Ubiquitin, lysosomes, and neurodegenerative diseases.
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Animals↗
Biomedical subjects
R J Mayer
Publications and source records attributed to R J Mayer.
Substrate specificities and properties of human phospholipases A2 in a mixed vesicle model.
Studies of the specificity of phospholipases A2 (PLA2s) for different substrates have usually been carried out in vesicles or mixed micelles, where differences in shape, size, or charge of vesicles formed with different phospholipids may give misleading results. Another factor is binding of the enzyme to the phospholipid surface, which has recently been addressed using vesicles of an anionic phospholipid, dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) to which some extracellular PLA2s were shown to bind with a very high affinity (Jain, M. K., and Berg, O. G. (1989) Biochem. Biophys. Acta 1002, 127-156). In the present report we have used a similar system to study the substrate preferences of two human PLA2s that are thought to be physiologically relevant in the metabolism of arachidonic acid: a recombinant form of the human synovial fluid (14 kDa) PLA2 and the cytosolic (85 kDa) PLA2 found in monocytic cells. It is shown that both human enzymes bind tightly to DMPM vesicles and follow the basic characteristics of processive hydrolysis in this model using analysis of progress curves and substrate competition experiments. Mixed vesicles containing DMPM with small amounts (3-5 mol%) of other phospholipids have been used to study the substrate selectivity of the two human isoenzymes. The synovial fluid PLA2 shows a clear preference (approximately 7-fold) for sn-glycero-3-phosphoethanolamine over sn-glycero-3-phosphocholine. Within glycerophosphocholines, this enzyme displays little preference for the sn-2 fatty acyl group, and a slight preference for phospholipids with sn-1-acyl versus sn-1-alkyl substituents. In contrast, the cytosolic PLA2 shows a marked selectivity for arachidonoyl in the sn-2 position and only minor differences in selectivity for the polar head group in the sn-3 position. This enzyme does not distinguish between sn-1-acyl and sn-1-alkyl subclasses of glycerophosphocholines.
Arachidonic Acid↗
Chemotherapy for metastatic colorectal cancer.
The systemic management of patients with colorectal cancer continues to focus on the use of 5-fluorouracil (5-FU). In the setting of metastatic disease, parenteral 5-FU has been shown to be superior to oral 5-FU; however, survival duration seems similar whether such parenteral 5-FU is administered in a "loading," weekly, or continuous infusion manner. The addition of other cytotoxic agents such as semustine, mitomycin C, or cisplatin to 5-FU does not appear to increase the response rate or prolong survival. The results of five randomized trials assessing the value of intraarterial hepatic infusions of 5-FU or floxuridine show that such regional chemotherapy increases the likelihood of hepatic response compared with systemic treatment but has little effect on survival and is associated with significant toxicity. Recent efforts in the management of patients with advanced disease have been directed at optimizing the activity of 5-FU by (1) enhancing the inhibition of DNA synthesis through the concomitant administration of folinic acid; (2) increasing drug incorporation into RNA through pretreatment with methotrexate or phosphonacetyl-L-aspartate; and (3) improving the drug's activity through the synergistic action of alpha-2a-interferon. Although the results of some of these investigations have been promising, only the 5-FU and folinic acid combination consistently has appeared to be superior to single-agent 5-FU. These different approaches to biochemical modulation are being compared in ongoing cooperative group trials.
Antineoplastic Agents↗
Protein processing in lysosomes: the new therapeutic target in neurodegenerative disease.
A little recognised feature of neurons is their large complement of lysosomes. Studies of the accumulation of the abnormal isoform of the prion protein (PrPSC) in the prion encephalopathies and the formation of beta/A4 protein from its precursor in Alzheimer's disease suggest that generation of these key proteins takes place in lysosome-related organelles. The release of hydrolytic enzymes from lysosomes may be a primary cause of neuronal damage. Although molecular genetic approaches have identified protein mutations central to the main neurodegenerative disease, cell biological observations are now beginning to unravel the intracellular pathways involved in the molecular pathogenesis of neurodegeneration: as a result, it is now appropriate to consider therapeutic manipulation of the lysosomal system as an approach to treatment.
Animals↗
A ubiquitin conjugating enzyme encoded by African swine fever virus.
The post-translational modification of proteins by covalent attachment of ubiquitin occurs in all eukaryotes by a multi-step process. A family of E2 or ubiquitin conjugating (UBC) enzymes catalyse one step of this process and these have been implicated in several diverse regulatory functions. We report here the sequence of a gene encoded by African swine fever virus (ASFV) which has high homology with UBC enzymes. This ASFV encoded enzyme has UBC activity when expressed in Escherichia coli since it forms thiolester bonds with [125I]ubiquitin in the presence of purified ubiquitin activating enzyme (E1) and ATP, and subsequently transfers [125I]ubiquitin to specific protein substrates. These substrates include histones, ubiquitin and the UBC enzyme itself. The ASFV encoded UBC enzyme is similar in structure and enzyme activity to the yeast ubiquitin conjugating enzymes UBC2 and UBC3. This is the first report of a virus encoding a functionally active UBC enzyme and provides an example of the exploitation of host regulatory mechanisms by viruses.
