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Biomedical subjects

N A Sharkey

Publications and source records attributed to N A Sharkey.

At least 55 records · Page 3Linked to original sources
PubMed1991

Biomechanical testing of new and old fixation devices for vertical shear fractures of the pelvis.

Malgaigne fractures of the pelvis have been treated with many different methods of fixation. We developed a plate for use on the anterior aspect of the sacroiliac (SI) joint using information obtained from cadaveric dissections and computed tomography (CT) scans of male (50) and female (50) pelvises. We tested each of six pelvises in the Instron, with five different fixation systems. Our results showed that the weakest system was the anterior quadrilateral frame plus two symphyseal plates. When comparing three posterior screws with the SI joint plate, the difference was not statistically significant. However, in both of these systems, a second symphyseal plate added to the overall stability.

Biomechanical Phenomena↗
PubMed1991

Anatomic and biomechanical assessment of transarticular screw fixation for atlantoaxial instability.

The purpose of this study is to elucidate anatomically the atlantoaxial transarticular screw fixation described by Magerl in 1979 and compare it biomechanically with Gallie wiring. Five human C1-C2 specimens were tested in flexion/extension and rotation intact, then after wiring and screw fixation. Mean screw length was 39 mm, 25 mm in the C2 lamina and 14 mm in the lateral mass. Angular displacement of screwed specimens was significantly less than control or wired groups. Stiffness at 0-0.5 Nm loads was significantly greater for screwed specimens than for wired or controls (101 +/- 49 Nm, 10.3 +/- 9.2 Nm, and 1.96 +/- 0.18 Nm, respectively). All specimens withstood 5 Nm in flexion and extension without failure. Screw fixation provides stability comparable to Gallie wiring and is stiffer at low-range forces and rotational angles.

Atlanto-Axial Joint↗
PubMed1989

Effects of bone ingrowth on the strength and non-invasive assessment of a coralline hydroxyapatite material.

The dependence of strength on the amount of bone growth into a hydroxyapatite material made from coral was investigated. Block and granular forms of the material were implanted into cortical and trabecular regions of the skeletons of 16 dogs. The results were examined after 4, 8, 12 and 16 wk, with four dogs in each experimental group. When implanted into cortical bone, the bending strength of the implant material was found to be highly correlated with the amount of pore space which had become occupied by bone (r = 0.92, P less than 0.005 for the block form; r = 0.84, P less than 0.005 for the granular form). Multiple regression analysis showed that six histomorphometric measures of ingrowth accounted for 96% of the variability in bending strength of the block material, and there were no significant differences between block and granular forms of the material. On the other hand, when implanted into trabecular bone, the block form of the material achieved greater compressive strength than the granular form. While both strength and ingrowth increased with time, there were poor correlations between these two variables. Finally, when the material is implanted into trabecular bone, it becomes stronger in compression than the surrounding bone; when implanted in cortical bone, linear modelling suggests that resorption and replacement of the implant would be required to approximate the bending strength of the surrounding bone.

Animals↗
PubMed1989

Comparison of octahydromezerein and mezerein as protein kinase C activators and as mouse skin tumor promoters.

Although mezerein resembles 12-O-tetradecanoylphorbol-13-acetate (TPA) in being a potent ligand for protein kinase C in vitro, its properties as a tumor promoter on mouse skin differ from those of TPA. Mezerein is a good second-stage promoter of papillomas, an inefficient complete promoter of papillomas, and an effective promoter of carcinomas. The mechanism and structural features responsible for the anomalous tumor promoting activity of mezerein are unknown. We have examined here the in vitro and in vivo activities of octahydromezerein (OHM) and compared them to those of TPA and mezerein. OHM was of interest because if it acted like mezerein it would afford a convenient route for radioactive labeling. Alternatively, if it functioned as a complete tumor promoter, it would implicate unsaturation as the critical structural feature of mezerein responsible for its altered promoting activity. Consistent with this latter possibility, we find that OHM was an effective complete tumor promoter for SENCAR mice. Moreover, the pattern and magnitude of papilloma induction, yielding a peak at 16-20 weeks followed by a decline by 30-32 weeks, resembled that for TPA; mezerein, in contrast, induced a gradual but steady increase in the number of papillomas which did not reach the OHM level by 32 weeks. The dosage of OHM for inducing a comparable degree of acute and chronic hyperplasia to that induced by mezerein was 3- to 10-fold higher. This difference agrees with the relative binding affinities to protein kinase C; the Ki for OHM was 2.7 nM, compared to 0.58 nM for mezerein.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1989

The fate of articular cartilage after transplantation of fresh and cryopreserved tissue-antigen-matched and mismatched osteochondral allografts in dogs.

