Infected non-union of a femur treated with a vascularized fibular graft.
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Adult↗
Biomedical subjects
W A Scott
Publications and source records attributed to W A Scott.
Human monocyte-endothelial cell interaction in vitro.
We have examined the interaction of freshly isolated human blood monocytes with cultured human umbilical vein endothelial cells in vitro. Purified monocytes incubated with confluent primary or passaged endothelial cells (EC) for 90 min at 37 degrees C bound at maximal densities of 6.5-7.0 X 10(3)/mm2 (8 or 9 per EC) without causing disruption of the monolayer. Monocyte-EC binding proceeded in the presence of plasma proteins or optimal phagocytic doses of opsonized zymosan particles. The avidity of attachment was not diminished by alternative monocyte isolation techniques. Monocyte attachment to EC was dependent upon the presence of divalent cations (magnesium greater than calcium) and was inhibited at 4 degrees C. Monocytes selectively bound to EC when incubated with monolayers composed of smooth muscle cells and EC. Neither EC monolayer confluence nor a variety of EC culture conditions affected the high levels of monocyte binding. In contrast, human neutrophils (less than 1 per EC) and lymphocytes (less than 2-3.5 per EC) bound at lower maximal densities under the same conditions, while platelet reactivity remained minimal. The distinctively higher affinity of human blood monocytes relative to other circulating white cells for binding to cultured human EC may have relevance to their function in vivo.
Cell Adhesion↗
Role of specific simian virus 40 sequences in the nuclease-sensitive structure in viral chromatin.
A nuclease-sensitive region forms in chromatin containing a 273-base-pair (bp) segment of simian virus 40 DNA encompassing the viral origin of replication and early and late promoters. We have saturated this region with short deletion mutations and compared the nuclease sensitivity of each mutated segment to that of an unaltered segment elsewhere in the partially duplicated mutant. Although no single DNA segment is required for the formation of a nuclease-sensitive region, a deletion mutation (dl45) which disrupted both exact copies of the 21-bp repeats substantially reduced nuclease sensitivity. Deletion mutations limited to only one copy of the 21-bp repeats had little, if any, effect. A mutant (dl135) lacking all copies of the 21- and 72-bp repeats, while retaining the origin of replication and the TATA box, did not exhibit a nuclease-sensitive region. Mutants which showed reduced nuclease sensitivity had this effect throughout the nuclease-sensitive region, not just at the site of the deletion, indicating that although multiple determinants must be responsible for the nuclease-sensitive chromatin structure they do not function with complete independence. Mutant dl9, which lacks the late portion of the 72-bp segment, showed reduced accessibility to BglI, even though the BglI site is 146 bp away from the site of the deletion.
Base Sequence↗
Implant-induced trabecular damage in cadaveric femoral necks.
After insertion of Newman's pins, Smith-Petersen nails and compression hip screws into human cadaveric proximal femora, the area of trabecular damage was measured. The nail and hip screw caused four times the area of damage seen with Newman's pin. The Smith Petersen nail caused significant peripheral damage which was undetectable radiographically.
Bone Nails↗
gamma-Interferon enhances the secretion of arachidonic acid metabolites from murine peritoneal macrophages stimulated with phorbol diesters.
Macrophage activation in vivo has been associated with qualitative and quantitative alterations in the release and metabolism of arachidonic acid. In the present study, we examined the effect of in vitro macrophage activation with recombinant gamma-interferon (IFN-gamma) on arachidonic acid secretion induced by exposure to a variety of stimulating agents. Secretion stimulated by challenge with unopsonized zymosan, insoluble immune complexes, the calcium ionophore A23187, or combinations thereof was unaltered in IFN-gamma-treated macrophages. However, when phorbol diesters active as tumor promoters were employed as challenge agents, arachidonate secretion was enhanced as much as 10-fold over that seen in nonactivated controls. The enhanced secretory response to PMA was detectable as early as 1 hr after exposure to IFN-gamma, reached a maximum within 3 to 6 hr, and subsequently declined to control levels even in the continued presence of the agent. Treatment with IFN-gamma did not alter the pattern of individual metabolites produced by macrophages challenged with either zymosan or PMA. Finally, the sensitivity to phorbol diesters was also increased by treatment with IFN-gamma (ED50 reduced from 35 ng/ml to 4 ng/ml). Thus, IFN-gamma could prime macrophages for a substantially amplified response to phorbol esters. Because the cellular mediator of PMA action has been identified as a Ca++, phospholipid-dependent protein kinase, a role for this enzyme in macrophage functional development is indicated.
