Edward A. Smuckler

A method for staining epoxy sections for light microscopy

A technique for staining sections of osmium-fixed, epoxy-embedded tissues for light microscopy is presented. The method employs aqueous toluidine blue at pH 11.1 and does not require prior removal of embedding medium. When stained with this technique and viewed with an oil immersion objective, the images are striking because of their great definition and resemblance to the images are striking because of their great definition and resemblance to of areas seen in the electron microscope; it also permits better utilization of the full resolving power of the light microscope.

The testicular morphology of individuals exposed to dibromochloropropane

Abstract Testicular biopsies from 10 individuals with histories of exposure to 1,2-dibromo-3-chloropropane (DBCP) were studied by light and electron microscopy. In seven subjects, spermatogenic activity was either significantly decreased or absent. There appeared to be a correlation between the diminution in spermatogenesis and the duration of industrial exposure to the agent.

Alterations of specific forms of cytochrome P-450 in rat liver during acute carbon tetrachloride intoxication

Male rats were pretreated with phenobarbital, 3-methylcholanthrene, or the corresponding vehicles, administered carbon tetrachloride (CCl4), and sacrificed 3 hr later. Hepatic microsomes were isolated and assayed for cytochrome P-450 content and mixed-function oxidase activity. The residual content of cytochrome P-450 after CCl4 intoxication was similar in saline and phenobarbital-pretreated animals. Substantially greater amount of cytochrome P-450 remained in animals pretreated with 3-methylcholanthrene and then challenged with CCl4. In saline-pretreated animals, the dealkylation of 7-ethoxycoumarin, but not benzphetamine or N,N-dimethylaniline, was decreased following CCl4 exposure. The enhanced dealkylation following phenobarbital, but not following by 3-methylcholanthrene, was also decreased after CCl4 poisoning. Microsomes were solubilized with sodium cholate; cytochrome P-450 was partially purified by ω-octylamino Sepharose 4B column chromatography; and cytochrome P-450-containing elements were separated by SDS-polyacrylamide gel electrophoresis. A decrease in the staining intensity of a band migrating with a molecular weight of 51,600 was noted following CCl4 treatment. Furthermore, the phenobarbital-induced component (MW 51,600), but not that appearing after 3-methylcholanthrene induction (MW 57,900), was also diminished. Microsomal proteins separated by SDS-polyacrylamide gel electrophoresis and stained for heme also showed a decrease in staining which was greater in those microsomal proteins from phenobarbital and saline than in 3-methylcholanthrene-induced animals. The data suggest that a specific cytochrome P-450(s) in the saline-treated animals as well as the cytochrome P-450(s) induced by phenobarbital, but not the 3-methylcholanthrene-induced cytochrome P-450, are susceptible to CCl4-induced destruction.

Carbon Tetrachloride Poisoning in Rats: Alteration in Ribosomes of the Liver

Microsomes from livers of albino rats treated with carbon tetrachloride were compared with those from normal rats with respect to their ability to incorporate amino acid. Acute carbon tetrachloride poisoning results in depressed capacity of microsomes to incorporate amino acid. From ultracentrifugal data, there is an apparent dissociation of 79S ribosomes into 54S components.

A comparison of the effects of partial surgical and partial chemical (CCl4) hepatectomy on microsomal cytochrome b5 and P450 and oxidative N-demethylation

SummaryThe effects of partial surgical hepatectomy and chemical (CCl4) hepatectomy on microsomal cytochromes b5 and P450, and oxidative N-demethylation were measured. CCl4-induced liver injury was associated with decreased levels of P450 and both steps in oxidative N-demethylation of dimethylaniline (N-oxide accumulation and formaldehyde release). Cytochrome b5 was rapidly restored to control levels. Surgical hepatectomy resulted in decreased cytochromes, decreased formaldehyde production, but increased N-oxide accumulation. These results were found in adrenalectomized animals, suggesting a direct effect of the hepatectomy.

Effects of SKF 525-A and adrenalectomy on the amino acid incorporation by rat liver microsomes from normal and CCl4-treated rats

Abstract Administration of SKF 525-A to rats is associated with an increase in the capacity of liver microsomes and ribosomes to incorporate labeled amino acids into protein. There is in addition a marked increase in the proportion of rapidly sedimenting ribosomal aggregates. Liver cell sap prepared from SKF-treated animals has also an increased activity in supporting amino acid incorporation by cell-free liver systems. These effects are fully developed 15–22 hours after the administration of the drug, and they are not dependent upon the presence of adrenals. Short-term (4 hours) treatment with SKF affords some protection of the liver against CCl4-induced depression of microsomal amino acid incorporation. With prolonged treatment (16–20 hours) there was a tendency towards potentiation of the CCl4 effect. Adrenalectomy made the rats more resistant to CCl4 challenge. Treatment of adrenalectomized rats with SKF for 16 hours or more reversed this protection. These findings and the observations of others suggest that adrenalectomy and SKF-treatment may interfere with the system responsible for the conversion of CCl4 into an active form, but also that the NADPH-dependent drug-metabolizing enzymes, strongly inhibited by SKF, are not immediately involved in this transformation.

