* Department of Environmental Health Sciences, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland;
Department of Preventive Medicine, School of Medicine, Soonchunhyang University, Chunan, Republic of Korea;
Department of Epidemiology, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland;
Department of Medicine, School of Medicine, The Johns Hopkins University, Baltimore, Maryland;
¶ Department of Community Medicine and Preventive Medicine, Mount Sinai Medical Center, New York, New York; and Departments of
|| Neurology, School of Medicine and
Neurotoxicology, Kennedy Krieger Institute, The Johns Hopkins University, Baltimore, Maryland
Received February 14, 2001; accepted May 7, 2001
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ABSTRACT |
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Key Words: blood lead; tibia lead; protein kinase C; back-phosphorylation.
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INTRODUCTION |
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The mechanism of lead toxicity may be due, in part, to disruption of calcium-mediated processes. This unique interaction between lead and calcium, and its relation to lead neurotoxicity, has been reviewed extensively (Bressler and Goldstein, 1991; Pounds, 1984
; Simons, 1993
). Lead displays an affinity for the calcium-binding protein, calmodulin, similar to the affinity of calcium (Goldstein and Ar, 1983
), and lead appears to activate calmodulin-dependent neurite outgrowth in hippocampal neurons (Kern and Audesirk, 1995
). Additionally, lead has a complex interaction with protein kinase C. pM concentrations of lead achieve levels of activity similar to nM concentrations of calcium in protein kinase C enzyme assays (Markovac and Goldstein, 1988b
). Interestingly, the maximal activity achieved with calcium is greater than the maximal activity observed with lead (Long et al., 1994
; Tomsig and Suszkiw, 1995
), suggesting that lead may act as a calcium agonist at low concentrations and an antagonist at higher concentrations. In addition to activating protein kinase C in enzymatic assays, exposure to lead induces translocation of protein kinase C from cytosol to the membrane in immature rat brain microvessels (Markovac and Goldstein 1988a
), rat brain astrocytes, and retinal endothelial cells (Laterra et al., 1992
). Finally, phosphorylation of erythrocyte membrane proteins by protein kinase C was observed in isolated human erythrocytes after exposure to lead (Belloni-Olivi et al., 1996
). Studies of protein kinase-C activity in rodents dosed with lead have confirmed the findings of these in vitro studies. Lead-induced deficits in learning were associated with changes in the activation of protein kinase C in the hippocampus (Chen et al., 1999
, 1997
). The neuronal specific isoform, protein kinase C-
, was also shown to undergo changes after chronic exposure to lead during development (Reinholz et al., 1999
).
To verify that protein kinase C is a target for lead, an association between activation of protein kinase C and exposure to lead should be demonstrated in humans. There are, however, several problems in studying associations between environmental exposures and biochemical changes. These include the need to use surrogate tissues because target tissues are inaccessible (e.g., brain, testis, kidney) and the need to use frozen samples. To overcome these problems, we measured protein kinase-C activity in erythrocytes, which are appropriate because previous studies have demonstrated activation of protein kinase C in erythrocytes after exposure to phorbol esters (Cohen and Foley, 1986; Palfrey and Waseem, 1985
). Notably, erythrocytes contain several proteins such as spectrin, adducin, band 4.1, and dematin, which have homologs in the brain (Bennett et al., 1988
; Bennett and Gilligan 1993
; Roof et al., 1997
; Yorifuji et al., 1989
). To overcome the problem of using frozen tissue, a back-phosphorylation assay was used, which measures the products of a protein kinase C catalyzed reaction (phosphoproteins). Protein kinase C activity was not measured directly because protein kinase C is very susceptible to proteolysis, and previous investigators suggested that tissue stored frozen should not be used as an enzyme source (Kikkawa et al., 1983
). Phosphoproteins are more stable and are often measured as an indicator of protein kinase activity.