Adjuvants, Immunologic↗
Salmonellosis in an African elephant.
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Animals↗
Stimulation of human endothelial cell prostacyclin synthesis by select leukotrienes.
Cultured endothelial cells from human umbilical cord labeled with [3H]20:4 release radiolabel when exposed to leukotrienes C or D (LTC or LTD). The major radiolabeled 20:4 metabolite recovered in the culture medium was prostacyclin. Both leukotrienes produced a dose-dependent synthesis of prostacyclin, with a maximal response at 10(-7) M leukotriene. LTC promoted a twofold greater response than did LTD at all concentrations tested (10(-9) to 10(-7) M). In contrast, no release of radiolabel above basal levels was evident with a challenge of LTE or LTB at the same concentrations. Endothelial cells metabolize approximately 40-50% of exogenously supplied LTC to LTD and LTE in 60 min. Levels of alpha-glutamyltranspeptidase (gamma-GTPase), the ectoenzyme reported to convert LTC or LTD, were detected in intact endothelial cells with the chromogenic substrate L-gamma-glutamyl-p-nitroanilide at levels sufficient to account for the observed rate of LTC metabolism. High concentrations of the gamma-GTPase inhibitors, glutathione and AT-125, blocked the metabolism of LTC by endothelium. These results suggest that degradation of leukotrienes by endothelium may be one mechanism for inactivation of these lipid mediators.
Arachidonic Acid↗
Endocytosis in Entamoeba histolytica. Evidence for a unique non-acidified compartment.
Our studies on endocytosis in Entamoeba histolytica trophozoites suggest that there are two vacuolar compartments in this organism. The first compartment consists of large vacuoles (greater than 2 microns diameter). As measured by the fluid phase markers, fluorescein isothiocyanate (FITC)-dextran and horseradish peroxidase (HRP), this compartment is a rapid equilibrium with the external milieu and is constantly exchanging (1-2 h) its contents with the external medium. The contents of these vacuoles are not acidified. This together with the absence of degradation of fluid phase markers clearly differentiates these vacuoles from lysosomes of eucaryotes. By labeling externally disposed peptides on the surface membrane of trophozoites with 125I, we could show that the surface membrane was rapidly internalized over a 2-h period and then reached a plateau. All major 125I surface proteins, with the exception of a set of peptides in the 40,000 molecular weight range, were interiorized and approximately 60% of the total radiolabel were found to be in the internal membrane fraction at any given time. The kinetics of this process were similar to those for the uptake of fluid phase markers and are best explained by cycling of the surface membrane into the vacuolar compartment(s) and then back to the cell surface. The second vacuolar compartment consisted of small vesicles (less than 2 microns diameter) with acidified contents as indicated by acridine orange uptake. The endocytic nature of these vesicles was shown by their slow (days) labeling with FITC-dextran, and spectral analysis of internalized FITC-dextran confirmed that this second compartment is acidified (pH 5.2).
Acridine Orange↗
The case for an open approach to tibial nailing.
Medullary nailing of the tibia without exposing the fracture site is a technically possible procedure. In order to test if it is a superior method to open nailing, a retrospective study of 55 cases was undertaken; 35 had been nailed by the closed technique and 20 by open operation. All fractures united. Knee movements were full unless there had also been other injuries. Complications were common; infection occurred in 20 per cent of open nailings and in 31 per cent of closed nailings . Twenty per cent of the tibias showed rotational deformities and 9 per cent of patients developed peroneal nerve palsy. Complications occurred less frequently in cases operated on by an open technique. It is suggested that opening the fracture site may reduce the complication rate.
Adolescent↗
The cyclooxygenase and lipoxygenase activities of platelet-depleted human monocytes.
The significant numbers of platelet contaminants bound to the surface of freshly isolated human monocytes can be removed during incubations in fresh human serum. We used cells purified by these means to examine the cyclooxygenase and lipoxygenase metabolites of arachidonic acid synthesized in response to various stimuli. Thromboxane is the single major product synthesized by human monocytes in response to inflammatory particles (opsonized zymosan or immunoglobulin G complex-coated beads). The molar amounts produced were equivalent to those by platelets stimulated with thrombin. In contrast, exposure to calcium ionophore A23187 led to greatly enhanced lipoxygenase metabolite synthesis. Under these conditions, levels of leukotriene B4 (30 to 70 pmol/10(6) cells) and leukotriene C4 (up to 20 pmol/10(6) cells) production were five- to tenfold higher than those observed with particulate stimuli. The circulating human monocyte is a source of a variety of vasoactive mediators derived from arachidonic acid.
