Frank L. Siegel

Hyperphenylalaninemia: Disaggregation of Brain Polyribosomes in Young Rats

A single injection of l-phenzylalanine in 7-day-old rats produtced disaggregation of brain polyribosomties and inhibition of in vitro protein synthesis in cell-free systems prepared from brain. Liver polyribosomes and in vitro protein synthesis in hepatic systems were not affected. In 4-week-old rats these effects on brain protein synthesis did not occur.

Effects of lead on rat kidney and liver: GST expression and oxidative stress

The effect of acute exposure to lead acetate on the expression of glutathione S-transferase (GST) subunits and the levels of reduced and oxidized glutathione (GSH) and malondialdehyde (MDA) in rat kidney and liver was determined. The purpose of this study was to determine if GSH depletion and/or oxidative stress were responsible for changes in the expression of some or all GSTs that followed lead exposure. In kidney, all GST subunits increased following injection of lead. The level of kidney GSH was not changed at either 0.5 or 1 h after lead exposure, but increased 3, 6, 12 and 24 h after a single injection of lead. MDA levels (a marker of lipid peroxidation) did not change in kidney following lead injection. Immunohistochemical markers of oxidative stress and nitric oxide production were also unchanged by lead administration. Therefore. we conclude that the increases in GST levels in kidney following lead exposure were not dependent on oxidative stress. In liver, lead injection caused GSH depletion (61% of control 12 h after lead treatment) and increased MDA production (2.5-fold increase 6 h after lead exposure), while GSTA1, GSTA2, GSTM1 and GSTM2 did not increase. Analysis of the effects of lead on GST mRNA and GST cellular localization were performed by Northern blot and immunohistochemical techniques. Immunoperoxidase light microscopy and immunogold electron microscopy revealed that the increase in kidney GSTM1 and GSTP1 occurred in nuclei, cytoplasm and microvilli of proximal tubules. Northern blot analysis of GSTA2 and GSTP1 mRNAs showed that their increase following lead exposure was inhibited by actinomycin D, suggesting transcriptional induction. This study demonstrates that acute lead exposure causes dramatic changes in the subcellular distribution and expression of rat kidney GSTs, and that these changes are not a result of oxidative stress.

Polyribosome disaggregation and cell-free protein synthesis in preparations from cerebral cortex of hyperphenylalaninemic rats.

Abstract— Seven‐day‐old rats were injected intraperitoneally with l‐phenylalanine (1 g/kg) and the time course of brain polyribosome disaggregation and changes in brain levels of phenylalanine, tryptophan and tyrosine were determined. Disaggregation of brain polyribosomes preceded the increase in levels of phenylalanine in brain, and followed the same time course as depletion of tryptophan from brain.

Regional, cellular and subcellular distribution of calcium-activated cyclic nucleotide phosphodiesterase and calcium-dependent regulator in porcine brain.

Abstract— Cyclic nucleotide phosphodiesterase activity (calcium‐dependent and calcium‐independent) and CDR (calcium‐dependent regulator protein of phosphodiesterase) are present in all ten brain regions examined, with the specific activity of both being highest in areas predominating in grey matter and lowest in areas consisting largely of white matter. Fractionation of cerebral cortex into neuronal and glial perikarya shows that cyclic nucleotide phosphodiesterase and CDR are present at approximately equal levels in both cell types. Subcellular fractionation reveals that calcium‐sensitive enzyme specific activity, as well as CDR are highly localized in the 100,000 g supernatant fraction. Regional, cellular and subcellular distribution studies indicate that the distribution of CDR closely parallels the distribution of enzyme specific activity, suggesting that the levels of cyclic nucleotide phosphodiesterase and CDR may be synchronized.

Regional protein alterations in rat kidneys induced by lead exposure.

Lead is a potent neuro‐ and nephrotoxin in humans and a renal carcinogen in rats. Previous studies have detected lead‐induced increases in the activities of specific detoxification enzymes in distinct kidney cell types preceding irreversible renal damage. While preferential susceptibility of the highly vascularized cortex to the effects of lead is clear, lead effects on the medullary region have remained unexplored. The present study was undertaken to investigate the extent to which regional renal protein expression differs and to determine which, if any, regionally distinct protein markers indicative of leads renotoxic mechanism might be detected in kidney cortical and medullary cytosols. We examined protein expression in these two functionally and anatomically distinct regions, and identified several proteins that are differentially expressed in those regions and were significantly altered by lead. Kidney cytosols from rats injected with lead acetate (114 mg/kg, three consecutive daily injections) were separated by two‐dimensional electrophoresis. Lead exposure significantly (P<0.001) altered the abundance (either ↑ or ↓) of 76 proteins in the cortex and only 13 in the medulla. Eleven of the proteins altered in the protein patterns were conclusively identified either by matrix‐assisted laser desorption/ionization mass spectrometry / electrospray ionization‐mass spectrometry (MALDI‐MS/ESI‐MS) analysis of peptide digests, immunological methods, or by gel matching. Several of the cortical proteins altered by lead were unchanged in the medulla while others underwent similar but lesser alterations. These observations reflect the complexity of leads nephrotoxicity and endorse the application of proteomics in mechanistic studies as well as biomarker development in a variety of toxicologic paradigms.

Proteomic analysis of simulated occupational jet fuel exposure in the lung.

