Steven E. Kornguth

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.

Paraneoplastic Retinopathy Associated with Andretinal Bipolar Cell Antibodies in Cutaneous Malignant Melanoma

Purpose: It has been shown previously that the sera, from patients with visual paraneoplastic syndrome associated with lung cancer, contain immunoglobulins that are reactive with the tumor and with photoreceptor and large retinal ganglion cells. The purpose of this study is to determine the retinal cell population that reacts with immunoglobulins in the sera of patients with melanoma-associated retinopathy. Methods: Clinical and electrophysiologic studies were used to determine the locus responsible for the visual defect in each patient. Sera from two patients with melanomaassociated retinopathy, from a patient with herpes zoster, and from a patient who had a colon tumor were obtained. The sera were incubated with sections of retina obtained from a healthy 3-year-old child who had died of asphyxiation. The tissue sections subsequently were incubated with biotin-labeled anti-human immunoglobulin G, and then with streptavidin-labeled peroxidase. Finally, the tissue sections were developed to show peroxidase activity in the targeted retinal cells. Results: Clinical and electrophysiologic studies were consistent with a defect in intraretinal transmission distal to the photoreceptors. The immunoglobulins from the patients with the melanoma-associated retinopathy reacted selectively with the bipolar cells of the retina; approximately 30% of the bipolar cells were immunoreactive. The sera from the other two patients were not reactive with any of the retinal cells examined. Conclusions: The sera of patients with the paraneoplastic syndrome, melanomaassociated retinopathy, contain high titer immunoglobulins that are reactive only with a subset of the bipolar retinal cells. The clinical, electrophysiologic, and immunologic studies are all consistent with an intra-retinal transmission defect at the level of the bipolar cells.

Autoimmune basis for visual paraneoplastic syndrome in patients with small cell lung carcinoma. Retinal immune deposits and ablation of retinal ganglion cells

Recently, patients with visual paraneoplastic syndrome (VPS) were described, a binocular loss of vision found in patients with small cell carcinoma of the lung (SCCL). The patients have serum antibodies against a small number of discrete antigens which are shared by the retina and small cell carcinoma cells, and which are associated with cells and processes of the ganglion cell layer of the retina. Pathologic findings are presented with regard to the presence of immunoglobulins in, and the nature of the lesions in, the central nervous system of a VPS patient. The patients blood‐brain barrier was shown to be compromised, as demonstrated by the finding of high immunoglobulin levels in the cerebrospinal fluid and immune deposits in the retina. It is further shown that within the central nervous system only the retina and optic nerve show any tissue damage with the specific loss of retinal ganglion cells and their processes. The findings support the hypothesis of an autoimmune cause for this remote effect of cancer.

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.

Isolation and partial characterization of a tubulin-like protein from human and swine synaptosomal membranes

Synaptic membranes from human and swine brains were solubilized with 8 M urea and the proteins were reduced and alkylated. A protein was isolated from both sources and had identical amino acid compositions and molecular weights as determined by electrophoresis on polyacrylamide-sodium dodecylsulfate gels and by ion-exchange chromatography and gel filtration on Bioglas 1000. The apparent molecular weight of the protein was 53 000 on the acrylamide-sodium dodecylsulfate gels. Neither neutral sugars nor sialic acid was a significant component of the protein. When the proteins were digested with trypsin and the resultant peptides subjected to chromatography (n-butanol/acetic acid/water) and electrophoresis (pH 3.7) the peptide maps were identical. The protein comprises 1-2 percent of the total synaptosomal protein. With regard to amino acid composition, molecular weight, peptide map characteristics, behavior on DEAE-cellulose columns, electrophoretic mobility and sugar content, the synaptic protein is quite similar to the monomer of swine tubulin.

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.

Biotechnology: Bridging Research and Applications,

Abstract : Biotechnology is advancing at a rapid pace with numerous applications in medicine, industry, agriculture and environmental remediation. Recognizing this, government, industrial and academic research and development investment in biotechnology has expanded rapidly. The past decade has seen the emergence of applications of this technology with a dual-use potential. Military applications focus on four major areas: biomedical technology, such as vaccine development and medical diagnostics; detection of toxins, chemicals and pathogens; material biotechnology; and biological decontamination, including biodegradation and bioremediation. This conference emphasizes the non-medical applications of biotechnology. The first two sessions focus on the synthesis and properties of molecules that may be used in detectors. The traditional approach to detection of chemical and biological agents relied on the development of specific assays or analyses for known agents. Advances in molecular biology have made possible the production of large quantities of toxins which were previously available in minute quantities, and the molecular engineering of toxins and pathogens with specific pharmacologic and physical-chemical properties.

Effect of polylysine on the cytology of Ehrlich ascites tumor cells

Abstract Studies with fluorescent polylysine have shown that the polylysine binds to the lipoprotein surface of Ehrlich ascites tumor cells and does not penetrate the cell membrane to any appreciable extent within 10 min. Following polylysine treatment the mitochondria and nuclei were found near the cytoplasmic membrane. This disorganization may cause the previously observed telophase inhibition and unipolar mitosis caused by polylysine in vivo. Cytoplasmic agglutination of RNA and protein was probably caused by the polyelectrolyte character of the polycation, and chromatin clumping was most likely caused by sulfhydryl-polylysine interaction. The morphological changes resulting from the interaction of polylysine with Ehrlich ascites tumor cells seem identical with those noted following the reaction of these cells with specific antibodies and complement.

Neuronal Proteins and Paraneoplastic Syndromes

AN early manifestation of small-cell carcinoma of the lung and ovarian carcinoma is peripheral or central nervous system dysfunction — the so-called paraneoplastic syndromes — which is frequently e...

Isolation of synaptic complexes in a caesium chloride density gradient: electron microscopic and immunohistochemical studies.

A technique is described for the improved isolation of synaptic complexes. Homogenates of guinea pig cerebellum and cerebral cortex were subjected to centrifugation, first in a discontinuous sucrose gradient, and secondly in a continuous caesium chloride gradient. Of the six bands that were obtained, band four contained a high proportion of synaptic complexes showing both pre‐ and postsynaptic elements attached to each other; myelin and free mitochondria were not contaminants of this fraction. Fluorescein‐labelled γ‐globulin prepared against band four reacted with synaptic regions of cerebellum and cerebral cortex, but not with cervical cord.