African Swine Fever Virus↗
alpha B crystallin expression in non-lenticular tissues and selective presence in ubiquitinated inclusion bodies in human disease.
alpha B crystallin is a lens protein which has homology with the small heat-shock proteins and is also expressed in non-lenticular tissues. Polyclonal antibodies have been raised to a synthetic peptide corresponding to residues 1-10 of alpha B crystallin. The antiserum detects a 20 kDa polypeptide on nitrocellulose replicas after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate of extracts of heart muscle known to be rich in alpha B crystallin. Staining of normal human tissues reveals immunoreactivity of lens capsular epithelium, skeletal muscle, cardiac muscle, smooth muscle, renal tubular epithelium, Schwann cells, and glial cells, as has been described by other workers. In addition, positive staining of normal thyroid epithelium, colonic epithelium, and stratified squamous epithelium was seen. Tissues known to contain ubiquitinated inclusion bodies were immunostained with the anti-alpha B-crystallin antiserum. Staining of cortical Lewy bodies, astrocytic Rosenthal fibres, and hepatic Mallory bodies was seen, but only a proportion of inclusions were positive. Neurones containing the ubiquitinated inclusions of Alzheimer's disease were only very rarely immunostained and the ubiquitinated inclusions of motor neurone disease were not detected by the antiserum. Reactive astrocytes in cerebral tissues were strongly immunostained. The results suggest that alpha B crystallin is involved in the formation of ubiquitinated inclusion bodies that have associated intermediate filaments and support previous observations on the localization of a brain-specific ubiquitin carboxy-terminal hydrolase which similarly divides ubiquitinated filamentous inclusions in the central nervous system into two main groups.
Alzheimer Disease↗
Immunoreactivity to ubiquitin-protein conjugates is present early in the disease process in the brains of scrapie-infected mice.
Brains from mice infected with either the 87V or the ME7 strains of mouse-passaged sheep scrapie were taken at stages during the disease process and immunostained to show the localization of ubiquitin-protein conjugates. In both models, conjugates were seen as fine, dot-like structures; as coarser, granular lesions within or adjacent to neurones; and in areas surrounding plaques. The dot-like structures were visible at 28 days post-ME7 infection and at 55 days in 87V-infected mice. In both models, the extent of immunoreactive changes increased as the disease progressed and terminal infection was as described earlier by us (Lowe et al., J. Pathol 1990; 162: 61-66). The patterns of development of these features were distinctive in two ways: progression from region to region was observable and the density of the pathological lesions grew exponentially as the clinical symptoms appeared. The earliest pathological dot-like structures corresponded temporally with the earliest detection of PrPSC by Western blotting, and immunogold electron microscopic investigation of the dot-like lesions indicated that they were the multi-vesicular, lysosome-related, dense bodies that we have described previously in terminal disease (Laszlo et al., J Pathol 1992; 166: 333-341). Until now, ubiquitin-protein conjugates were seen mainly in inclusion bodies associated with the terminal stages of a range of human degenerative diseases. This study establishes that ubiquitin-protein conjugates accumulate in lysosome-related bodies very early and appear to be intimately related to the pathological processes in the animal disorders that we have studied.
Animals↗
Production and characterization of a polyclonal antibody to the c-erbB-3 protein: examination of c-erbB-3 protein expression in adenocarcinomas.
A polyclonal rabbit antibody was raised to the c-erbB-3 protein using a synthetic peptide corresponding to amino acids 1229-1241 of the predicted protein sequence of c-erbB-3. In Western blot analysis this antibody detects a single band at approximately 165 kD in a c-erbB-3 transfected (293/HER-3) human cell line. c-erbB-3 protein expression was then examined in a variety of adenocarcinomas. Expression of c-erbB-3 protein was indicated by membrane and/or cytoplasmic tumour cell immunoreactivity in formalin-fixed, paraffin-embedded tissue sections. c-erbB-3 protein was detected in a series of 13 out of 14 primary breast carcinomas, 3 of 5 gastric adenocarcinomas, 8 of 9 colonic adenocarcinomas, 2 of 9 prostatic adenocarcinomas, 0 of 6 renal cell carcinomas, 1 of 4 primary lung adenocarcinomas, and 5 of 7 endometrial adenocarcinomas. Immunohistochemical expression of the c-erbB-3 protein appears to be a relatively common event in adenocarcinomas, and further studies are now warranted to establish the role of the c-erbB-3 protein in neoplasia.
