGF-HRP as a primary antigen, and a covalent complex of EGF with rhodamine-labelled lactalbumin. All of the light and electron microscopic labels showed similar patterns of binding. EGF initially bound to diffusely distributed cell surface receptors at 4 degrees C. The EGF-receptor complexes clustered into clathrin-coated pits on the cell surface only when the temperature was raised to 37 degrees C. In KB and Swiss 3T3 cells, this was followed by rapid internationalization into receptosomes, compartmentalization into the Golgi system, clustering in the clathrin-coated regions of the Golgi, and finally delivery into lysosomes from the Golgi. This general pathway was seen in Swiss 3T3 cells which have a low number of EGF receptors, KB cells which have a moderate number of receptors and A431 cells that have a high number of receptors. However, the ruffling activity induced in A431 cells by EGF produced some internalization through macropinosomes, making the pathway of entry more difficult to evaluate. Double label experiments showed that EGF is internalized together with alpha 2-macroglobulin and adenovirus particles. These data clarify the route of entry of EGF in different cell types using multiple labels, and shows that it enters cells through the same coated pit entry pathway as most other ligands previously examined.     

The effect of concanavalin A on egestion of food vacuoles in Tetrahymena

The ability of concanavalin A (conA) to disrupt food vacuole elimination at the cytoproct of Tetrahymena pyriformis, strain GL-C, was investigated using fluorescence microscopy and thin section electron microscopy. ConA was found to induce "tails" in Tetrahymena. These tails were specifically stained by fluorescent conA. Thin section observations of conA-treated cells revealed that these tails were the result of abnormal egestion of food vacuole contents at the cytoproct. Tail formation appears to result from an inhibition of endocytosis of food vacuole membrane during egestion. Instead, the food vacuole membrane appears to be cast out of the cell, along with the contents of the vacuole. The mechanism of this inhibition may be related to an apparent absence of microtubules or microfilamentous mat in the cytoproct region of conA-treated cells. Although conA is ingested into food vacuoles in large amounts, conA appears to affect endocytosis only from outside the cell; ingested conA does not appear to be effective. ConA may exert its influence by binding to the cytoproct region. The ability of conA to induce tail formation is inhibited by sugars specific to it. Numerous membranous vesicles are found in association with the oral cilia and cytoproct region of conA-treated cells. These vesicles may be the conA-binding material reported to be secreted by Tetrahymena.