Physician support and rewards: developing a support system.
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Adaptation, Psychological↗
Biomedical subjects
D Burdick
Publications and source records attributed to D Burdick.
Preferential adsorption, internalization and resistance to degradation of the major isoform of the Alzheimer's amyloid peptide, A beta 1-42, in differentiated PC12 cells.
A central question in Alzheimer's disease (AD) is the role of amyloid in pathogenesis. Recent discoveries implicating the longer A beta 1-42 form of amyloid in pathogenesis led us to characterize the interaction of A beta with cells to elucidate differences that might account for these observations. We characterized the adsorption, internalization and degradation of radiolabeled A beta in NGF-differentiated PC12 cells under conditions that are not acutely toxic. All A beta peptides examined absorb to the surface of PC12 cells and are internalized; however the adsorption and internalization of A beta 1-42 is significantly greater than that of A beta 1-40 and A beta 1-28. The adsorption of A beta 1-42 is decreased by treatment of the cells with neuraminidase, but not heparitinase. The fate of the internalized A beta 1-42 is also very different than shorter A beta peptides; a fraction of the internalized A beta 1-42 accumulates intracellularly and is resistant to degradation for at least 3 days while A beta 1-40 and shorter peptides are eliminated with a half life of about 1 h. A beta 1-42 does not appear to inhibit lysosomal hydrolases, since A beta 1-28 is degraded at the same rate in the presence or absence of A beta 1-42. The intracellular A beta 1-42 is located in a dense organellar compartment and colocalizes with the lysosomal markers Lucifer Yellow and horseradish peroxidase. These data indicate that there are significant differences in the cell surface adsorption, internalization and catabolism of A beta 1-42 compared to A beta 1-40 and A beta 1-28. These differences may be important for the preferential accumulation of the longer A beta 1-42 isoform and its association with AD pathogenesis.
Adsorption↗
Vesicular transport: implications for cell polarity.
In polarized cells intracellular sorting of plasma membrane proteins occurs to a large extent at the trans-Golgi network, giving rise to vesicles destined for distinct plasma membrane domains. This review discusses the several pathways, both direct and indirect, which lead to protein incorporation into the correct cell surface, as well as the mechanisms involved. Proteins contain signals which direct their incorporation into the distinct vesicles destined for plasma membrane microdomains. Specific coat proteins are involved in vesicle assembly and are likely to play a role in the generation of discrete vesicle populations. Molecules involved in vesicle docking and fusion may also add specificity to the targeting process.
Amino Acid Sequence↗
Cultured rat microglia express C1q and receptor for C1q: implications for amyloid effects on microglia.
Senile plaques, the pathological hallmark of Alzheimer's disease (AD), are associated with complement components, including C1q. Reactive microglia appear to be involved in the later stages of plaque development. Since tissue macrophages are known to synthesize C1q, cultured rat microglia were examined for C1q immunoreactivity. Anti-C1q staining was detected, particularly in process-bearing microglia, indicating constitutive expression of C1q. Thus, microglia could provide a source of C1q for plaques even before becoming reactive. Since it has been previously shown that C1q binds beta 1-42, the major constituent of senile plaques, and since beta 1-42 is toxic to microglia in vitro, we asked if preincubation of beta 1-42 with C1q alters either metabolic indices of amyloid-induced degeneration in microglial cultures or the formation of amyloid deposits on these cells. While electron microscopic analysis of negatively stained amyloid fibrils confirmed that pre-incubation with C1q induced the association of C1q with the fibrils, no effect of the binding of C1q to beta 1-42 on beta 1-42 toxicity in microglia was observed. Interestingly, immunoreactivity for the C1q receptor that is known to modulate phagocytosis was found and was up-regulated in non-process-bearing microglia by interferon-gamma. While these data exclude a role for the C1q receptor in beta 1-42 toxicity in microglia, the observed expression and up-regulation of C1q receptor on microglia by interferon-gamma would be consistent with a role for C1q in complement-mediated inflammatory responses in AD and as a potential activator of microglial function in plaques.
