Analysis of hemostasis in the zebrafish.
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Animals↗
Biomedical subjects
J P Sheehan
Publications and source records attributed to J P Sheehan.
Identification and characterization of zebrafish thrombocytes.
To analyse primary haemostasis in the zebrafish we have identified and characterized the zebrafish thrombocyte by morphologic, immunologic and functional approaches. Novel methods were developed for harvesting zebrafish blood with preservation of thrombocytes, and assaying whole blood adhesion/aggregation responses in microtitre plates. Light and electron microscopy of the thrombocyte illustrated morphological characteristics including the formation of aggregates, pseudopodia, and surface-connected vesicles analagous to the platelet canalicular system. Immunostaining with polyclonal antisera versus human platelet glycoproteins demonstrated the presence of glycoprotein Ib and IIb/IIIa-like complexes on the thrombocyte surface. Whole blood assays for adhesion/aggregation and ATP release showed ristocetin-induced adhesion without ATP release, and platelet agonist (collagen, arachidonic acid) induced aggregation with ATP release. Blood harvested from zebrafish treated with aspirin demonstrated inhibition of arachidonic acid induced aggregation and agonist induced ATP release, consistent with at least partial dependence on an intact cyclo oxygenase pathway. The combined morphologic immunologic and functional evidence suggest that the zebrafish thrombocyte is the haemostatic homologue of the mammalian platelet. Conservation of major haemostatic pathways involved in platelet function and coagulation suggests that the zebrafish is a relevant model for mammalian haemostasis and thrombosis.
Animals↗
Factors associated with hydrocephalus after aneurysmal subarachnoid hemorrhage.
OBJECTIVE: Associations among various factors and the occurrence of hydrocephalus after aneurysmal subarachnoid hemorrhage (SAH) were evaluated retrospectively in 897 patients enrolled in the North American study of tirilazad mesylate. METHODS: Patients were assessed for hydrocephalus in a blinded fashion. Assessment of hydrocephalus was made on the basis of 3-month follow-up computed tomographic studies or, for those without a 3-month follow-up scan, on the basis of the latest computed tomographic studies obtained at least 10 days after SAH. Criteria indicating the occurrence of hydrocephalus were the presence of significantly enlarged temporal horns or prior placement of a ventricular shunt. Univariate analysis was performed to assess relationships among various factors and hydrocephalus. Factors statistically associated with the occurrence of hydrocephalus were analyzed further using logistic regression analysis. RESULTS: Overall, 25.9% of the 897 patients developed hydrocephalus. Statistically significant associations among the following factors and hydrocephalus were observed (P value; risk coefficient): 1) severity of 3-month post-SAH Glasgow Outcome Scale (0.0001; 2.00); 2) increased ventricular size at admission (0.0001; 2.78); 3) neurological grade severity at admission (0.0274; 1.26); 4) preexisting hypertension (0.0284; 1.66); 5) alcoholism (0.0066; 2.30); 6) female sex (0.0056; 0.49); 7) increased aneurysm size (0.0239; 0.56); 8) pneumonia (0.0299; 1.78); 9) meningitis (0.0290; 5.86); and 10) intraventricular hemorrhage at admission (0.0414; 1.64). CONCLUSION: Hydrocephalus seems to have a multifactorial etiology. Knowledge of risk factors related to the occurrence of hydrocephalus may help guide neurosurgeons in the long-term care of patients who have experienced aneurysmal SAH.
Adult↗
If you injure your back on the job.
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Accidents, Occupational↗
Phosphorothioate oligonucleotides inhibit the intrinsic tenase complex.