Here, we report associations of lead exposure and dose measures, including blood lead, tibia lead measured by X-ray fluorescence, zinc protoporphyrin, and exposure duration with protein kinase C activation assessed with the back phosphorylation assay. The data revealed inverse associations supporting conclusions from studies in cell culture (Belloni-Olivi et al., 1996; Laterra et al., 1992
; Markovac and Goldstein 1988a
) and rats (Chen et al., 1999
, 1997
; Reinholz et al., 1999
) that have shown activation of protein kinase C after exposure to lead.
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MATERIALS AND METHODS |
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Study design and subject selection.
From October 1997 to August 1999, 803 lead workers and 135 non-exposed control subjects were recruited in the Republic of Korea for a 4-year prospective study of the health effects of lead (Schwartz et al., in press). We enrolled 212 consecutive subjects during April to July 1998, 156 men and 56 women, to study protein kinase C activity from 4 lead storage battery companies during April to July 1998. The current study is a cross-sectional analysis of data from the first year of the prospective study. The study subjects were examined at Soonchunhyang University, Institute of Industrial Medicine, in Chunan, Korea. All subjects provided written, informed consent and participation was voluntary. The study was approved by Institutional Review Boards at the Johns Hopkins School of Hygiene and Public Health and Soonchunhyang University School of Medicine.
Data collection.
Subjects completed a standardized questionnaire; a medical history questionnaire; an occupational history interview; and provided a 10-ml blood specimen that was stored at 70°C as whole blood, plasma, and red blood cells.
An occupational physician obtained the medical history and a trained psychologist and a registered nurse conducted the neurobehavioral testing. Tibia lead was measured by K-shell 109Cd x-ray fluorescence (XRF) and chelatable lead burden was estimated as 4-h urinary lead excretion after oral administration of 10 mg/kg meso-2,3-dimercaptosuccinic acid (DMSA). The standardized questionnaire assessed demographic characteristics, education, tobacco and alcohol use, detailed medical history, subjective symptoms related to lead exposure, and complete job history.
Biologic measures of lead exposure and dose.
Blood zinc protoporphyrin (ZPP) levels were measured at the site with 12 drops of fresh whole blood and a portable hematofluorimeter (Aviv Model 206; Blumberg et al., 1977). Hemoglobin was assayed by the cyanmethemoglobin method with use of a Coulter Model AcTB, and hematocrit was measured by the capillary centrifugation method. Blood-lead levels (µg/dl) were analyzed with a Zeeman background-corrected atomic absorption spectrophotometer (Hitachi Z-8100 model) with the NIOSH standard addition method (Kneip and Crable, 1988) at Soonchunhyang University Institute of Industrial Medicine, a certified reference laboratory for lead in Korea. Tibia lead was assessed (in units of µg Pb/g of bone mineral) after a 30-min measurement at the left mid-tibia shaft by using 109Cd K-shell x-ray fluorescence as described previously (Schwartz et al., 1999
; Todd et al., 1992
).
Erythrocyte membrane preparation.
Venous blood was collected in a heparinized vacutainer tube. Each sample was centrifuged at 1000 x g for 20 min and the plasma and white cells were removed by aspiration. The erythrocytes were washed 3 times with equal volumes of saline. Erythrocytes were preserved by rapidly freezing in a cryopreservative (Rowe et al., 1968). Briefly, an equal amount of cryoprotective additive solution (28% glycerol, 3% mannitol, and 0.65% NaCl) was added to packed erythrocytes and thoroughly mixed. Erythrocytes were transferred under dry ice to the Kennedy Krieger Institute, Baltimore, Maryland, and then stored at 70°C.
The method for isolating the erythrocyte membrane is critical to measuring protein kinase-C activation, because the membrane composition depends upon the preparation method. Briefly, approximately 34 ml of glycerolized erythrocytes was thawed on wet ice (Dodge et al., 1963). Osmotic lysis was initiated by adding 20 mM sodium phosphate buffer at pH 7.4 to the erythrocytes, to achieve an erythrocyte:buffer ratio of 1:15 v/v. After 20 min on ice, the hemolysate was centrifuged at 20,000 x g for 40 min. The supernatant fraction was decanted and the pellet containing the membrane proteins was washed 4 or 6 times with phosphate buffer until the supernatant fraction was colorless. The membrane proteins were suspended in phosphate buffer and the protein content of the membranes was determined (Bradford, 1976
). Aliquots of membranes were stored at 70°C until use.