Alzheimer Disease↗
beta-Amyloid activates complement by binding to a specific region of the collagen-like domain of the C1q A chain.
beta-amyloid peptides that accumulate within the brain of individuals with Alzheimer's disease bind to C1q and activate the classical C pathway via a specific interaction with a site within the collagen-like domain of C1q (C1q-CLF). Synthetic analogues of beta-amyloid peptides, beta 1-42 and beta 1-40, bound to C1q and were strong activators of C as assessed by both total C consumption and C4 consumption. beta 1-42 was significantly more effective than beta 1-40 in binding to C1q and triggering C activation, whereas beta 1-28 demonstrated little or no binding or C activation. This C-activating capacity seems to be largely correlated with the assembly of the beta 1-42 into low speed sedimentable aggregates and/or macromolecular fibrils. Radiolabeled C1q and C1q-CLF bind specifically to these aggregates or amyloid fibrils. In addition, using synthetic C1q peptides in a solid phase binding assay, the major binding site of beta 1-42 to C1q was localized to the C1q A chain collagen-like residues 14-26, a region previously described as a novel interaction site for Ab-independent activators of C1. C1q A chain peptide 14-26 blocked the ability of beta-amyloid peptides to activate the classical C pathway, providing evidence that this relatively unrecognized mechanism of C activation (via binding to the C1q-CLF) may have crucial physiologic consequences. Finally, these observations provide further support for the hypothesis that C activation and inflammation may be a component in the pathogenesis of AD and suggest possibilities for modulating the progression of AD.
Alzheimer Disease↗
Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state.
The progressive neurodegeneration of Alzheimer's disease has been hypothesized to be mediated, at least in part, by beta-amyloid protein. A relationship between the aggregation state of beta-amyloid protein and its ability to promote degeneration in vitro has been previously suggested. To evaluate this hypothesis and to define a structure-activity relationship for beta-amyloid, aggregation properties of an overlapping series of synthetic beta-amyloid peptides (beta APs) were investigated and compared with beta AP neurotoxic properties in vitro. Using light microscopy, electrophoresis, and ultracentrifugation assays, we found that few beta APs assembled into aggregates immediately after solubilization, but that over time peptides containing the highly hydrophobic beta 29-35 region formed stable aggregations. In short-term neuronal cultures, toxicity was associated specifically with those beta APs that also exhibited significant aggregation. Further, upon the partial reversal of beta 1-42 aggregation, a concomitant loss of toxicity was observed. A synthetic peptide derived from a different amyloidogenic protein, islet amyloid polypeptide, exhibited aggregation but not toxicity, suggesting that beta AP-induced neurotoxicity in vitro is not a nonspecific reaction to aggregated protein. The correlation between beta AP aggregation and neurotoxicity was also observed in long-term neuronal cultures but not in astrocyte cultures. These data are consistent with the hypothesis that beta-amyloid protein contributes to neurodegeneration in Alzheimer's disease.
Amino Acid Sequence↗
Intracellular accumulation and resistance to degradation of the Alzheimer amyloid A4/beta protein.
