Role of plasminogen, plasmin, and plasminogen activators in the migration of fibroblasts into plasma clots

Human diploid fibroblasts were seeded onto or into plasma clots and different aspects of cell adhesion and migration were measured. The roles of plasminogen activators and plasmin were studied by either the removal of plasminogen from plasma prior to clotting or by the addition of 10 mM epsilon-aminocaproic acid, which brings about an inhibition of plasmin in this system. When cells were seeded onto the surface of plasma clots, rates of attachment, spreading, and migration were unaffected by plasminogen depletion or plasmin inhibition. In contrast, when cells were seeded into plasma clots, then, although the rates of cells spreading were unaffected, cell migration was abolished by plasminogen depletion or by plasmin inhibition. When cells were seeded onto the surface of plasma clots and the rate of migration into the clots was measured, there was an absolute requirement for plasmin activity; while fibroblasts migrated rapidly into the fibrin lattice of control clots, in the case of plasminogen-depleted clots, cells failed to penetrate the lattice. Focussing through a plasma clot revealed that fibroblasts do not migrate through the fibrin lattice but instead, localized areas of fibrinolysis are generated and cells migrate over the surface of the area of lysis.     

Measurement of serum glycosylated proteins and glycosylated low density lipoprotein fraction in diabetes mellitus

A simple method was developed for estimating serum glycosylated protein levels using gel filtration with Bio-Gel P6 by determining the protein and sugar content in the void volume fraction. The glycosylated protein levels (GSP) correlated well with fasting blood sugar levels and glycosylated albumin level (G-ALB) determined by affinity chromatography with Blue Sepharose CL6B. The glycosylation level of heparin-citrate precipitable fraction of serum which predominantly contained low density lipoprotein (G-LDL) also correlated well with GSP and LDL-cholesterol levels. Significantly different values were obtained for GSP, G-ALB, and G-LDL between normals and diabetics.     

Factors Involved in Hydrolysis of Microcrystalline Cellulose by Acetivibrio cellulolyticus

Acetivibrio cellulolyticus cellulase obtained by the water elution of residual cellulose from the growth medium was compared with the cellulase activity present in culture supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that water elution released most of the protein bands which adhered to undigested cellulose from the culture medium. The enzyme in the culture supernatant and that eluted from residual cellulose had specific activities for Avicel hydrolysis that were 20- to 40-fold greater than that of Trichoderma reesei cellulase. However, Ca²⁺ and a reducing agent such as dithiothreitol were required for maximum Avicel hydrolysis rates by these A. cellulolyticus enzyme preparations. The effect of these agents on p-nitrophenyl lactopyranoside hydrolysis suggested that they were required by an exoglucanase component. Supernatant enzyme preparations contained large amounts of carbohydrate which was separated from most of the cellulase protein by phenyl-Sepharose chromatography. Removal of this carbohydrate, which interfered with protein fractionations, allowed for an activity stain analysis of the supernatant enzyme.     

Rat liver mitochondria contain two immunologically distinct dihydrolipoamide dehydrogenases

We have raised antisera against dihydrolipoamide dehydrogenase. One antigen was isolated from purified bovine kidney pyruvate dehydrogenase complex (PDC). The other antigen was a commercial preparation of porcine heart dihydrolipoamide dehydrogenase (E3) which did not first involve purification of the alpha-keto acid dehydrogenase complex(es). Both antibody preparations cross-reacted with the E3 components of PDC, alpha-ketoglutarate dehydrogenase complex, and branched-chain keto acid dehydrogenase complex. This demonstrates the immunological identity of the E3 components. These sera totally precipitated E3 activity from the purified complexes, from purified preparations of E3, and from extracts of rat heart and kidney mitochondria. The two sera vary in their reaction with rat liver mitochondrial extracts: the anti PDC-E3 serum left residual E3 activity (approximately 50% of the original) that was precipitable by the anti-E3 anti-serum. This indicates that liver contains two immunologically distinct forms of E3. Metabolic assays measuring the differential effects of the two sera on the glycine decarboxylation reaction suggest that the form which is immunologically nonreactive with the anti-PDC-E3 serum could represent the E3 involved in the glycine cleavage system.     

Concanavalin A prevents phorbol-mediated redistribution of protein kinase C and beta-adrenergic receptors in rat glioma C6 cells

Exposure of rat glioma C6 cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused an activation of protein kinase C wherein the enzyme rapidly became membrane-bound (T 1/2 of 15 min). This translocation of protein kinase C from cytosol to membrane was followed by a sequestration of cell surface beta-adrenergic receptors and a loss of isoproterenol-stimulated adenylate cyclase activity. We had reported previously that prior exposure of rat glioma cells to concanavalin A prevents the TPA-mediated sequestration of receptors and desensitization of adenylate cyclase (Kassis et al., 1985). We now show that the concanavalin A treatment also prevents the translocation and activation of protein kinase C. These results are further evidence that in the TPA-treated cells, sequestration of beta-adrenergic receptors is mediated by membrane-bound protein kinase C.     

Restriction endonucleases for pulsed field mapping of bacterial genomes.

