Biomedical subjects
E S Johnson
Publications and source records attributed to E S Johnson.
A proteolytic pathway that recognizes ubiquitin as a degradation signal.
Previous work has shown that a fusion protein bearing a "nonremovable" N-terminal ubiquitin (Ub) moiety is short-lived in vivo, the fusion's Ub functioning as a degradation signal. The proteolytic system involved, termed the UFD pathway (Ub fusion degradation), was dissected in the yeast Saccharomyces cerevisiae by analyzing mutations that perturb the pathway. Two of the five genes thus identified, UFD1 and UFD5, function at post-ubiquitination steps in the UFD pathway. UFD3 plays a role in controlling the concentration of Ub in a cell: ufd3 mutants have greatly reduced levels of free Ub, and the degradation of Ub fusions in these mutants can be restored by overexpressing Ub. UFD2 and UFD4 appear to influence the formation and topology of a multi-Ub chain linked to the fusion's Ub moiety. UFD1, UFD2, and UFD4 encode previously undescribed proteins of 40, 110, and 170 kDa, respectively. The sequence of the last approximately 280 residues of Ufd4p is similar to that of E6AP, a human protein that binds to both the E6 protein of oncogenic papilloma viruses and the tumor suppressor protein p53, whose Ub-dependent degradation involves E6AP. UFD5 is identical to the previously identified SON1, isolated as an extragenic suppressor of sec63 alleles that impair the transport of proteins into the nucleus. UFD5 is essential for activity of both the UFD and N-end rule pathways (the latter system degrades proteins that bear certain N-terminal residues). We also show that a Lys --> Arg conversion at either position 29 or position 48 in the fusion's Ub moiety greatly reduces ubiquitination and degradation of Ub fusions to beta-galactosidase. By contrast, the ubiquitination and degradation of Ub fusions to dihydrofolate reductase are inhibited by the UbR29 but not by the UbR48 moiety. ufd4 mutants are unable to ubiquitinate the fusion's Ub moiety at Lys29, whereas ufd2 mutants are impaired in the ubiquitination at Lys48. These and related findings suggest that Ub-Ub isopeptide bonds in substrate-linked multi-Ub chains involve not only the previously identified Lys48 but also Lys29 of Ub, and that structurally different multi-Ub chains have distinct functions in Ub-dependent protein degradation.
Detection of antibodies to avian leukosis/sarcoma viruses (ALSV) and reticuloendotheliosis viruses (REV) in humans by ELISA.
We used a modified commercial ELISA kit to test for antibodies to avian leukosis/sarcoma and reticuloendotheliosis viral antigens in the sera of 45 poultry workers and their matched controls. We found that 42% of sera from poultry workers had anti-avian leukosis Sarcoma viruses (anti-ALSV) and 20% had anti-reticuloendotheliosis viruses (anti-REV), antibody titers that were higher than the highest titer recorded in control subjects, and hence were regarded as positive. To determine the specificity of these reactions, selected sera were absorbed with ALSV or REV antigens alone, or with chick embryo fibroblasts (CEF) alone, or with both CEF and ALSV/REV, and then retested. In each case, absorption resulted in a statistically significant reduction in absorbance, which was greatest for the combined CEF and ALSV/REV absorption, thus suggesting that the reactions involved viral as well as chicken antigens. However, definitive tests such as Western Blot analyses are needed to confirm whether indeed antibodies to these viruses were specifically elicited in human sera.
In vitro and in vivo activities of reduced-size antagonists of luteinizing hormone-releasing hormone.
A novel series of octapeptide LHRH antagonists was designed on the basis of the structure of the (2-9) fragment of a LHRH agonist. By adopting a systematic SAR study, we were able to improve first the in vitro activity and then the in vivo LH suppression, raising them up to the range of the decapeptide antagonists NalGlu (51) and A-75998 (50), resulting in A-76154 (49). The octapeptide antagonist A-76154 is the most potent reduced-size LHRH antagonist reported. It suppresses LH in the castrated rat by over 80% for a period of 4 h following sc bolus administration of 30 micrograms/kg.
Current serum levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin in phenoxy acid herbicide applicators and characterization of historical levels.