The A4 or beta protein is a peptide that constitutes the major protein component of senile plaques in Alzheimer disease. The A4/beta protein is derived from a larger, transmembrane amyloid precursor protein (APP). The putative abnormal processing events leading to amyloid accumulation are largely unknown. Here we report that a 42-residue synthetic peptide, beta 1-42, corresponding to one of the longer forms of the A4/beta protein, accumulates in cultured human skin fibroblasts and is stable for at least 3 days. The peptide appears to accumulate intracellularly, since it does not accumulate under conditions that prevent endocytosis and accumulation is correlated with the acquisition of resistance to removal by trypsin digestion. This intracellular accumulation is also correlated with the ability of the peptide to aggregate as determined by SDS/polyacrylamide gel electrophoresis. At low concentrations of the beta 1-42 peptide, which favor the nonaggregated state, no accumulation is observed. Shorter peptide analogs (28 or 39 residues) that are truncated at the C terminus, which lack the ability to aggregate in SDS gels, fail to accumulate. The accumulated intracellular beta 1-42 peptide is in an aggregated state and is contained in a dense organellar compartment that overlaps the distribution of late endosomes or secondary lysosomes. Immunofluorescence of the internalized peptide in permeabilized cells reveals that it is contained in granular deposits, consistent with localization in late endosomes or secondary lysosomes. Sequence analysis indicates that some of the internalized peptide is subject to N-terminal trimming. These results suggest that the aggregated A4/beta protein may be resistant to degradation and suggest that the A4/beta protein may arise, at least in part, by endosomal or lysosomal processing of APP. Our results also suggest that relatively nonspecific proteolysis may be sufficient to generate the A4/beta protein if this part of APP is selectively resistant to proteolysis.
Amino Acid Sequence↗
Assembly and aggregation properties of synthetic Alzheimer's A4/beta amyloid peptide analogs.
The amyloid A4 or beta peptide is a major component of extracellular amyloid deposits that are a characteristic feature of Alzheimer's disease. We synthesized a series of peptide analogs of the A4/beta peptide which are progressively longer at their carboxyl termini, including 42- and 39-residue peptides which represent the major forms of the A4/beta peptide in senile plaque and the hereditary cerebral hemorrhage with amyloidosis form, respectively. All peptides tested, beta 1-28 through beta 1-42, formed amyloid-like fibrils and previously unreported thin sheet-like structures which stained with thioflavin T and Congo Red. The solubility of beta 1-42 and shorter peptides was pH and concentration dependent, with a broad insolubility profile in the pH range of 3.5-6.5 and at concentrations above 0.75 mg/ml. Only peptides of 42 residues or longer were significantly insoluble at pH 7.4. beta 1-47 and beta 1-52 peptides are highly insoluble in aqueous media but are soluble at 40 mg/ml in the alpha helix-promoting solvent, 1,1,1,3,3,3-hexafluoro-2-propanol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the beta 1-42 peptide migrates as a series of higher molecular mass aggregates whereas shorter peptides migrate as monomers. Aggregation is also dependent on pH, peptide concentration, and time of incubation in aqueous medium. These results indicate that the length of the hydrophobic carboxyl terminus of the A4/beta peptide is important in determining the solubility and aggregation properties of the A4/beta peptide and that acid pH environment, high peptide concentration, and long incubation time would be predicted to be important factors in promoting amyloid deposition.
Alzheimer Disease↗
Nerve growth factor stimulation of GAP-43 phosphorylation in intact isolated growth cones.
Phosphorylation of the nervous system-specific growth cone protein GAP-43 by kinase C in vivo occurs exclusively in growth cones and distal axons, and the onset of this phosphorylation is delayed relative to the onset of axonogenesis, with the delay predicted on the time needed for axons to reach the vicinity of their targets (Meiri et al., 1991). We have used a subcellular fraction of intact growth cones (IGCs) to investigate whether this induction of GAP-43 phosphorylation can be influenced by target-derived substances, and show here that increased phosphorylation of GAP-43 can be both stimulated and maintained by NGF at concentrations of 2 x 10(-10) M. This low concentration of NGF and the subsequent phosphorylation of GAP-43 are both consistent with the interpretation that phosphorylation is due to the binding of NGF to a biologically active high-affinity receptor. Second, we used the monoclonal antibody 2G12 to show that the NGF-stimulated phosphorylation of GAP-43 occurs on serine, the kinase C phosphorylation site, consistent with the results seen in vivo. Levels of phosphorylated GAP-43 in the intact IGCs are also modulated by calcium-stimulated dephosphorylation that could be inhibited by EGTA but not okadaic acid and that therefore resembled the calcineurin-stimulated dephosphorylation reported in vitro. The results suggest that the spatial and temporal regulation of GAP-43 phosphorylation that occurs during axonogenesis in vivo can be regulated by target-derived neurotropic molecules, specifically NGF.