Fundamental to many bacterial genome mapping strategies currently under development is the need to cleave the genome into a few large DNA fragments that can be resolved by pulsed field gel electrophoresis. Identification of endonucleases that infrequently cut a genome is of key importance in this process. We show that the tetranucleotide CTAG is extremely rare in most bacterial genomes with G+C contents above 45%. As a consequence, most of the sixteen bacterial genomes we have tested are cleaved less than once every 100,000 base pairs by one or more endonucleases that have CTAG in their recognition sequences: Xba I (TCTAGA), Spe I (ACTAGT), Avr II (CCTAGG) and Nhe I (GCTAGC). Similarly, CCG and CGG are the rarest trinucleotides in many genomes with G+C content of less than 45%. Thus, Sma I (CCCGGG), Rsr II (CGGWCCG), Nae I (GCCGGC) and Sac II (CCGCGG) are often suitable endonucleases for producing fragments that average over 100,000 base pairs from such genomes. Pulsed field gel electrophoresis of the fragments that result from cleavage with endonucleases that cleave only a few times per genome should assist in the physical mapping of many prokaryotic genomes.     

Expression of engrailed proteins in arthropods, annelids, and chordates

engrailed is a homeobox gene that has an important role in Drosophila segmentation. Genes homologous to engrailed have been identified in several other organisms. Here we describe a monoclonal antibody that recognizes a conserved epitope in the homeodomain of engrailed proteins of a number of different arthropods, annelids, and chordates; we use this antibody to isolate the grasshopper engrailed gene. In Drosophila embryos, the antibody reveals engrailed protein in the posterior portion of each segment during segmentation, and in a segmentally reiterated subset of neuronal cells during neurogenesis. Other arthropods, including grasshopper and two crustaceans, have similar patterns of engrailed expression. However, these patterns of expression are not shared by the annelids or chordates we examined. Our results provide the most comprehensive view that has been obtained of how expression patterns of a regulatory gene vary during evolution. On the basis of these patterns, we suggest that engrailed is a gene whose ancestral function was in neurogenesis and whose function was co-opted during the evolution of segmentation in the arthropods, but not in the annelids and chordates.     

Drosophila neuroglian: a member of the immunoglobulin superfamily with extensive homology to the vertebrate neural adhesion molecule L1

Drosophila neuroglian is an integral membrane glycoprotein that is expressed on a variety of cell types in the Drosophila embryo, including expression on a large subset of glial and neuronal cell bodies in the central and peripheral nervous systems and on the fasciculating axons that extend along them. Neuroglian cDNA clones were isolated by expression cloning. cDNA sequence analysis reveals that neuroglian is a member of the immunoglobulin superfamily. The extracellular portion of the protein consists of six immunoglobulin C2-type domains followed by five fibronectin type III domains. Neuroglian is closely related to the immunoglobulin-like vertebrate neural adhesion molecules and, among them, shows most extensive homology to mouse L1. Its homology to L1 and its embryonic localization suggest that neuroglian may play a role in neural and glial cell adhesion in the developing Drosophila embryo. We report here on the identification of a lethal mutation in the neuroglian gene.     

Skinned muscle fibers of Aplysia: potentiation of contraction by "background" calcium and lack of effect of cyclic AMP

1. Aplysia buccal muscle E1 can be skinned with saponin in a low ionic strength medium. Pulses of calcium, which were ineffective at causing contraction in intact fibers, elicited contraction in skinned fibers. 2. Tension in skinned fibers increased at [Ca2+] greater than 10(-7) M and was maximal at 6 x 10(-7) M. 10(-5) M [Ca2+] caused irreversible damage to the fibers. 3. Fibers did not exhibit "catch", i.e. they relaxed quickly upon removal of calcium. 4. Optimal pH for tension was 7.0. 5. Contractile responses to calcium pulses were increased by raising "background" [Ca2+] to 10(-7) M. 6. Cyclic AMP (10(-4) and 10(-3) M) had no effect on tension.     

Central noradrenergic activity in intact and sinoaortic denervated Dahl rats

Lesions in forebrain areas richly innervated by noradrenergic terminals and involved in cardiovascular function reduce or prevent hypertension in the Dahl salt-sensitive (S) rats fed a high (H) salt diet. This led us to examine two questions. (1) Is the noradrenergic activity altered in discrete forebrain and brainstem areas of SH rats? (2) Are these changes in noradrenergic activity eliminated by sinoaortic denervation (SAD)? Studies were done in 10-week-old female SH and Dahl salt-resistant (RH) rats. Half of the rats in each group had SAD surgery 1 week prior to study. An index of norepinephrine (NE) turnover was determined by measuring the decline in tissue NE concentration 8 h after administering alpha-methyl-p-tyrosine, a NE synthesis blocker, to animals from each of four groups: sham-RH, SAD-RH, sham-SH, and SAD-SH (n = 18-20 per group). Various discrete brain areas were obtained using the "punch technique." In SH rats the index of NE turnover was increased in the median preoptic nucleus and decreased in the paraventricular nucleus compared with RH rats regardless of SAD. In contrast, in SH rats the index of NE turnover was increased in the supraoptic nucleus and locus ceruleus compared with RH rats; however, SAD-RH had greater turnover of NE at these sites than SAD-SH. In summary, changes in noradrenergic activity in the median preoptic nucleus and the paraventricular nucleus may be related to genetic predisposition to hypertension in SH rats. In contrast, changes in the locus ceruleus and the supraoptic nucleus of SH rats may be related to impaired baroreflexes and thereby contribute to hypertension.