BACKGROUND: Workers who sprayed phenoxy acid herbicides, especially those who sprayed before 1975, may have been exposed to significant amounts of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent animal carcinogen present in herbicide preparations as a contaminant. PURPOSE: The aims of this study were (a) to determine serum levels of TCDD in a representative sample of workers occupationally exposed to the agent during the spraying of phenoxy acid herbicides; (b) to compare serum levels in workers exposed before 1965, when concentrations in herbicide products were unregulated and high, with levels in workers exposed after 1974, when concentrations were lower as a result of government regulations worldwide; and (c) to examine the correlation, if any, between serum levels and duration of employment in spraying. METHODS: Thirty-seven subjects were randomly selected from a group of 654 men who had sprayed the herbicides 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4-dichlorophenoxyacetic acid (2,4-D) in Australia for at least 12 months. The workers were classified as follows: eight who sprayed only before 1965, nine who sprayed only during the period after 1964 and before 1975, and 20 who sprayed during the period after 1974 and before 1991. Serum from the workers was analyzed for TCDD by high-resolution gas chromatography and high-resolution mass spectrometry at a detection limit of 0.6 parts per trillion (ppt) on a lipid-weight basis. In addition, rates of exposure to TCDD were estimated, as were TCDD serum concentrations at termination of employment and intensity of herbicide use. RESULTS: Only one worker, with a serum TCDD level of 34 ppt, had a serum level higher than the maximum level of 26 ppt reported for the general population. Assuming a half-life of 7.1 years, we estimated the mean exposure rates to be 2.7, 2.3, and 0.06 ppt/mo for the three epochs, respectively. We found the highest serum level of TCDD at the time of cessation of employment to be 329 ppt. Calendar period and intensity of use of 2,4,5-T and 2,4-D were statistically significant determinants of rate of exposure to TCDD, but 2,4-D was associated with exposure rate only for the pre-1975 periods. Estimated rates prior to 1965 were more than an order of magnitude higher than those after 1974. CONCLUSION: The highest estimated exposure rate was 20.7 ppt/mo, which suggests that some sprayers may have been exposed to levels comparable with those that produce cancer in laboratory animals.
Perspectives on risk assessment impact of recent reports on benzene.
Very sensitive methods that can detect the benzene metabolite muconic acid (MA) in the urine of virtually all members of the general population have recently become available and have been used in a few occupational studies as a marker of benzene exposure. Preliminary findings from these studies suggest that urinary MA may be a reliable marker of occupational exposure to greater than 5 ppm benzene. It was also consistently observed that a certain proportion of the general population have urinary MA levels compatible with those seen in persons occupationally exposed to greater than 1 ppm benzene. It is unlikely that these elevated levels can be explained solely as being artifactual. The frequency with which they occur for a given individual, and the duration with which they are maintained, are not known. Information on these two factors is needed in order to adequately assess whether or not these levels present a significant risk for a segment of the general population.
Ubiquitin as a degradation signal.
For many short-lived eukaryotic proteins, conjugation to ubiquitin, yielding a multiubiquitin chain, is an obligatory pre-degradation step. The conjugated ubiquitin moieties function as a 'secondary' signal for degradation, in that their posttranslational coupling to a substrate protein is mediated by amino acid sequences of the substrate that act as a primary degradation signal. We report that the fusion protein ubiquitin--proline--beta-galactosidase (Ub-P-beta gal) is short-lived in the yeast Saccharomyces cerevisiae because its N-terminal ubiquitin moiety functions as an autonomous, primary degradation signal. This signal mediates the formation of a multiubiquitin chain linked to Lys48 of the N-terminal ubiquitin in Ub-P-beta gal. The degradation of Ub-P-beta gal is shown to require Ubc4, one of at least seven ubiquitin-conjugating enzymes in S.cerevisiae. Our findings provide the first direct evidence that a monoubiquitin moiety can function as an autonomous degradation signal. This generally applicable, cis-acting signal can be used to manipulate the in vivo half-lives of specific intracellular proteins.
Increasing radiation dose intensity using hyperfractionation in patients with malignant glioma. Final report of a prospective phase I-II dose response study.