Adenocarcinoma↗
Ubiquitin, lysosomes and neurodegenerative diseases.
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Brain↗
A role for lysosomes in scrapie pathogenesis.
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Animals↗
Ballooned neurons in several neurodegenerative diseases and stroke contain alpha B crystallin.
alpha B crystallin is a protein which has homology with the small cell stress proteins. A characterized antibody to residues 1-10 of alpha B crystallin was used to immunostain tissues containing ballooned (chromatolytic, achromasic) neurons. The tissues included two cases of classical Pick's disease, one case of dementia with swollen achromasic neurons in the cortex, two cases of Alzheimer's disease with large numbers of ballooned neurons, two cases of motor neuron disease, four cases of cortico-basal degeneration, and four cases with areas of brain showing swollen neurons adjacent to recent cerebral infarcts. The anti-alpha B crystallin showed strong diffuse cytoplasmic immunoreactivity of swollen cortical neurons in all the diseases. Astrocytes and oligodendroglial cells were also stained in normal tissues as previously described. Weak diffuse immunoreactivity with an antibody to ubiquitin-conjugates was also seen in the swollen neurons from cases of neurodegenerative disease but not following infarction. Ballooned neurons have been shown to contain phosphorylated neurofilament epitopes not normally present in the perikaryonal region. The presence of alpha B crystallin in ballooned neurons, together with previous data which also indicate its close association with intermediate filaments, suggest that alpha B crystallin may be involved in aggregation and remodelling of neurofilaments in disease. The presence of alpha B crystallin in neurons at the edge of areas of cerebral infarction is likely to reflect cells which are regenerating following damage; its detection may therefore be a marker for such cells. On a practical level, the antibody greatly facilitates the localization of such abnormal neurons in diagnostic histology.
Alzheimer Disease↗
2,5-Hexanedione-induced intermediate filament aggregates contain ubiquitin-protein conjugate immunoreactivity and resemble Rosenthal fibres.
A number of chronic degenerative disorders including cerebellar astrocytomas and Parkinson's disease are characterized by the presence of cytosolic inclusions which contain intermediate filament (IF) aggregates and ubiquitin-protein conjugate immunoreactivity. In cerebellar astrocytomas these inclusions are known as Rosenthal fibres. 2,5-hexanedione (HD) treatment is known to induce IF aggregates in cells in culture. HD-induced aggregates have therefore been studied as a potential model for the clinical inclusions. Exposure of astrocyte cultures to 2 mM HD for 2 or 4 weeks led to the formation of aggregates of the IFs (glial fibrillary acidic protein and vimentin). The aggregates contained ubiquitin-protein conjugates, which, on electron microscopy appeared to be localized in a peripheral shell. In addition, ubiquitin mRNA levels were found to be elevated approximately threefold by HD treatment. HD-induced inclusions and Rosenthal fibres were found to share a number of features. HD administration, therefore, appears to be a suitable model for the production of pathological inclusions.
Animals↗
Etoposide, doxorubicin, and cisplatin chemotherapy for advanced gastric adenocarcinoma: results of a phase II trial.
PURPOSE: A phase II study of etoposide, doxorubicin, and cisplatin (EAP) therapy in patients with advanced gastric carcinoma was performed in an attempt to confirm encouraging results reported by German investigators using an identical EAP regimen. PATIENTS AND METHODS: Thirty-six consecutive, previously untreated patients with surgically unresectable, measurable gastric carcinoma were treated every 28 days with etoposide (120 mg/m2, days 4, 5, and 6), doxorubicin (20 mg/m2, days 1 and 7), and cisplatin (40 mg/m2, days 2 and 8). A total of 108 courses of treatment was given. RESULTS: Therapy was associated with myelosuppression (median granulocyte nadir, 239/microL; median platelet nadir, 81,000/microL), which reached its maximum 14 days after the start of therapy and necessitated hospitalization after 24 of 108 (22%) treatment courses. Four of 36 (11%) patients died of treatment-related toxicity: three from sepsis and one from hemorrhage. Objective responses were observed in 12 of 36 (33%) patients; three (8%) patients experienced a clinical complete response. The median time to progression was 4 months for all 36 patients and 8 months for the 12 responding patients. Of the 13 patients with localized but unresectable disease, five subsequently underwent surgical resection; only one of these five was rendered disease-free. CONCLUSION: The EAP regimen is highly toxic and results in response rates and survival times no better than those of more easily tolerated treatment programs.
Adenocarcinoma↗
Risk factors for high-dose cytarabine neurotoxicity: an analysis of a cancer and leukemia group B trial in patients with acute myeloid leukemia.