We analyzed protein expression in the cytosolic fraction prepared from whole lung tissue in male Swiss‐Webster mice exposed 1 h/day for seven days to aerosolized JP‐8 jet fuel at concentrations of 1000 and 2500 mg/m3, simulating military occupational exposure. Lung cytosol samples were solubilized and separated via large scale, high resolution two‐dimensional electrophoresis (2‐DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant quantitative and qualitative changes in tissue cytosol proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass fingerprinting, confirmed by sequence tag analysis, and related to impaired protein synthetic machinery, toxic/metabolic stress and detoxification systems, ultrastructural damage, and functional responses to CO2 handling, acid‐base homeostasis and fluid secretion. These results demonstrate a significant but comparatively moderate JP‐8 effect on protein expression and corroborate previous morphological and biochemical evidence. Further molecular marker development and mechanistic inferences from these observations await proteomic analysis of whole tissue homogenates and other cell compartment, i.e., mitochondria, microsomes, and nuclei of lung and other targets.

EFFECTS OF ACUTE HYPERTHERMIA ON POLYRIBOSOMES, IN VIVO PROTEIN SYNTHESIS AND ORNITHINE DECARBOXYLASE ACTIVITY IN THE NEONATAL RAT BRAIN

—Acute hyperthermia produces in situ disaggregation of brain polyribosomes in infant rats, as determined by electron microscopy. Protein synthesis is inhibited in infant, but not weanling, rat brain by 45 min of hyperthermia; this inhibition is reversed during a 2 h recovery period at normothermic conditions. Hepatic protein synthesis was inhibited less than that of brain. Acute hyperthermia also leads to a profound loss of ornithine decarboxylase activity in brain; during recovery the activity of this enzyme overshoots to values greater than those of normothermic control rats. This increase is blocked by cycloheximide administration. In testis, a tissue with high ornithine decarboxylase activity, enzyme activity was not affected by hyperthermia and recovery, indicating tissue specificity for these effects.

Isolation and purification of a calcium-binding protein from electroplax of Electrophorus electricus

An acidic calcium-binding phosphoprotein has been isolated from a cholinergic tissue, electroplax from Electrophorus electricus. The purification procedures included (NH4)2SO4 fractionation, boiling treatment, ECTEOLA-cellulose chromatography, and gel filtration on Sephadex G-100. Experiments were performed to compare this protein and a calcium-binding protein isolated from mammalian brain, adrenal medulla, and testis. These experiments showed that the two proteins were identical in terms of molecular weight (14 000), calcium-binding dissociation constant (kd=2.1-10(-5) M), electrophoretic mobility at pH 8.7 in 15% polyacrylamide gels, and phosphorus content (1 mol phosphorus per mol protein). In addition, the two proteins had similar amino acid compositions and peptide maps. Although the electroplax protein was not present in eel skeletal muscle, preliminary experiments indicated that small amounts of the protein were present in other eel tissues, namely brain, liver and spleen. These results suggest an identity between the electroplax and mammalian calcium-binding proteins and extend the findind of comparatively large amounts of the protein from mammalian nervous tissue to a cholinergic nervous tissue, electroplax. The close similarity of the proteins suggests a conservation of structure during evolution which may be required to fulfill a role in neuronal function.

The effects of hyperthermia on polyribosomes and amino acid levels in infant rat brain

Abstract Brain polyribosomes prepared from infant rats exposed to elevated ambient temperatures (39.5°C for 45 min) are highly disaggregated. Disaggregation is evident 15 min after the animals have entered the hot environment and is nearly maximal after 25 min. Substantial recovery of polyribosomes is seen 20 min after the hyperthermic rats are transferred from 39.5 to 33°C ambient temperature, indicating that heat-induced disaggregation is quickly reversible. During hyperthermia the levels of certain amino acids in the brain, particularly essential amino acids, rise markedly. The increases in amino acid levels are reversed within 1 h after the rats are transferred from the hot environment to 33°C ambient temperature. The levels of amino acids in blood plasma also rise during hyperthermia; however, the increases are relatively smaller and less selective than in the brain but are also reversed by returning the animals to 33°C ambient temperature. Injection of phenylalanine into infant rats causes hyperphenylalaninemia, elevated brain phenylalanine levels, and brain polyribosome disaggregation ( Aoki & Siegel , 1970 ). The present observations show that elevated brain amino acid levels and polyribosome disaggregation are also linked in hyperthermic infant rats, but it is not yet clear if the two phenomena are causally related. Although the mechanisms of the increases of blood and brain amino acid levels remain to be elucidated, they may have a significant bearing on the functional status of the protein synthetic machinery of the brain, on the levels of neurotransmitter substances in the brain which are derived from amino acid precursors, and on the intermediary metabolism of the brain.

Purification of a calcium-binding protein from pig brain☆

Abstract Proteins from pig brain were screened for high affinity calcium binding by gel filtration of protein fractions previously incubated with calcium-45. Only two proteins from brain exhibited calcium binding in this assay. One of these proteins has been isolated and purified to homogeneity as judged by electrophoresis as a single band in acrylamide gels. The purification procedure employed extraction of a pig brain acetone powder with 0.05 m Tris-HCl, pH 7.4, and fractionation of the proteins in this aqueous extract by ammonium sulfate precipitation, chromatography on ECTEOLA-cellulose, gel filtration on Sephadex G-75, and chromatography on DEAE-cellulose. The molecular weight of the calcium-binding protein was found to be 13,000 as determined by gel filtration. The electrophoretic behavior of this protein was not influenced by calcium or 8 m urea, which do produce electrophoretic heterogeneity of the S-100 protein, which also binds calcium. Amino acid analysis revealed that the protein is rich in glutamic and aspartic acids, threonine, serine, leucine, glycine and lysine. Fractionation of pig liver acetone powder demonstrated that this calcium-binding protein is not present in liver.