The long-term success of massive osteochondral allografts depends not only on the incorporation of the transplanted articular cartilage. Osteochondral allografts are immunogenic, and, once an immune response is stimulated by exposure to donor cellular antigens, the cartilage becomes vulnerable to direct injury by cytotoxic antibodies or by lymphocytes, or to indirect injury by inflammatory mediators and enzymes induced by the immune response. To clarify the role of histocompatibility antigen-matching on the health of transplanted articular cartilage, we orthotopically implanted canine leukocyte antigen-matched and mismatched proximal osteochondral allografts of the radius, both fresh and cryopreserved, in beagles. Four groups of dogs received: (1) canine leukocyte antigen-mismatched frozen allografts, (2) canine leukocyte antigen-mismatched fresh allografts, (3) canine leukocyte antigen-matched fresh allografts, or (4) canine leukocyte antigen-matched frozen allografts. In twelve of the dogs, the contralateral leg was subjected to a sham operation, and in ten of the dogs, the proximal part of the radius was removed and replaced as an autogenous graft control. All animals were followed for eleven months after the operation and then were killed. The cartilage of the grafts was evaluated grossly, histologically, and biochemically. The biochemical analysis consisted of measurement of dry weight, content of glycosaminoglycan and hydroxyproline, and galactosamine-to-glucosamine ratios. Analyses of variance were used to study the effect of tissue antigen-matching and freezing on degradation of cartilage. During the study, no dog had grossly obvious clinical abnormalities, all host-graft interfaces healed, and no joints dislocated. The gross appearance of the cartilage was normal for both the joints that had an autogenous graft and those that were subjected to the sham operation. The cartilage of all allografts was thinned, dull, and roughened. The synovial membrane of all of the joints that had been operated on was mildly fibrotic and hyperplastic, but only that of the dogs that had an allograft was severely fibrotic and hyperplastic and demonstrated an inflammatory response. The inflammatory response was most severe in joints that had received a fresh canine leukocyte antigen-mismatched allograft. Invasive pannus was more frequent in joints that had received a fresh graft, particularly those that had received a canine leukocyte antigen-mismatched allograft, and cartilage was sometimes eroded to subchondral bone. Freezing was harmful to the cartilage. Very few cells survived the freezing procedure, and frozen grafts received s significantly worse histological scores had significantly less glycosaminoglycans and had a lower ratio of galactosamine to glucosamine than fresh grafts.

Animals↗
PubMed1988

An apparatus to measure flexor tendon excursion and angular motion of the distal interphalangeal joint in a rabbit model.

Investigators evaluating flexor tendon adhesions have utilized visual, histological, and gross mechanical methods in reporting their results. We have developed a device to measure tendon excursion and distal interphalangeal joint motion for a small animal model. The device was tested for accuracy and reproducibility using the rabbit hindfoot. There was no statistical difference between the right and the left in 30 pairs of feet. This device provides an accurate method to quantify tendon excursion and relative adhesion formation in a rabbit model without destroying the specimen.

Animals↗
PubMed1988

Modeling of the bryostatins to the phorbol ester pharmacophore on protein kinase C.