Systemic administration of ISIS 2302, a 20-mer antisense phosphorothioate oligonucleotide targeting human intercellular adhesion molecule-1 mRNA, causes prolongation of plasma clotting times in both monkey and human studies. The anticoagulant effects of ISIS 2302 were investigated with both in vitro coagulation assays in human plasma and purified enzyme systems. At high oligonucleotide plasma concentrations (>100 microgram/mL), prolongation of the prothrombin and thrombin times was observed. In a thrombin time assay using purified components, high concentrations of ISIS 2302 inhibited thrombin clotting activity both by stimulating inhibition by heparin cofactor II and directly competing with fibrinogen for binding to anion binding exosite I. In contrast, low concentrations of ISIS 2302 (<100 microgram/mL) showed a selective, linear prolongation of the activated partial thromboplastin time (PTT). The rate limiting effect of 50 microgram/mL ISIS 2302, which prolonged the PTT to 1.5 times control, was identified by sequential modification of the clotting assay. Delaying addition of oligonucleotide until after contact activation failed to correct prolongation of the PTT. The calcium-dependent steps of the intrinsic pathway were individually assessed by adding sufficient activated coagulation factor to correct the PTT in plasma deficient in that specific factor. Addition of factor XIa, IXa, VIIIa, or Va failed to correct the PTT in the presence of ISIS 2302. In contrast, 0.2 nmol/L factor Xa corrected prolongation of the PTT in factor X-deficient plasma with or without oligonucleotide present. ISIS 2302 (50 microgram/mL) did not prolong a modified Russel viper venom time, suggesting no significant inhibition of prothrombinase. Thus, 50 microgram/mL ISIS 2302 prolonged the PTT by selectively inhibiting intrinsic tenase activity. ISIS 2302 showed partial inhibition of intrinsic tenase activity (to approximately 35% of control) at clinically relevant oligonucleotide concentrations in a chromogenic assay. This activity was oligonucleotide sequence-independent but required the phosphorothioate backbone, suggesting that inhibition of intrinsic tenase is a general property of this class of oligonucleotides. These results are relevant to both the therapeutic use of phosphorothioate oligonucleotides and the potential design of inhibitors of the intrinsic tenase complex, a novel target for anticoagulation.
Animals↗
Mitochondria in sporadic amyotrophic lateral sclerosis.
Mitochondria are abnormal in persons with amyotrophic lateral sclerosis (ALS) for unknown reasons. We explored whether aberration of mitochondrial DNA (mtDNA) could play a role in this by transferring mitochondrial DNA (mtDNA) from ALS subjects to mtDNA-depleted human neuroblastoma cells. Resulting ALS cytoplasmic hybrids (cybrids) exhibited abnormal electron transport chain functioning, increases in free radical scavenging enzyme activities, perturbed calcium homeostasis, and altered mitochondrial ultrastructure. Recapitulation of defects previously observed in ALS subjects and ALS transgenic mice by expression of ALS mtDNA support a pathophysiologic role for mtDNA mutation in some persons with this disease.
1-Methyl-4-phenylpyridinium↗
The role of preoperative adjuvant treatment in the management of esthesioneuroblastoma: the University of Virginia experience.
OBJECTIVE: Multidisciplinary management of esthesioneuroblastoma has effected markedly increased survival during the past 20 years. The potential for radical craniofacial surgery for complete en bloc resection, the availability of advanced neuroimaging modalities, and the incorporation of neoadjuvant therapy into treatment strategies for tumor remission have all contributed to this accomplishment. However, a standard protocol for the management of these lesions has not been accepted; preoperative radiation and chemotherapy have been advocated, but neither radiographic nor clinical response has been quantified. METHODS: Thirty-four consecutive patients with biopsy-proven esthesioneuroblastoma treated at one institution from 1976 to 1994 were reviewed to determine the effects of preoperative radiation therapy, with or without chemotherapy, on tumor size and long-term survival. RESULTS: In a multivariate regression analysis, advanced age was predictive of decreased disease-free survival (P=0.008), whereas advanced Kadish stage was associated with a borderline higher rate of disease-related mortality (P=0.056). Two-thirds of the patients showed a significant reduction in tumor burden with adjuvant therapy. Patients with response to neoadjuvant therapy demonstrated a significantly lower rate of disease-related mortality (P=0.050). In this series, the overall 5- and 10-year survival rates were 81.0 and 54.5%, respectively. CONCLUSION: Preoperative neoadjuvant therapy provides a valuable complement to radical craniofacial resection, leading to reduction in tumor burden. Patients experiencing reduction in tumor volume by neoadjuvant therapy demonstrate an improved prognosis.