We attempted to show a dose effect relationship for radiation therapy by treating patients harbouring malignant glioma with increasing doses of radiation in a step-wise fashion. We postulated that no increase in delayed toxicity would be seen because we used hyperfractionation technique. Between January 1981 and December 1988 we treated 280 patients three times daily at 4 hour intervals. 100 patients received a total dose of 6141 cGy, 73 patients received 7120 cGy, and 107 patients received 8000 cGy. CCNU was given at the time of tumor progression following radiotherapy. Median time to tumor progression was 28 weeks for patients who received 6141 cGy, 27 weeks for patients who received 7120 cGy and 36 weeks for patients who received 8000 cGy. Median survival was 46 weeks for patients who received 6141 cGy, 38 weeks for patients who received 7120 cGy and 45 weeks for patients who received 8000 cGy. There was no statistically significant difference in either time to tumor progression or survival among the three treatment arms and no dose response effect was seen. There was no increase in delayed radiation toxicity when the total radiation dose was increased up to 8000 cGy.
Nested case-control study of lung cancer in the meat industry.
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Distribution and sampling of Aedes taeniorhynchus (Diptera: Culicidae) eggs in a Florida mangrove forest.
The distribution of Aedes taeniorhynchus (Wiedemann) eggs in a Florida mangrove basin forest was quantified and used to design a sampling plan. Eggs were found in detritus-rich soil with the highest densities in a band at elevations 0.1-0.2 m above the water line. Dispersion indices (k and Taylor's b) indicated that the eggs were aggregated; 14 of 16 populations tested fit the negative binomial distribution. A fixed-size sampling plan using systematic sampling was designed from these data.
Aedes taeniorhynchus (Diptera: Culicidae) oviposition patterns in a Florida mangrove forest.
The association of Aedes taeniorhynchus eggs and several variables was studied in a Florida mangrove forest. Eggs were limited to stands of red mangrove (Rhizophora mangle L.) that were embedded within a black mangrove (Avicennia germinans L.) forest. The occurrence of eggs was related significantly to elevation and the amount of detritus. Field and laboratory studies indicated that grazing on black mangrove detritus by the snail Melampus coffeus L. may have limited detritus accumulation and soil organic content, thus restricting mosquito oviposition to the red mangrove stands. Eggshells were concentrated in the same habitat as eggs, suggesting that eggshells may be used to identify oviposition patterns.
Human exposure to 2,3,7,8-TCDD and risk of cancer.
Most of the evidence for the carcinogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in humans has centered around whether it causes malignant lymphomas (ML) and soft-tissue sarcomas (STS). A critical review of the literature indicates that the evidence does not support a causal role for TCDD in the etiology of ML. For STS, the evidence does not specifically incriminate TCDD either, although there is room for doubt. Cancers of other sites, particularly of the respiratory system and thyroid which were found to be statistically significantly in excess in either of the two largest studies of combined cohorts of occupationally exposed workers, were identified as candidate tumors for which a possible etiological role of TCDD might need investigation in future studies.
cis-trans recognition and subunit-specific degradation of short-lived proteins.
The N-end rule, a code that relates the metabolic stability of a protein to the identity of its amino-terminal residue, is universal in that different versions of the N-end rule operate in mammals, yeast and bacteria (unpublished data). The N-end rule-based degradation signal comprises a destabilizing amino-terminal residue and a specific internal lysine residue. We now show that, in a multisubunit protein, these two determinants can be located on different subunits and still target the protein for destruction. Moreover, in this case (trans recognition) only the subunit that bears the lysine determinant is actually degraded. Thus an oligomeric protein can contain both short-lived and long-lived subunits. These insights have functional and practical implications.
Association between soft tissue sarcomas, malignant lymphomas, and phenoxy herbicides/chlorophenols: evidence from occupational cohort studies.