Back phosphorylation assay to determine protein kinase-C activation in vivo.
We adapted the in vitro back-phosphorylation assay to measure protein kinase-C activation in vivo (Walaas et al., 1988). The principle of this assay is that proteins that were phosphorylated by protein kinase C in vivo do not undergo phosphorylation in an in vitro assay in the presence of exogenously added protein kinase C. Therefore, a decrease in the level of phosphorylation of an erythrocyte membrane protein in the back-phosphorylation assay indicates an increase in protein kinase C activation in vivo. The assay has been used to measure protein phosphorylation in different tissues, including human cerebrospinal fluid (Gandy et al., 1990
), human heart (Schwinger et al., 1998
; Zakhary et al., 1999
), rat brain (Barone et al., 1994
; Hoffman and Janis, 1993
), and rat heart (Bartel et al., 1993
). In adapting the assay, the range of concentrations of erythrocyte membrane protein that resulted in a linear relation with protein phosphorylation was found to be between 0.1 µg to 3 µg of erythrocyte membrane. The concentration of exogenous protein kinase C was chosen on the basis of the sensitivity of the assay and cost.
In the back-phosphorylation assay, erythrocyte membrane proteins were thawed at 4°C, and sonicated for 30 s on wet ice. Membrane proteins were diluted with phosphate buffer to a concentration of 0.16 mg/ml. Phosphatidylserine (PS) was prepared by drying down under nitrogen gas on wet ice, and resuspending the lipid by sonicating for 30 s on ice in 50 mM Tris/HCl (pH 7.4). Phorbol-12,13-dibutyrate (PDBu) (1 mg/ml) was added to PS to obtain a concentration of 12.5 µg/ml and 0.25 mg/ml, respectively. A solution of cofactors was made by taking equal volumes of PS/PDBu, membrane protein, and 12 mM CaCl2 and 30-mM dithiothreitol (DTT), both in 50-mM Tris/HCl (pH 7.5).
[-32P]ATP was made at 0.08 mCi/ml in 45-mM MgCl2/150 µM ATP fresh for each assay. Protein kinase C was prepared at 0.1 unit/25 µl. To initiate the reaction, 25 µl of cofactor solution, 25 µl of [
-32P] ATP, and 25 µl of protein kinase C were mixed. The final concentrations of reactants were 1 µg of membrane protein, 0.1 unit of protein kinase C, and 2.6 µCi of [
-32P]ATP. Reaction mixtures were incubated at 30°C for 30 min in a heat block, and the reactions were stopped by adding an equal amount of ice-cold acetone. The supernatant fraction was removed by centrifugation at 20,000 x g for 20 min at 4°C, and the pellet was resuspended in 30 µl of loading buffer. Samples were boiled for 3 min and subjected to SDSPAGE. Gels were stained with 0.005% Coomassie brilliant blue solution (in 40% methanol, 7% acetic acid) overnight at room temperature, then destained and dried.
Back-phosphorylation was determined by measuring 32P incorporation into membrane proteins with a bio-imaging analyzer (Fuji BAS 2500, Mac BAS version 2.5) after exposing the dried gel for 30 min to an imaging plate (Fuji, BAS-IIIs). The amount of back-phosphorylation was expressed as units of photostimulated luminescence (PSL).
Spectrin staining was measured by laser densitometry to control for possible differences in the amount of membrane proteins. Spectrin is the major erythrocyte skeletal membrane protein and is composed of and ß subunits with molecular masses of 240 and 230 kDa, respectively. Because the subunits have similar molecular masses on the gels and could not be distinguished, we measured spectrin in the 2 subunits combined. We used the optical density measurements to adjust the regression models of back-phosphorylation levels for differences in the quantity of membrane protein in the assay.