Animals↗
Engineering subtilisin BPN' for site-specific proteolysis.
A combination of protein engineering and substrate optimization was used to create variants of the serine protease, subtilisin BPN', which efficiently and specifically cleave a designed target sequence in a fusion protein. The broad substrate specificity of wild-type subtilisin BPN' is greatly restricted by substitution of the catalytic histidine-containing of the catalytic histidine 64 with alanine (H64A) so that certain histidine-containing substrates are preferentially hydrolysed (Carter, P., Wells, J.A. Science 237:394-399, 1987). The catalytic efficiency, (kcat/Km), of this H64A variant was increased almost 20-fold by judicious choice of substrate and by installing three additional mutations which increase the activity of wild-type subtilisin. The most favorable substrate sequence identified was introduced as a linker in a fusion protein between a synthetic IgG binding domain of Staphylococcus aureus protein A and Escherichia coli alkaline phosphatase. The fusion protein (affinity purified on an IgG column) was cleaved by the prototype H64A enzyme and its improved variant, efficiently and exclusively at the target site, to liberate an alkaline phosphatase product of the expected size and N-terminal sequence. Several features of H64A variants of subtilisin make them attractive for site-specific proteolysis of fusion proteins: they have exquisite substrate specificity on the N-terminal side of the cleavage site and yet are broadly specific on the C-terminal side; they can be produced in large quantities and remain highly active even in the presence of detergents, reductants (modest concentrations), protease inhibitors, at high temperatures, or when specifically immobilized on a solid support.
Amino Acid Sequence↗
Heating by microwave, hot air, and flowing steam to eliminate inoculated Salmonella from poultry feed.
Poultry feed samples (mash) obtained from a commercial mill were inoculated with either 100 or 5,000 cells/g of a marker strain of Salmonella montevideo. Samples (125 g) were then placed in a microwave oven at power settings of 1.5 or 2.0 kW for 60, 90, or 120 sec; a hot air oven at 99 C (210 F) or 110 C (230 F) for 15, 30, 45, or 60 min; or a flowing steam chamber for 1, 5, 10, or 20 min. Temperature, moisture, and presence of the inoculated S. montevideo were determined in all samples before and after treatment. Feed samples placed in the microwave oven reached a maximum temperature of 186 C (367 F) and had a final moisture content as low as .8%. About 25% of the samples inoculated with 100 cells/g and about 40% of those inoculated with 5,000 cells/g remained positive after treatment. In the hot air oven, feed reached a maximum temperature of 93 C (200 F) with a moisture level after heating of about 4%. Salmonellae were recovered from 35 and 60% of the low and high inoculum level samples, respectively. In flowing steam, maximum feed temperature was 90 C (194 F) with a final moisture level of 15%. After 1, 5, 10, and 20 min of flowing steam, 100, 90, 60, and 0% of the samples were found to contain salmonellae, respectively. All treatments occasionally eliminated salmonellae from the samples, but results were inconsistent and unpredictable.
Air↗
Peutz-Jeghers syndrome. A clinicopathologic study of a large family with a 27-year follow-up.
A family group of ten patients with the Peutz-Jeghers syndrome has been followed for a 27-year period. Eight members of the family had one or more manifestations of the syndrome. One member of the family died following a bypass procedure of a nonresectable carcinoma of the jejunum. Postmortem examination revealed this to be an adenocarcinoma of the jejunum arising in a Peutz-Jeghers polyp and metastases were present in the mesenteric lymph nodes. Two members of the family developed breast carcinomas, one arising in a fibroadenoma; both patients died, one of metastatic breast carcinoma, the other of a second primary malignancy (adenocarcinoma of the jejunum arising in a Peutz-Jeghers polyp). Three family members had benign ovarian tumors, one patient had a benign breast tumor and another patient had a benign colloid thyroid nodule. While the authors of this report believe that they have added a documented case of an adenocarcinoma of the jejunum arising in a Peutz-Jeghers polyp to the literature, the exact risk of intestinal cancer in the Peutz-Jeghers syndrome is unknown, but probably very small. Gastrointestinal surgery should continue to be performed in symptomatic patients and all patients should be followed closely at regular intervals.