Adolescent↗
Calcium homeostasis and reactive oxygen species production in cells transformed by mitochondria from individuals with sporadic Alzheimer's disease.
Alzheimer's disease (AD) is associated with defects in mitochondrial function. Mitochondrial-based disturbances in calcium homeostasis, reactive oxygen species (ROS) generation, and amyloid metabolism have been implicated in the pathophysiology of sporadic AD. The cellular consequences of mitochondrial dysfunction, however, are not known. To examine these consequences, mitochondrially transformed cells (cybrids) were created from AD patients or disease-free controls. Mitochondria from platelets were fused to rho0 cells created by depleting the human neuroblastoma line SH-SY5Y of its mitochondrial DNA (mtDNA). AD cybrids demonstrated a 52% decrease in electron transport chain (ETC) complex IV activity but no difference in complex I activity compared with control cybrids or SH-SY5Y cells. This mitochondrial dysfunction suggests a transferable mtDNA defect associated with AD. ROS generation was elevated in the AD cybrids. AD cybrids also displayed an increased basal cytosolic calcium concentration and enhanced sensitivity to inositol-1,4, 5-triphosphate (InsP3)-mediated release. Furthermore, they recovered more slowly from an elevation in cytosolic calcium induced by the InsP3 agonist carbachol. Mitochondrial calcium buffering plays a major role after this type of perturbation. beta-amyloid (25-35) peptide delayed the initiation of calcium recovery to a carbachol challenge and slowed the recovery rate. Nerve growth factor reduced the carbachol-induced maximum and moderated the recovery kinetics. Succinate increased ETC activity and partially restored the AD cybrid recovery rate. These subtle alterations in calcium homeostasis and ROS generation might lead to increased susceptibility to cell death under circumstances not ordinarily toxic.
Adenine Nucleotides↗
MPP+ induced apoptotic cell death in SH-SY5Y neuroblastoma cells: an electron microscope study.
PD is a common, late-onset neurodegenerative disorder that results in part from the gradual loss of dopaminergic neurons in the substantia nigra pars compacta. The neurotoxin MPTP can induce PD-like clinical symptomatology and neuropathological destruction and, thus, has been used as a PD model. The human neuroblastoma cell line SH-SY5Y possesses many of the qualities of human neurons and, as such, has served as a model for them. Apoptosis is the mode of cell death induced in SH-SY5Y cells by MPTP, and this was confirmed with nick end labeling and bisbenzimide staining. Transmission electron microscopic analysis of the ultrastructural changes occurring in neurotoxin exposed SH-SY5Ys revealed many morphological characteristics consistent with apoptosis. These changes included plasmalemmal blebbing, altered cytosolic density, nuclear condensation and fragmentation, pronounced vacuole formation, ribosomal dispersion, and the disappearance of the golgi complex, microtubules, and smooth endoplasmic reticulum. Limited amounts of rough endoplasmic reticulum and mitochondria exhibited normal morphology throughout the apoptotic changes but then were disrupted during secondary necrotic changes. The in vitro induction of apoptosis by a parkinsonism neurotoxin might be reflective of the mechanisms of in vivo nigral degeneration occurring during PD.
1-Methyl-4-phenylpyridinium↗
Altered calcium homeostasis in cells transformed by mitochondria from individuals with Parkinson's disease.
Parkinson's disease may be linked to defects in mitochondrial function. Mitochondrially transformed cells (cybrids) were created from Parkinson's disease patients or disease-free controls. Parkinson's disease cybrids had 26% less complex I activity, but maintained comparable basal calcium and energy levels. Parkinson's disease cybrids recovered from a carbachol-induced increase in cytosolic calcium 53% more slowly than controls even with lanthanum and thapsigargin blockade. Inhibition of complex I with the Parkinson's disease-inducing metabolite 1-methyl-4-phenylpyridinium (MPP+) similarly reduced the rate of recovery after carbachol. This MPP(+)-induced reduction in recovery rates was much more pronounced in control cybrids than in Parkinson's disease cybrids. Parkinson's disease cybrids had less carbonyl cyanide m-chlorophenylhydrazone-releasable calcium. Bypassing complex I with succinate partially restored Parkinson's disease cybrid, and MPP+ suppressed control cybrid recovery rates. The subtle alteration in calcium homeostasis of Parkinson's disease cybrids may reflect an increased susceptibility to cell death under circumstances not ordinarily toxic.