Assay variability and adjustment.
Some variability in the level of back-phosphorylation was observed even in the same preparations of membrane. To control for this variability, a single-standard sample was included each time experimental samples were assayedon seven different assay dates. The intra-day coefficient of variation (CV) ranged from 10.9 to 27.6%, with a mean of 16.3%. The inter-day coefficient of variation of the standard sample, however, was 31.2%. Thus, all samples were adjusted by the ratio of the standard sample on the date of the assay to the mean of all standard samples. This method of adjustment controlled for inter-day variability. The means of the 2 results were calculated from samples that were assayed in duplicate on separate gels. The correlation between duplicates was high (Pearson's r = 0.87, p < 0.001) and the mean percent difference between duplicates was 25.2%.
Statistical analysis.
Data analysis was performed with the Stata statistical software program (Stata Release 5.0, College Station, Texas). Variables examined in relation to back-phosphorylation were age, sex, tobacco and alcohol use, body-mass index, hemoglobin, and hematocrit. Separate linear regression models were constructed for each phosphorylated protein. Blood lead, tibia lead, ZPP, and duration of exposure were added separately to each model. Variables that were considered potential confounders were added in a forward stepwise procedure and were retained in the final model if they were found to be predictors of back-phosphorylation levels, or if, in our judgement, they had an important impact on the size of the ß coefficient of the lead exposure and dose term in the model. For ease of presentation of results, and because the phosphorylation of each of the 3 proteins was due to a similar mechanism, the final regression models of back-phosphorylation were selected with the same set of confounding variables (i.e., age, sex, and spectrin optical density) for each lead exposure and dose variable. All regression models were evaluated for violation of the assumptions of linear regression. Regression diagnostics such as residual plots, added variable plots, and variance inflation factors were evaluated to examine whether influential points, multi-colinearity, non-linearity, departures from normality, or non-homogeneous variance could account for the study results.
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RESULTS |
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Predictors of Back-phosphorylation Levels
In the total population of subjects, age was not associated with levels of back-phosphorylation (Table 2). In an analysis of only males, a significant correlation was observed between age and back-phosphorylation of the 52 kDa and 48 kDa bands. The Pearson's correlation coefficients were 0.25 (p < 0.01) and 0.19 (p = 0.02) for the 52 kDa and 48 kDa bands, respectively. The mean levels of back-phosphorylation of the 240230 kDa, the 52 kDa, and the 48 kDa bands were significantly higher in current drinkers (p < 0.01, p = 0.04, and p = 0.06, respectively) in males, compared to non-drinkers, but in females, differences by alcohol consumption status were not observed. Neither body mass index (BMI) nor hematocrit were correlated with levels of back-phosphorylation.
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Linear regression was used to model the back-phosphorylation levels of the 230240 kDa, the 52 kDa, and 48 kDa bands (a separate model for each band) to control for confounding variables (Table 3). All models included one lead exposure or dose variable (e.g., blood lead, tibia lead, ZPP, or exposure duration), controlling for age, sex, and spectrin optical density. The influence of tobacco and alcohol consumption was evaluated in the models but neither was found to be a predictor of back-phosphorylation levels, nor were they important confounding variables, and thus, tobacco and alcohol consumption were not retained in the final models. After controlling for age, sex, and spectrin optical density, tibia lead and exposure duration were consistent negative predictors of back-phosphorylation levels of all 3 erythrocyte membrane proteins, which was the expected direction of the associations (Table 3
and Figs. 2 and 3
). Because of concern that the 3 highest values of tibia lead (tibia lead > 190 µg/g, see Fig. 2
) could have had a disproportionate influence on the regression results, the linear regression model was evaluated without these 3 points. After elimination of these points, the association of tibia lead with spectrin back-phosphorylation levels became stronger and more significant (the tibia lead ß coefficient decreased from 1.40 to 2.87 PSL units per µg/g, and the p value declined from 0.02 to < 0.001). Indeed, examination of residual-residual plot with a lowess line supported the conclusion that the association of tibia lead levels with spectrin back-phosphorylation was present along the entire range of tibia lead levels, and was not confined to just the higher tibia lead levels (not shown; Cleveland, 1979).