PURPOSE: We analyzed pretreatment characteristics of patients with postremission acute myeloid leukemia (AML) treated with high-dose cytarabine (HIDAC) during a recent Cancer and Leukemia Group B (CALGB) trial to determine risk factors associated with HIDAC neurotoxicity. PATIENTS AND METHODS: One hundred seventy-six patients received at least one course of HIDAC as part of a CALGB protocol designed to determine the optimal dose of cytarabine (ara-C) for postremission treatment of AML. HIDAC consisted of 3 g/m2 ara-C infused over 3 hours at 12-hour intervals on days 1, 3, and 5. The pretreatment characteristics of 170 patients were available for risk analyses. RESULTS: Eighteen patients (10%) experienced neurotoxicity. Univariate analyses demonstrated associations between the occurrence of neurotoxicity and elevated serum creatinine, age, and alkaline phosphatase (AP). Multivariate analysis showed that these variables were independent risk factors. These findings were used to construct a risk model with the following parameters: creatinine greater than or equal to 1.2 mg/dL, age greater than or equal to 40 years, and AP greater than or equal to 3 x normal. Seventeen of 46 (37%) patients with two or more of these criteria developed neurotoxicity compared with one of 124 (1%) patients with one or none. The sensitivity and specificity of this model were 94% and 81%, respectively. CONCLUSION: We conclude that patients with two or more of the following parameters may be at increased risk for HIDAC neurotoxicity: (creatinine greater than or equal to 1.2 mg/dL, age greater than or equal to 40, and AP greater than or equal to 3 x normal). However, this model should be confirmed by analysis of additional groups of patients treated with HIDAC.
Acute Disease↗
Intensive post-remission therapy with Ara-C in adults with acute myeloid leukemia: initial results of a CALGB phase III trial. The Cancer and Leukemia Group B.
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Acute Disease↗
Effects of a novel lanosterol 14 alpha-demethylase inhibitor on the regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in Hep G2 cells.
A 32-carboxylic acid derivative of lanosterol (SKF 104976) was found to be a potent inhibitor of lanosterol 14 alpha-demethylase (14 alpha DM). 14 alpha DM activity in a Hep G2 cell extract was inhibited 50% by 2 nM SKF 104976. Exposure of intact cells to similar concentrations of the compound resulted in the inhibition of incorporation of [14C]acetate into cholesterol with concomitant accumulation of lanosterol as well as a 40-70% decrease in 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity. SKF 104976 did not effect low density lipoprotein uptake and degradation in Hep G2 cells, suggesting that HMGR and low density lipoprotein receptor activity were not coordinately regulated under these conditions. Reduction of the flux of carbon units in the sterol synthetic pathway by as much as 80% did not alter the suppressing effect of SKF 104976 on HMGR activity. However, under conditions where sterol synthesis was almost completely blocked by lovastatin, HMGR activity was not suppressed by SKF 104976. Mevalonate, at concentrations that did not decrease HMGR activity, was able to restore the inhibiting effect of SKF 104976 on HMGR activity. The rapid inhibition (2-3 h) of HMGR activity by SKF 104976 to 30-60% of the level in controls was not dependent on the initial amount of HMGR enzyme present. These findings suggest that upon inhibition of 14 alpha DM by SKF 104976, a mevalonate-derived precursor regulates HMGR activity, even when the sterol synthetic rate is considerably reduced and when HMGR protein levels are very high. In Hep G2 cells, formation of oxylanostenols from [3H]mevalonate reached a maximum between 1 and 10 nM SKF 104976 and was negligible at higher concentrations. This result suggests that oxylanostenols are not the key mediators of the modulation of HMGR in Hep G2 cells upon 14 alpha DM inhibition.
Carcinoma, Hepatocellular↗
Ubiquitin-protein conjugates and alpha B crystallin are selectively present in cells undergoing major cytomorphological reorganisation in early chicken embryos.
Ubiquitin-protein conjugates and alpha B crystallin are detected immunohistochemically in cells undergoing extensive morphological reorganisation in early chicken embryos. Cytoplasmic ubiquitinated proteins and alpha B crystallin are coordinately found in cells of the lens, notochord and myotome. The antigens appear in the myotome cells precisely at the point at which the cells begin to migrate from the dorsomedial lip of the dermamyotome. The findings indicate that ubiquitin and alpha B crystallin may have a coordinate role in the extensive architectural remodeling which occurs in these developing tissues in the early chick embryo. Some form of functional association between protein ubiquitination and alpha B crystallin in cells may explain why alpha B crystallin is found with ubiquitin-protein deposits in some neurodegenerative diseases.
Amino Acid Sequence↗