1-Methyl-4-phenylpyridinium↗
Effect of high fiber intake in fish oil-treated patients with non-insulin-dependent diabetes mellitus.
The short-term effect of high fiber intake on fish-oil treatment in 15 free-living, non-insulin-dependent diabetic patients was evaluated by using a controlled, sequential study design. During an 8-wk fish-oil-treatment period when patients received 20 g fish oil/d, the usual daily fiber intake was increased with a 15-g pectin supplement at midpoint. Fish oil alone lowered triacylglycerol and very-low-density-lipoprotein-cholesterol concentrations by 41% and 36%, respectively (both P < 0.01 by the end of the treatment period) with unchanged mean total, low-density-, and high-density-lipoprotein-cholesterol concentrations. When the fiber intake was increased, however, total and low-density-lipoprotein-cholesterol concentrations decreased significantly (P < 0.001 and < 0.05, respectively) with fish-oil treatment. The cholesterol ester fraction of plasma lipids was reduced by 34% when compared with fish oil alone (P < 0.05). The plasma triacylglycerol fraction decreased further by 44% (P < 0.001). Other beneficial effects observed included a 30% decline in the fatty acid fraction (P < 0.002) by end of the treatment period. Diabetic control was maintained during the 12-wk study. In conclusion, a high fiber intake may be beneficial in fish oil-treated diabetic patients.
Adult↗
Utilization of type I collagen gel, demineralized bone matrix, and bone morphogenetic protein-2 to enhance autologous bone lumbar spinal fusion.
Autologous bone grafts are currently considered "gold standard" material for achieving long-term spinal arthrodesis. The present study was performed to determine whether demineralized bone matrix (DBM), type I collagen gels, or bone morphogenetic protein-2 (BMP-2) can improve autologous bone spinal fusions. Using a unilateral decompression-contralateral fusion technique in dogs, each of these materials was added to an autologous bone graft. Volumetric analysis, histological analysis, and biomechanical testing were performed to assess the effectiveness of each material. The DBM had an inhibitory effect on solid bone fusion of the spine, whereas the type I collagen gels improved the bony interface between the graft and the host spine. The BMP-2 strongly enhanced the amount of bone deposition at the fusion site and increased the number of intervertebral levels that were solidly fused. This study strongly supports the use of BMP-2 as an additive to autologous bone grafts in spine stabilization.
Animals↗
Safeguard your license: the disciplinary process.
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Humans↗
Origin and functional consequences of the complex I defect in Parkinson's disease.
The mitochondrial electron transport enzyme NADH:ubiquinone oxidoreductase (complex I), which is encoded by both mitochondrial DNA and nuclear DNA, is defective in multiple tissues in persons with Parkinson's disease (PD). The origin of this lesion and its role in the neurodegeneration of PD are unknown. To address these questions, we created an in vitro system in which the potential contributions of environmental toxins, complex I nuclear DNA mutations, and mitochondrial DNA mutations could be systematically analyzed. A clonal line of human neuroblastoma cells containing no mitochondrial DNA was repopulated with mitochondria derived from the platelets of PD or control subjects. After 5 to 6 weeks in culture, these cytoplasmic hybrid (cybrid) cell lines were assayed for electron transport chain activities, production of reactive oxygen species, and sensitivity to induction of apoptotic cell death by 1-methyl-4-phenyl pyridinium (MPP+). In PD cybrids we found a stable 20% decrement in complex I activity, increased oxygen radical production, and increased susceptibility to 1-methyl-4-phenyl pyridinium-induced programmed cell death. The complex I defect in PD appears to be genetic, arising from mitochondrial DNA, and may play an important role in the neurodegeneration of PD by fostering reactive oxygen species production and conferring increased neuronal susceptibility to mitochondrial toxins.