Arachidonate Lipoxygenases↗
Effect of leukotrienes on endothelium and the transendothelial migration of neutrophils.
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Animals↗
A selective defect in arachidonic acid release from macrophage membranes in high potassium media.
Murine peritoneal macrophages cultured in minimal essential medium (alpha-MEM; 118 mM Na+, 5 mM K+) released arachidonic acid (20:4) from phospholipids on encountering a phagocytic stimulus of unopsonized zymosan. In high concentrations of extracellular K+ (118 mM), 3H release from cells prelabeled with [3H]20:4 was inhibited 80% with minimal reduction (18%) in phagocytosis. The inhibitory effect of K+ on 20:4 release was fully reversed on returning cells to medium containing Na+ (118 mM). Preingestion of zymosan particles by macrophages maintained in high K+ medium resulted in cells being "primed" for 20:4 release, which was only effected (without the further addition of particles) by changing the medium to one containing Na+. In contrast, 20:4 release from cells stimulated with the calcium ionophore A23187 was unimpaired by the elevated K+ medium, suggesting no direct effect of high K+ on the phospholipase. Macrophages stimulated with zymosan in alpha-MEM metabolized the released 20:4 to prostacyclin, prostaglandin E2 (PGE2), and leukotriene C (LTC). The smaller quantity of released 20:4 in high K+ medium was recovered as 6-Keto-PGF1 alpha, the breakdown product of prostacyclin, and PGE2. No LTC was synthesized. In high K+, resting (no zymosan) macrophages synthesized hydroxyeicosatetraenoic acids from exogeneously supplied 20:4 in proportions similar to cells maintained in alpha-MEM. These findings and the similarity of products (including LTC) produced by A23187 stimulated cells in alpha-MEM and high K+ medium indicated that the cyclooxygenase and lipoxygenase pathway enzymes were not directly inhibited by high extracellular K+. We conclude that high concentrations of extracellular K+ uncouple phagocytosis of unopsonized zymosan from the induction of the phospholipase responsible for the 20:4 cascade and suggest that the lesion is at the level of signal transduction between the receptor-ligand complex and the phospholipase.
Animals↗
Barriers to nuclease Bal31 digestion across specific sites in simian virus 40 chromatin.
A portion of the nucleoprotein containing viral DNA extracted from cells infected by simian virus (SV40) is preferentially cleaved by endonucleases in a region of the genome encompassing the origin of replication and early and late promoters. To explore this nuclease-sensitive structure, we cleaved SV40 chromatin molecules with restriction enzymes and digested the exposed termini with nuclease Bal31. Digestion proceeded only a short distance in the late direction from the MspI site, but some molecules were degraded 400 to 500 base pairs in the early direction. By comparison, BglI-cleaved chromatin was digested for only a short distance in the early direction, but some molecules were degraded 400 to 450 base pairs in the late direction. These barriers to Bal31 digestion (bracketing the BglI and the MspI sites) define the borders of the same open region in SV40 chromatin that is preferentially digested by DNase I and other endonucleases. In a portion of the SV40 chromatin, Bal31 could not digest through the nuclease-sensitive region and reached barriers after digesting only 50 to 100 base pairs from one end or the other. Chromatin molecules that contain barriers in the BglI to MspI region are physically distinct from molecules that are open in this region as evidenced by partial separation of the two populations on sucrose density gradients.
Chromatin↗
DNA replication and chromatin structure of simian virus 40 insertion mutants.
Insertion of DNA segments into the nuclease-sensitive region of simian virus 40 alters both replication efficiency and chromatin structure. Mutants containing large insertions between the simian virus 40 origin of replication (ori site) and the 21-base-pair repeated sequences replicated poorly when assayed by transfection into COS-1 cells. Replication of mutants with shorter insertions was moderately reduced. This effect was cis-acting and independent of the nucleotide sequence of the insert. The nuclease-sensitive chromatin structure was retained in these mutants, but the pattern of cleavage sites was displaced in the late direction from the ori site. New cleavage sites appeared within the inserted sequences, suggesting that information specifying the nuclease-sensitive chromatin structure is located on the late side of the inserts. Accessibility to BglI (which cleaves within the ori site) was reduced in the larger insertion mutants. These results support the conclusion that efficient function of the viral origin of replication is correlated with its proximity to an altered chromatin structure.
Animals↗
Club foot. Observations on the surgical anatomy of dorsiflexion.