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DISCUSSION |
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Although nearly 95% of the lead in blood is in the erythrocyte, we did not observe a relation between blood lead and erythrocyte protein kinase C activity. A possible explanation for this result is that lead in the erythrocytes does not activate protein kinase C, because most of it is bound to hemoglobin (Barltrop and Smith, 1972),
-aminolevulinic acid dehydratase (Bergdahl et al., 1997
) and other lead-binding proteins (Church et al., 1993
). These proteins have been shown to bind lead and their levels in erythrocytes are much greater than the level of protein kinase C. Rather, we observed a significant association between tibia lead and erythrocyte protein kinase C activity, suggesting that tibia lead is a better predictor of protein kinase C activation than blood lead. Similarly, tibia lead has been shown to be a better predictor of serum lead than is blood lead (Cake et al., 1996
), and serum lead is also the likely primary source of lead that is taken up by different tissues. It is possible that the lead entering the cells is not bound to protein and is therefore available to interact with plasma membranes and associated enzymes such as protein kinase C.
The 230240-kDa erythrocyte membrane phosphoprotein can be tentatively identified as spectrin, and the 52-kDa and 48-kDa proteins as the 2 subunits of dematin, using molecular mass as the criteria (Husain-Chishti et al., 1989). Similar observations have been made in vitro, where increases in phosphorylation of dematin (also referred to as band 4.9) were observed in erythrocytes after treatment with phorbol ester activators of protein kinase C (Al and Cohen, 1993
). Homologs of spectrin (Bennett and Gilligan 1993
) and dematin (Faquin et al., 1988
) are found in the brain, where they are involved in maintaining cell shape, suggesting that lead exposure also increases the phosphorylation of these proteins in the brain. However, direct analysis of protein phosphorylation in the brain is needed because of the obvious differences between erythrocytes and brain. Interestingly, even in the rat brain, the types of proteins that undergo phosphorylation have not been identified, even though activation of protein kinase C is associated with exposure to lead in rat brain (Chen et al., 1999
, 1997
; Reinholz et al., 1999
). Identification of these proteins may help to elucidate the mechanisms of lead neurotoxicity.
Our study is the first that has shown a relation between erythrocyte protein kinase C and exposure to lead in vivo in humans. Because it is the first, other studies are not available to help us identify potential confounding variables. It is thus possible that uncontrolled and unrecognized factors could account for these relations. Also, we did not establish the temporal relation between lead exposure and changes in erythrocyte protein kinase C activity because the study was cross-sectional in design. Therefore, studies in other human populations, and also longitudinal studies are needed to verify the associations reported here in erythrocyte protein kinase C activity and lead exposure.