1-Methyl-4-phenylpyridinium↗
Molecular methods of enhancing lumbar spine fusion.
OBJECTIVE: An optimal method for spinal fusion would induce rapid growth of bone via an osteoconductive and osteoinductive implant. This study examines the spinal fusion enhancement potential of some osteoconductive and osteoinductive biomaterials. METHODS: Four similar canines received unilateral posterolateral fusions on the left side at T13-L1 and L4-L5 and on the right side at L2-L3 and L6-L7. The experiments were grouped as follows: Group A, autogenous bone harvested from the iliac crest; Group B, autogenous bone and collagen; Group C, no implant; and Group D, autogenous bone, collagen, and recombinant human bone morphogenetic protein-2. Radiographic assessment, three-dimensional computed tomographic volumetric analysis, and biomechanical testing were performed at each level. RESULTS: For Groups A and B, the fusions demonstrated moderate bone formation at 6 and 12 weeks postoperatively. Group D fusions exhibited earlier and more dramatic increases in volume and radiodensity and eventually were comparable in size to the vertebral bodies. Average fusion volumes computed from three-dimensional computed tomographic analysis were: Group A = 1.243 cc, Group B = 0.900 cc, Group C = 0.000 cc, and Group D = 6.668 cc (P = 0.003 compared to Group A). Group D exhibited flexion and extension biomechanical properties much greater than controls. The addition of recombinant human bone morphogenetic protein-2 consistently yielded the strongest fused segments and, on average, enhanced extension stiffness by 626% and flexion stiffness by 1120% over controls. CONCLUSION: The most advantageous spinal fusion implant matrix consisted of recombinant human bone morphogenetic protein-2, autogenous bone, and collagen. Future investigators, however, need to examine the appropriate quantities of the individual components and clarify the efficacy of the matrix for the various types of spinal fusion approaches.
Animals↗
Safeguard your license: avoid these pitfalls.
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Defensive Medicine↗
Heparin cofactor II is regulated allosterically and not primarily by template effects. Studies with mutant thrombins and glycosaminoglycans.
Besides its critical role in hemostasis, the serine protease thrombin also participates in wound healing, inflammation, and atherosclerosis. Thrombin is inhibited by the serpins antithrombin and heparin cofactor II (HCiI) in reactions that are accelerated markedly by specific glycosaminoglycans. Following vascular injury, thrombin must be inhibited at both intravascular and extravascular sites that impose different constraints on the recognition of thrombin by these inhibitors. The present study examines the role of anion-binding exosite II of thrombin in the interaction with glycosaminoglycans and HCII. Acceleration of thrombin inhibition by serpins in the presence of glycosaminoglycans is proposed to occur by a template mechanism, in which inhibitor and protease bind simultaneously to the same glycosaminoglycan chain, facilitating their interaction. According to the template model, disruption of protease binding to glycosaminoglycan should significantly reduce acceleration of the inhibition. Specific mutations in exosite II (R89E, R245E, K248E, and K252E) disrupted thrombin binding to both dermatan sulfate and heparin, indicating that both glycosaminoglycans bind to a common site in exosite II. The same mutations markedly decreased the rate constant for thrombin inhibition by antithrombin-heparin (up to 100-fold) but had little effect on the rate constant for thrombin inhibition by HCII-heparin (7-fold maximal reduction) and no effect on the rate constant for thrombin inhibition by HCII-dermatan sulfate. These results are incompatible with a template model for thrombin inhibition by HCII and dermatan sulfate. In the presence of glycosaminoglycan, HCII and antithrombin interact with opposing thrombin exosites and use distinct mechanisms of glycosaminoglycan catalysis. Antithrombin employs a template mechanism that requires heparin to interact with thrombin exosite II, whereas HCII employs an allosteric mechanism that requires thrombin exosite I but is largely independent of exosite II. These findings have potential implications for glycosaminoglycan therapy and for the respective physiologic roles of HCII and antithrombin.
Allosteric Regulation↗