The bryostatins are macrocyclic lactones that represent an additional structural class of potent activators of protein kinase C. These marine animal biosynthetic products are of unusual interest because they induce only a subset of the biological responses induced by the phorbol esters. We have now determined the binding affinities of naturally occurring and semisynthetic bryostatins for protein kinase C by competition analysis with [26-3H]bryostatin 4 as the radioactive ligand. Esterification of the hydroxyl group at C26 caused dramatic loss of activity as did inversion of the asymmetric center at this position. In contrast, neither of the ester groups at C7 and C20 had a major influence on activity. Computer modeling of the phorbol esters, related diterpenes, and indole alkaloids suggested that the C20, C9, and C4 oxygens of phorbol represented critical elements of the phorbol ester pharmacophore. The C26 oxygen of the bryostatins, together with the C1 and C19 oxygens, gave an excellent spatial correlation with this model, with a root-mean-square deviation of 0.16 A (compared to 0.10-0.35 A among phorbol-related diterpenes). The extension of the phorbol ester pharmacophore model to the bryostatins and its agreement with the structure-activity relations for the bryostatin class of compounds provide additional support for the validity of the model.

Animals↗
PubMed1988

Effects of estrogen deficiency on the growth of tissue into porous titanium implants.

The effect of systemic deficiency of estrogen on the growth of tissue into porous titanium-fiber implants was studied in ovariectomized Beagle dogs. Five dogs were ovariectomized and five dogs had a sham operation. After waiting four months to allow the levels of circulating estrogen to decline, a titanium-alloy implant that contained four surface pads of titanium-alloy mesh was implanted in the proximal part of the humerus of each dog bilaterally. Two months later, the implants were harvested and subjected to a mechanical push-out test and quantitative histological study. The push-out strength of the implants from the ovariectomized dogs was 31 per cent less than in the control animals. Ovariectomy caused no difference in the amount of ingrowth of bone but resulted in a significant increase in the amount of fibrous connective tissue within the porous pads. The presence of this fibrous tissue appeared to have an important effect on bone-implant fixation: in the control dogs, strength correlated positively with ingrowth of bone and negatively with ingrowth of fibrous tissue, whereas in the ovariectomized dogs, strength correlated positively with ingrowth of fibrous tissue and not at all with ingrowth of bone.

Animals↗
PubMed1987

Efficacy of bone staples for fixation.

The pull-out strength of two types of bone staples (A, Techmedica; B, Richards) was evaluated in a canine model and compared with a control of 6.5 mm cancellous bone screw (Synthes). Fifteen adult mongrel dogs were divided into three groups of five animals each. The implants were placed in the lateral femoral condyle and proximal humerus on the right side at time zero, and on the left side 3 weeks later. The dogs were killed at 6 weeks. In addition a third site, the tibial tubercle, was used to examine the effectiveness of each device for patellar tendon reattachment. The strength of the reattached patellar tendon was measured and compared with a control intact patellar tendon insertion. The pull-out loads from bone of the two staple types are not statistically different. The displacement to failure, however, was found to be 71% greater (p less than 0.007), and the work to failure 128% greater (p less than 0.010) for staple A as compared with staple B. This may be of clinical relevance in avoiding staple back-out. Both staples were inferior to the screw for pull-out load displacement and work to failure (p less than 0.0001). All reattached tendons were significantly weaker (mean 202 N) than the intact tendon (mean 1,448 N) regardless of implant type. The dissected specimens uniformly showed necrosis of the tendon under the implant. Healing was by massive scar formation surrounding the implant.

Animals↗
PubMed1986

Analysis of the phorbol ester pharmacophore on protein kinase C as a guide to the rational design of new classes of analogs.

The diterpene diester phorbol 12-myristate 13-acetate and the alkaloid teleocidin B are structurally unrelated natural products that display similar potent irritant and tumor-promoting activities. Computer modeling of these and other structural classes of tumor promoters show a marked similarity in the relative positions of certain heteroatoms and hydrophobic groups. For phorbol this mapping consists of the C-4, C-9, and C-20 hydroxyl groups as well as a hydrophobic region filled by a long-chain acyl functionality attached to either the C-12 or the C-13 positions. Diacylglycerols, thought to be the endogenous activators of the major phorbol ester receptor protein kinase C likewise fit this model in a stereospecific fashion. As an initial test of the utility of the model, members of a new and simplified class of activators were synthesized that possess the predicted essential structural features. These compounds all inhibited specific phorbol ester binding to protein kinase C, albeit with low affinity (10-60 microM); further analysis of one derivative, decylhydroxylindole, confirmed that the inhibition of phorbol ester binding was competitive. This same derivative inhibited epidermal growth factor binding in intact Swiss 3T3 cells and studies with another derivative showed phosphorylation of a 40-kDa protein in platelets. Both of these in vivo responses are characteristic of phorbol esters.