In conclusion, the data suggest that chronic adult lead exposure is related to erythrocyte protein kinase C activity, as measured by in vitro back-phosphorylation, in erythrocytes among lead-exposed workers. Blood lead, a measure of current exposure, was not a predictor of protein kinase C activation, whereas tibia-lead levels and exposure duration, measures of cumulative exposure, were independent predictors of erythrocyte protein kinase C activity. Because lead is associated with neurobehavioral decrements in children (Bellinger et al., 1987; Needleman et al., 1990
) and adults (Chia et al., 1997; Stewart et al., 1999; Schwartz, 2001, in press), and protein kinase C plays important roles in many neuronal and synaptic processes (Routtenberg 1991
), our results may have implications for the mechanism of lead neurotoxicity in adults. Further exploration of this hypothesis requires that phosphorylation of erythrocyte membrane proteins be evaluated as a predictor of neurobehavioral function.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Alexander, B. H., Checkoway, H., van Netten, C., Muller, C. H., Ewers, T. G., Kaufman, J. D., Mueller, B. A., Vaughan, T. L., and Faustman, E. M. (1996). Semen quality of men employed at a lead smelter. Occup. Environ. Med. 53, 411416.[Abstract]
Barltrop, D., and Smith, A. (1972). Lead binding to human haemoglobin. Experientia 28, 7677.[ISI][Medline]
Barone, P., Morelli, M., Popoli, M., Cicarelli, G., Campanella, G., and Di Chiara, G. (1994). Behavioural sensitization in 6-hydroxydopamine-lesioned rats involves the dopamine signal transduction: Changes in DARPP-32 phosphorylation. Neuroscience 61, 867873.[ISI][Medline]
Bartel, S., Karczewski, P., and Krause, E. G. (1993). Protein phosphorylation and cardiac function: Cholinergic-adrenergic interaction. Cardiovasc. Res. 27, 19481953.[ISI][Medline]
Bellinger, D., Leviton, A., Waternaux, C., Needleman, H., and Rabinowitz, M. (1987). Longitudinal analyses of prenatal and postnatal lead exposure and early cognitive development. N. Engl. J. Med. 316, 103743.[Abstract]
Belloni-Olivi, L., Annadata, M., Goldstein, G. W., and Bressler, J. P. (1996). Phosphorylation of membrane proteins in erythrocytes treated with lead. Biochem. J. 315, 401406.[ISI][Medline]
Bennett, V., Gardner, K., and Steiner, J. P. (1988). Brain adducin: A protein kinase C substrate that may mediate site-directed assembly at the spectrin-actin junction. J. Biol. Chem. 263, 58605869.
Bennett, V., and Gilligan, D. M. (1993). The spectrin-based membrane skeleton and micron-scale organization of the plasma membrane. Annu. Rev. Cell. Biol. 9, 2766.[ISI]
Bergdahl, I. A., Grubb, A., Schutz, A., Desnick, R. J., Wetmur, J. G., Sassa, S., and Skerfving, S. (1997). Lead binding to -aminolevulinic acid dehydratase (ALAD) in human erythrocytes. Pharmacol. Toxicol. 81, 153158.[ISI][Medline]
Blumberg, W. E., Eisinger, J., Lamola, A. A., and Zuckerman, D. M. (1977). Zinc protoporphyrin level in blood determined by a portable hematofluorometer: A screening device for lead poisoning. J. Lab. Clin. Med. 89, 712723.[ISI][Medline]
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248254.[ISI][Medline]
Bressler, J. P., and Goldstein, G. W. (1991). Mechanisms of lead neurotoxicity. Biochem. Pharmacol. 41, 479484.[ISI][Medline]
Cake, K. M., Bowins, R. J., Vaillancourt, C., Gordon, C. L., McNutt, R. H., Laporte, R., Webber, C. E., and Chettle, D. R. (1996). Partition of circulating lead between serum and red cells is different for internal and external sources of lead. Am. J. Ind. Med. 29, 440445.[ISI][Medline]
Chen, H. H., Ma, T., and Ho, I. K. (1999). Protein kinase C in rat brain is altered by developmental lead exposure. Neurochem. Res. 24, 415421.[ISI][Medline]
Chen, H. H., Ma, T., Paul, I. A., Spencer, J. L., and Ho, I. K. (1997). Developmental lead exposure and two-way active avoidance training alter the distribution of protein kinase C activity in the rat hippocampus. Neurochem. Res. 22, 11191125.[ISI][Medline]
Chia, S. E., Chia, H. P., Ong, C. N., and Jeyaratnam, J. (1997). Cumulative blood-lead levels and neurobehavioral test performance. Neurotoxicology 18, 793803.[ISI][Medline]
Church, H. J., Day, J. P., Braithwaite, R. A., and Brown, S. S. (1993). Binding of lead to a metallothionein-like protein in human erythrocytes. J. Inorg. Biochem. 49, 5568.[ISI][Medline]
Cleveland, W. S. (1979). Robust, locally-weighted regression and smoothing scatter plots. J. Am. Stat. Assoc. 74, 829836.[ISI]
Cohen, C. M., and Foley, S. F. (1986). Phorbol ester- and Ca2+-dependent phosphorylation of human red cell membrane skeletal proteins. J. Biol. Chem. 261, 77017709.