Adenocarcinoma↗
Issues in screening and cancer communications: overview of conference issues.
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Health Education↗
Transient positive postictal computed tomographic scan.
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Brain↗
Nitrogen balance responses and abomasal lysine levels in wethers fed supplemental polymerized L-lysine-HCl.
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Abomasum↗
Nitrogen balance, abomasal crude protein and amino acids in wethers fed formaldehyde-treated coastal bermudagrass and infused with methionine, glucose or monensin.
A 5 X 5 Latin square design was used to study the effects on nitrogen balance, wool growth and plasma amino acids of infusing methionine and (or) glucose into the abomasum of growing wethers fed formaldehyde-treated Coastal bermudagrass (F-CBG) or untreated Coastal bermudagrass hay. Daily treatments were: (1) CBG; (2) F-CBG + 3 g methionine (met); (4) F-CBG + 96 g glucose and (5) F-CBG + 3 met + 96 g glucose. Glucose infusion decreased (P < .005) moles/100 g of total essential amino acids in plasma; moles/100 g of lysine, valine, leucine and isoleucine were also reduced (P < .005). Moles/100 g of methionine in plasma were increased significantly by methionine infusion, but nitrogen retention was not affected (P > .10). The grams digestible protein to megacalorie digestible energy (DE) ratio was 36.6, 35.9 and 32.4 for CBG, F-CBG and F-CBG + glucose, respectively. The decrease (36.6 to 32.4) resulted in increased nitrogen retained (P < .05), increased percentage nitrogen intake retained (P < .05) and increased percentage digested nitrogen retained (P < .01). Urinary nitrogen excretion was lower (P < .01) in wethers receiving the F-CBG + glucose treatment than in those receiving the F-CBG treatment (3.60 vs 5.09 g/day). Effects of formaldehyde treatment of CBG and rumen infusion of monensin on quantities of various crude protein (CP) components reaching the abomasum were also determined in a 4 X 4 Latin square design experiment. Total CP reaching the abomasum increased (P < .01) with formaldehyde treatment and resulted in increased quantities of amino acids reaching the abomasum (P < .05). Monensin had no effect on these two parameters, and no interaction between monensin and formaldehyde was observed. Dietary CP reaching the abomasum increased with formaldehyde treatment (P < .005) and with infusion of monensin (P < .025). Total microbial protein (MP) and MP reaching the abomasum per 100 g dry matter digested were not influenced by either formaldehyde or monensin.
Abomasum↗
A stochastic compartment model of stomach cancer with correlated waiting time distributions.
The incidence and growth rate of stomach cancer in the US population is modelled, for each sex, as a partially observed, discrete state stochastic process. Explicit evaluation of the transition rates between the states of the model is made possible by identifying them as specific functions of the time spent within each state. The functions used in the model were selected from the medical and epidemiological literature. With the model it was found possible to obtain fits to the age distribution of deaths due to stomach cancer for white males in 1975 and for selected age ranges for white females. These results suggested that the natural history of stomach cancer is different for females above and below age 65.
Adult↗
Evaluation of technetium scanning for myocardial contusion.
Myocardial contusion is a serious problem not easily diagnosed. Laboratory and clinical studies carried out on myocardial infarction suggested that Technetium scanning might clarify this diagnostic dilemma. Radionuclide imaging with Technetium was carried out in 29 patients suspected of having myocardial contusion. Of 13 patients in whom contusion was confirmed electrocardiographically, scan was positive in only two. Use of the technique in diagnosis of myocardial contusion is not recommended.
Adolescent↗