Blood Platelets↗
PubMed1986

Comparison of the activity of phorbol 12-myristate 13-acetate and the diglyceride glycerol 1-myristate 2-acetate.

Diglycerides function as analogs of the phorbol ester tumor promoters. We compare here the activity of glycerol 1-myristate 2-acetate (GMA) with the corresponding phorbol 12-myristate 13-acetate (PMA). GMA inhibited phorbol ester binding to reconstituted protein kinase C, stimulated protein kinase C enzymatic activity, and, upon addition to intact 3T3 cells, inhibited [125I]EGF binding. Its potency was much less than that of PMA, however (2.9 X 10(4)-fold less for phorbol ester binding, greater than 1.2 X 10(5)-fold less for inhibition of EGF binding), and its activity on the intact cells was more transient. The marked difference in potency contrasts with the much smaller differences found previously for the corresponding pairs of dilaurate and dioleate derivatives.

Animals↗
PubMed1986

Purification of stable protein kinase C from mouse brain cytosol by specific ligand elution using fast protein liquid chromatography.

The Ca2+- and phospholipid-dependent protein kinase (protein kinase C) has been purified to electrophoretic homogeneity from mouse brain cytosol. [20-3H]phorbol 12,13-dibutyrate binding activity was found to coelute quantitatively with the protein kinase activity throughout the purification procedure. The crude extract was first run over a DE52 column. Fractions containing peak activities were then chromatographed using a fast protein liquid chromatography system with a Mono Q column followed by chromatography on the same column run in the presence of 1 mM adenosine triphosphate. The adenosine triphosphate specifically shifted the elution position of the Ca2+- and phospholipid-dependent protein kinase, providing a powerful step in the purification procedure. The remaining minor contaminants were removed by hydrophobic chromatography on a TSK-phenyl-5-PW column. This purification procedure required less than 2 days after the initial large batch DE52 column chromatography. The molecular weight of the purified receptor was estimated to be 81,000 by its mobility on sodium dodecyl sulfate/polyacrylamide gels, in agreement with the published values. Optimal conditions for the storage of the purified receptor were sought. Both protein kinase and phorbol ester binding activities were stable for 2 mo when stored in the presence of 0.01% Triton X-100 at -70 degrees C. Polyclonal antibodies to the purified receptor have been prepared from rabbits. These antibodies recognized the purified receptor in electroblotting assays and were able to immunoprecipitate the purified receptor.

Animals↗
PubMed1985

Kinetic evidence that 1,2-diolein inhibits phorbol ester binding to protein kinase C via a competitive mechanism.

Diacylglycerols inhibit binding of [20-3H]phorbol 12,13-dibutyrate ([3H]PDBu) to protein kinase C (the phorbol ester receptor). This inhibition could reflect competitive binding by the diglyceride. Alternatively, it might simply represent perturbation of the lipid environment required for binding activity. As predicted for a competitive mechanism, we report here that inhibitory concentrations of the diglyceride 1,2-diolein do not affect the off-rate of [3H]PDBu from its receptor. This behavior contrasts with that of arachidonic acid, which appears to interact via a mixed mechanism.

Arachidonic Acid↗
PubMed1985

Receptors for the phorbol ester tumour promoters.