Dodge, J. T., Mitchel, C., and Hanahan, D. J. (1963). The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrcytes. Arch. Biochem. Biophys. 100, 119130.[ISI]
Faquin, W. C., Husain, A., Hung, J., and Branton, D. (1988). An immunoreactive form of erythrocyte protein 4.9 is present in non-erythroid cells. Eur. J. Cell. Biol. 46, 168175.[ISI][Medline]
Gandy, S. E., Grebb, J. A., Rosen, N., Albert, K. A., Devinsky, O., Blumberg, H., Anderson, N., Cedarbaum, J. M., Porter, R. J., Sedvall, G., and et al. (1990). General assay for phosphoproteins in cerebrospinal fluid: A candidate marker for paraneoplastic cerebellar degeneration. Ann. Neurol. 28, 829833.[ISI][Medline]
Goldstein, G. W., and Ar, D. (1983). Lead activates calmodulin-sensitive processes. Life Sci. 33, 10011006.[ISI][Medline]
Hoffman, F. J., Jr., and Janis, R. A. (1993). Effects of calcium channel antagonists on the phosphorylation of major protein kinase-C substrates in the rat hippocampus. Biochem. Pharmacol. 46, 677681.[ISI][Medline]
Husain-Chishti, A., Faquin, W., Wu, C. C., and Branton, D. (1989). Purification of erythrocyte dematin (protein 4.9) reveals an endogenous protein kinase that modulates actin-bundling activity. J. Biol. Chem. 264, 89858591.
Kern, M., and Audesirk, G. (1995). Inorganic lead may inhibit neurite development in cultured rat hippocampal neurons through hyperphosphorylation. Toxicol. Appl. Pharmacol. 134, 111123.[ISI][Medline]
Kikkawa, U., Minakuchi, R., Takai, Y., and Nishizuka, Y. (1983). Calcium-activated, phospholipid-dependent protein kinase (protein kinase C) from rat brain. Methods Enzymol. 99, 288298.[ISI][Medline]
Kneip, T. J., and Crable, J. V. (1988). Methods for Biological Monitoring: A Manual for Assessing Human Exposure to Hazardous Substances. American Public Health Association, Washington, DC.
Lanphear, B. P. (1998). The paradox of lead poisoning prevention (published erratum appears in Science 282). Science 281, 16171618.
Laterra, J., Bressler, J. P., Indurti, R. R., Belloni-Olivi, L., and Goldstein, G. W. (1992). Inhibition of astroglial-induced endothelial differentiation by inorganic lead: A role for protein kinase C. Proc. Natl. Acad. Sci. U.S.A. 89, 1074810752.[Abstract]
Long, G. J., Rosen, J. F., and Schanne, F. A. (1994). Lead activation of protein kinase C from rat brain. Determination of free calcium, lead, and zinc by 19F NMR. J. Biol. Chem. 269, 834837.
Markovac, J., and Goldstein, G. W. (1988a). Lead activates protein kinase C in immature rat brain microvessels. Toxicol. Appl. Pharmacol. 96, 1423.[ISI][Medline]
Markovac, J., and Goldstein, G. W. (1988b). Picomolar concentrations of lead stimulate brain protein kinase C. Nature 334, 7173.[ISI][Medline]
Needleman, H. L., Schell, A., Bellinger, D., Leviton, A., and Allred, E. N. (1990). The long-term effects of exposure to low doses of lead in childhood. An 11-year follow-up report. N. Engl. J. Med. 322, 8388.[Abstract]
Palfrey, H. C., and Waseem, A. (1985). Protein kinase C in the human erythrocyte. J. Biol. Chem. 260, 1602116029.