Some case-control studies have reported a significant association between occupational use of phenoxy herbicides and chlorophenols and soft-tissue sarcomas and malignant lymphomas. However, persons who spray or apply these substances are concomitantly exposed to other potentially carcinogenic chemicals and oncogenic viruses, which have been found or suspected to play a role in the etiology of these tumors. No study has thoroughly controlled for these other exposures, some of which have been shown to be independently associated with these tumors even after controlling for exposure to phenoxy acids or chlorophenols. On the other hand, it has been found that an observed risk from exposure to phenoxy herbicides disappeared on controlling for some of these concomitant exposures in the rare instance this was attempted. Also, on several occasions, an association has been observed with occupations in which exposure to phenoxys and chlorophenols may occur, but not with the compounds themselves. Accordingly, a detailed review of the evidence from occupational cohort studies was conducted, to see if it corroborates that from case-control studies. It was found that the evidence does not unequivocally incriminate phenoxys and chlorophenols as a cause of these tumors. The results obtained with cohort studies of sprayers and applicators do not corroborate the association reported among this occupational group, in case-control studies. It is possible that the suspected association may well be due, partly or wholly, to one or more of the other concomitant exposures. However, in view of the fact that the majority of the cohorts need further follow-up to be informative, it is concluded that further studies of these cohorts are required before it can be determined whether or not these tumors are caused by exposure to phenoxy acids and chlorophenols.
Bias on withdrawing lost subjects from the analysis at the time of loss, in cohort mortality studies, and in follow-up methods.
Cohort mortality studies may differ from morbidity and other follow-up studies in that, in many of the methods of follow-up employed, the identification or follow-up process and the process of determination of outcome (death) are essentially the same. Furthermore, some of the methods may involve preferential identification of deaths over live persons, or vice versa. This latter area of difference is particularly problematic in situations in which the comparison group (the general population) has not been subjected to tracing, and in which certain persons who are being traced are not covered by the universe defined by the follow-up method. Because of these peculiarities, it is shown that, during the analysis, the usual practice of withdrawing persons lost to follow-up at the time of loss can lead to seriously biased results when follow-up rate is not very high.
Novel astrocytic protein in multiple sclerosis plaques.
Monoclonal antibody J1-31 (MAb J1-31, isotype IgG 2b) was raised against crude homogenate of brain tissue from a multiple sclerosis (MS) patient (autopsy sample; Malhotra et al.: Microbios Letters 26:151-157, 1984). In human brain, MAb J1-31 recognizes an intracellular protein antigen (J1-31 antigen), which bands at approximately 30,000 daltons under reducing conditions for sodium dodecyl sulfate gel electrophoresis (Singh et al.: Bioscience Reports 6:73-79, 1986). By immunofluorescence microscopy, MAb J1-31 stains those cells that are also stained by antiserum to glial fibrillary acidic protein (GFAP), namely astrocytes, retinal Müller cells, and tanycytes in the ependyma (Predy et al.: Bioscience Reports 7:491-502, 1987). In addition, MAb J1-31 stains ciliated ependymal cells that do not express GFAP. Using a model system for gliosis (laceration-type injury of rat spinal cord), we were able to show that astrocytes responding to central nervous system injury exhibit greatly enhanced staining for J1-31 antigen (Predy et al.: Journal of Neuroscience Research 19:397-404, 1988; Predy and Malhotra: Brain Research Bulletin in press, 1989). In this article, we demonstrate that immunofluorescence staining owing to MAb J1-31 is greatly enhanced in MS plaques, as compared to adjacent "apparently normal" white matter. (This is consistent with previous results as MS plaques characteristically show an astroglial response [reactive gliosis] leading to the formation of a glial scar [McKhann: Annual Review of Neuroscience 5:219-239, 1982].) In addition, we present further evidence that J1-31 antigen is distinct from GFAP, although these two proteins may be associated spatially with one another.
LH-RH antagonists: design and synthesis of a novel series of peptidomimetics.
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Active reduced-size hexapeptide analogues of luteinizing hormone-releasing hormone.
A series of reduced-size hexapeptide analogues of LH-RH were synthesized that contain the residues corresponding to amino acid positions 4-9 and are linked to various carboxylic acids in place of residue 3. These compounds were tested in vitro in the rat pituitary receptor binding and LH release assays. A wide range of binding affinities was obtained up to and exceeding that of LH-RH. Both agonists and antagonists were obtained. From the SAR studies, it appears that a very precise size, length, and shape of the substituent at position 3 is required to achieve agonist activity, whereas the structural requirements for antagonist activity appear to be much less stringent. Depending on the nature of the substituent at positions 6 and 4, the biological response switches from antagonist to agonist or vice versa. The results suggest that conformational changes at position 6 or 4 feed back to the substituent at position 3, which induces the change from agonist to antagonist. The most potent compounds in the series were tested in vivo and found to be active.