The phorbol esters are potent tumour promotors in mouse skin and have profound effects on a wide variety of biological systems. Using the derivative [20-3H]phorbol 12,13-dibutyrate ([3H]PDBu), we demonstrated that specific receptors for the phorbol esters exist which mediate many of these biological effects. The receptors represent a complex between phospholipids and protein kinase C, an enzyme first identified by Nishizuka and coworkers. Considerable evidence indicates heterogeneity in the pharmacology of the biological responses to the phorbol esters. Likewise, multiple subclasses of binding sites have been observed. Differences in the phospholipids associated with protein kinase C may account for this heterogeneity, affecting both absolute and relative affinities. An endogenous analogue of the phorbol esters had been predicted from the high evolutionary conservation of the receptor. Nishizuka and coworkers have reported that diacylglycerols appear to be natural activators of protein kinase C. We find that diacylglyerols competitively inhibit phorbol ester binding, consistent with their acting at the same site on the enzyme. Likewise, by binding analysis, we can demonstrate a 1:1 stoichiometry between D-1-2-diacylglycerol and the receptor. The potencies of diacylglycerols for binding reflect their local concentration in the phospholipids. The K1 for diolein is approximately 0.1%. Relative to the corresponding phorbol esters, the potencies of the diacylglycerols are somewhat (17-fold) to markedly (30 000-fold) lower. We conclude that factors affecting phospholipid-receptor interactions or diacylglycerol production are of potential importance in the promotion process.

Animals↗
PubMed1985

Activation of the human neutrophil nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase by protein kinase C.

A variety of phagocytosable and soluble agonists stimulate the human neutrophil respiratory burst enzyme, NADPH-oxidase, an activity required for normal microbicidal function. Of these agonists, the phorbol esters, which stimulate diverse systems by their ability to substitute for diacylglycerol to activate protein kinase C (the major phorbol ester receptor), have now been shown to directly stimulate NADPH-oxidase through this same receptor. Almost 90% of the specific receptors for phorbol 12,13-dibutyrate (PDBu) were found in the cytosol upon subcellular fractionation. The dissociation constant for [3H]PDBu was 1.2 nM. No significant difference was found in the distribution of the receptor between subcellular fractions from resting as compared with phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils. On the basis of these binding studies, we were able to establish a reconstituted system in which PMA activated dormant NADPH-oxidase in a light membrane fraction when cytosol, NADPH, phosphatidylserine, or phosphatidylinositol and ATP were added. The calcium chelator, EGTA, inhibited the activation, which suggested a requirement for calcium at low concentrations. The half-maximally effective PMA dose was 1.1 nM, as predicted from the receptor content in these preparations. Reconstitution of oxidase activity was rapid, peaking within 1 min of incubation. Purified protein kinase C was able to substitute for the cytosol fraction, and accounted for 80% of the cytosol activity. These studies demonstrate that phorbol esters stimulate the neutrophil respiratory burst through activation of cytosolic protein kinase C, which in turn activates either a regulatory constituent or the NADPH-oxidase directly in the plasma membrane to generate an active O-2-generating system.

Caenorhabditis elegans Proteins↗
PubMed1985

Highly lipophilic phorbol esters as inhibitors of specific [3H]phorbol 12,13-dibutyrate binding.

We examined the ability of a series of highly lipophilic phorbol esters to inhibit [20-3H]phorbol 12,13-dibutyrate binding to the cytosolic aporeceptor from mouse brain. If added in the usual fashion directly into the aqueous phase of the assay mixture, phorbol 12,13-distearate, phorbol 12,13-dioleate, and phorbol 12,13-dimyristate showed very weak inhibitory activities, with apparent inhibitor equilibrium dissociation constant values above 4 microM. In contrast, if incorporated directly into the liposomes used to reconstitute the aporeceptor, all three derivatives inhibited binding with high apparent affinities, 7.4 to 34 nM. The less lipophilic derivative phorbol 12,13-didecanoate showed a similar high affinity, 2.4 to 3.2 nM, by either route of addition. Consistent with the activity of the lipophilic derivatives being masked by an inability to transfer from the aqueous to the lipid phase, phorbol 12,13-distearate added to the aqueous phase inhibited efficiently (apparent inhibitor equilibrium dissociation constant, 14 nM) in the presence of 0.03% Triton X-100. The results suggest that the phorbol ester receptor recognizes phorbol esters which are inserted into the lipid bilayer. They indicate, moreover, that the apparent low activity of the more lipophilic phorbol esters is strongly influenced by factors other than equilibrium binding affinities.

Animals↗