Pounds, J. G. (1984). Effect of lead intoxication on calcium homeostasis and calcium-mediated cell function: A review. Neurotoxicology 5, 295331.[ISI][Medline]
Reinholz, M. M., Bertics, P. J., and Miletic, V. (1999). Chronic exposure to lead acetate affects the development of protein kinase C activity and the distribution of the PKC isozyme in the rat hippocampus. Neurotoxicology 20, 609617.[ISI][Medline]
Roof, D. J., Hayes, A., Adamian, M., Chishti, A. H., and Li, T. (1997). Molecular characterization of abLIM, a novel actin-binding and double zinc-finger protein. J. Cell. Biol. 138, 575588.
Routtenberg, A. (1991). A tale of two contingent protein kinase-C activators: Both neutral and acidic lipids regulate synaptic plasticity and information storage. Prog. Brain Res. 89, 249261.[ISI][Medline]
Rowe, A. W., Eyster, E., and Kellner, A. (1968). Liquid nitrogen preservation of red blood cells for transfusion; a low glycerol-rapid freeze procedure. Cryobiology 5, 119128.[ISI][Medline]
Schwartz, B. S., and Stewart, W. F. (2000). Different associations of blood lead, meso 2,3-dimercaptosuccinic acid (DMSA)-chelatable lead, and tibial lead levels with blood pressure in 543 former organolead manufacturing workers. Arch. Environ. Health 55, 8592.[ISI][Medline]
Schwartz, B. S., Stewart, W. F., Bolla, K. I., Simon, P. D., Bandeen-Roche, K., Gordon, P. B., Links, J. M., and Todd, A. C. (2000). Past adult lead exposure is associated with longitudinal decline in cognitive function. Neurology 55, 11441150.
Schwartz, B. S., Stewart, W. F., Todd, A. C., and Links, J. M. (1999). Predictors of dimercaptosuccinic acid chelatable lead and tibial lead in former organolead manufacturing workers. Occup. Environ. Med. 56, 2229.[Abstract]
Schwinger, R. H., Bolck, B., Munch, G., Brixius, K., Muller-Ehmsen, J., and Erdmann, E. (1998). cAMP-dependent protein kinase A stimulated sarcoplasmic reticulum function in heart failure. Ann. NY Acad. Sci. 853, 240250.
Simons, T. J. (1993). Lead-calcium interactions in cellular lead toxicity. Neurotoxicology 14, 7785.[ISI][Medline]
Stewart, W. F., Schwartz, B. S., Simon, D., Bolla, K. I., Todd, A. C., and Links, J. (1999). Neurobehavioral function and tibial and chelatable lead levels in 543 former organolead workers. Neurology 52, 16101617.
Stollery, B. T., Broadbent, D. E., Banks, H. A., and Lee, W. R. (1991). Short-term prospective study of cognitive functioning in lead workers. Br. J. Ind. Med. 48, 739749.[ISI][Medline]
Todd, A. C., McNeill, F. E., Palethorpe, J. E., and et al. (1992). In vivo x-ray fluroescence of lead in bone using K x-ray excitation with 109Cd sources: Radiation dosimetry studies. Environ Res 57, 117132.[ISI][Medline]
Tomsig, J. L., and Suszkiw, J. B. (1995). Multisite interactions between Pb2+ and protein kinase C and its role in norepinephrine release from bovine adrenal chromaffin cells. J. Neurochem. 64, 26672673.[ISI][Medline]
Walaas, S. I., Browning, M. D., and Greengard, P. (1988). Synapsin Ia, synapsin Ib, protein IIIa, and protein IIIb, four related synaptic vesicle-associated phosphoproteins, share regional and cellular localization in rat brain. J. Neurochem. 51, 12141220.[ISI][Medline]
Yorifuji, H., Kanda, K., Sobue, K., and Hirokawa, N. (1989). Localization of 4.1 related proteins in cerebellar neurons. Eur. J. Cell. Biol. 48, 104115.[ISI][Medline]
Zakhary, D. R., Moravec, C. S., Stewart, R. W., and Bond, M. (1999). Protein kinase A (PKA)-dependent troponin-I phosphorylation and PKA regulatory subunits are decreased in human dilated cardiomyopathy. Circulation 99, 505510.