Dorsiflexion has been studied in three normal feet and in three feet with talipes equinovarus to determine the anatomical features which might contribute to the failure of operative treatment to correct the deformity. In the normal feet the movement of dorsiflexion was found to be essentially rotatory in nature and not simply hinging; as dorsiflexion proceeds the fibula moves forwards relative to the os calcis and the calcaneal tendon. In the club feet a posterolateral tether was found; this prevented fibular movement and blocked dorsiflexion. As a result of this study a posterior and lateral release is advocated for the operative correction of the hindfoot in a child with a club foot deformity, particularly under the age of a year.
Clubfoot↗
Arachidonic acid metabolism by human monocytes. Studies with platelet-depleted cultures.
Purified human monocytes release and metabolize endogenous arachidonic acid (20:4) from phospholipid stores when challenged with particulate inflammatory stimuli or the calcium ionophore A23187. Using radiolabeled cultures, the percentage of total [3H]20:4 released was similar with each type of stimulus. However, the spectrum of 20:4 metabolites differed. With opsonized zymosan (OpZ) or Sephadex beads coated with IgG immune complexes (Ig-beads), the predominant product was thromboxane (25% of the total) together with smaller amounts of other cyclo-oxygenase products and lipoxygenase metabolites. Levels of thromboxane synthesis by monocytes were comparable to those by platelets, as measured by radioimmunoassay. In contrast, exposure to the nonspecific agent A23187 led to mainly lipoxygenase products (70% of the total). Monocytes isolated from mononuclear cell fractions of peripheral blood contain platelets specifically rosetted to their surfaces. These platelet contaminants were removed by sequential incubations of monocytes in serum and EDTA followed by adherence and detachment from tissue culture vessels. The presence of platelets in routinely isolated monocytes presented a major difficulty in the study of human monocyte 20:4 metabolism since platelets also synthesize thromboxane. Loss of 12-HETE synthesis (16-fold reduction relative to 5-HETE) in A23187-stimulated cultures provided a convenient measure of platelet depletion. This together with the response to monocyte-specific stimuli (OpZ and Ig-beads) allowed for the distinction between monocyte and platelet 20:4 metabolism.
Arachidonic Acid↗
Cyclic AMP levels in relation to membrane phospholipid variations in Neurospora crassa.
The correlation between membrane phospholipid composition and total cyclic AMP levels was investigated by using Neurospora lipid auxotrophs under various supplementation conditions. The lipid composition of the supplemented cultures was determined, and the intracellular and extracellular cyclic AMP levels were measured at various stages of the culture growth. Kinetic parameters and the thermostability of adenylate cyclase and of cyclic AMP-dependent phosphodiesterase were measured under all supplementation conditions. In inositol deficient inl cultures the levels of intracellular cyclic AMP decreased exponentially towards the end of the log phase and thereafter. In chol-l; chol-2 cultures, grown in N-monomethylethanolamine and low choline supplementation, the level of intracellular cyclic AMP decreased as function of decreasing exogenous choline supplement. Rates of cyclic AMP extrusion in all cultures were comparable on dry weight basis, and thus not affected by the mycelial lipid composition. Adenylate cyclase activity and thermostability decreased under those supplementation conditions resulting in reduction of cyclic AMP. Cyclic AMP-dependent phosphodiesterase was insensitive to phospholipid changes. Accordingly, it is suggested that specific perturbations in cellular phospholipid composition affect the membrane-bound adenylate cyclase and hence the cyclic AMP synthesis in vivo.
3',5'-Cyclic-AMP Phosphodiesterases↗
Leukotriene C promotes prostacyclin synthesis by human endothelial cells.
Cultured endothelial cells from human umbilical vein were labelled with [3H]arachidonic acid for 16 hr. The radiolabel was localized primarily in phospholipids (93%) and 73% was distributed equally between phosphatidylcholine and phosphatidylethanolamine. Leukotriene C (10-1,000 nM) promoted a dose-dependent release of radiolabel into the culture medium. This response was 3.3 times control values at 100 nM. The major arachidonic acid metabolite synthesized was prostacyclin, which was 33% of the total released radiolabel. Endothelial cells also released small amounts of prostaglandin F2 alpha (6.1%), unidentified lipoxygenase products (14.8%), and unreacted arachidonic acid (33%). The 30-min time course of release was independent of the leukotriene C concentration used. Leukotriene D at similar concentrations also promoted endothelial cells to release primarily prostacyclin and unreacted arachidonic acid. The release of prostacyclin, a potent vasodilator agent, may be an important mediator in slow reacting substance effects on the vasculature.
Arachidonic Acid↗