Analysis of the phospholipid of the nuclear envelope and endoplasmic reticulum of liver cells by high pressure liquid chromatography

A method is described for the separation and analysis of phospholipids from rat-liver nuclear envelope and endoplasmic reticulum. The procedure employs a liquid environment, to which antioxidants can be added, and results in separation of NL, PE, PI, PS, and PC in 99% purity in 12 min; analytical columns and a radial compression system may be employed. The procedure results in phospholipids with a large proportion of highly unsaturated fatty acids; some differences in fatty acid distributions were found when nuclear envelope phospholipid fractions were compared with the corresponding fractions from endoplasmic reticulum.

Subcellular localization of human placental aryl hydrocarbon hydroxylase

Abstract Homogenates of human placentas were fractionated by a variety of differential centrifugation techniques. Prepared subfractions were assayed by ultrastructure analysis and marker enzyme quantitation. The lowest ratios of cytochrome oxidase/aryl hydrocarbon hydroxylase activities were observed in a 6.24 × 10 6 g -min sediment fraction following sequential precentrifugations at 1.5 × 10 4 , 9.75 × 10 4 , 2.18 × 10 5 , 3.03 × 10 5 and 4.64 × 10 5 g -min. Investigations of subfractions with respect to acid phosphatase, glucose-6-phosphatase, nucleoside disphosphatase, glucose-6-phosphate dehydrogenase, and aryl sulfatases A, B, and C also tended to support a concept of localization of the mixed-function oxidase system in the endoplasmic reticulum of human placental cells. Consistent with these chemical observations was the enrichment of membranous components seen in electron micrographs of the respective homogenate subfractions.

Structure of the rat alpha 1-acid glycoprotein gene.

The complete nucleotide sequence of the rat alpha 1-acid glycoprotein gene has been determined from an isolated lambda recombinant bacteriophage. Southern blot analysis and DNA sequencing indicate that there is only one gene per genome; it contains six exons and is located within a 3,200-base-pair fragment starting from a TATA box and extending to the polyadenylation signal AATAAA. Transcription starts 37 base pairs upstream from the beginning of the translation codon ATG. The TATA box (TATAAA) lies 26 base pairs upstream from this site. The gene contains several potential glucocorticoid receptor-binding sites, both inside and outside the structural gene.

Nucleocytoplasmic RNA transport

SummaryA number of closely related post-transcriptional facets of RNA metabolism show nuclear compartmentation, including capping, methylation, splicing reactions, and packaging in ribonucleoprotein particles (RNP). These nuclear ‘processing’ events are followed by the translocation of the finished product across the nuclear envelope. Due to the inherent complexity of these interrelated events,in vitro systems have been designed to examine the processes separately, particularly so with regard to translocation.A few studies have utilized nuclear transplantation/ microinjection techniques and specialized systems to show that RNA transport occurs as a regulated phenomenon. While isolated nuclei swell in aqueous media and dramatic loss of nuclear protein is associated with this swelling, loss of RNA is not substantial, and most studies on RNA translocation have employed isolated nuclei. The quantity of RNA transported from isolated nuclei is related to hydrolysis of high-energy phosphate bonds in nucleotide additives. The RNA is released predominantly in RNP: messenger-like RNA is released in RNP which have buoyant density and polypeptide composition similar to cytoplasmic messenger RNP, but which have distinctly different composition from those in heterogeneous nuclear RNP. Mature 18 and 28S ribosomal RNA is released in 40 and 60S RNP which represent mature ribosomal subunits. RNA transport proceeds with characteristics of an energy-requiring process, and proceeds independently of the presence or state of fluidity of nuclear membranes. The energy for transport appears to be utilized by a nucleoside triphosphatase (NTPase) which is distributed mainly within heterochromatin at the peripheral lamina. Photoaffinity labeling has identified the pertinent NTPase as a 46 kD polypeptide which is associated with nuclear envelope and matrix preparations. The NTPase does not appear to be modulated via direct phosphorylation or to reflect kinase-phosphatase activities. A large number of additives (including RNA and insulin) produce parallel effects upon RNA transport and nuclear envelope NTPase, strengthening the correlative relationship between these activities. of particular interest has been the finding that carcinogens induce specific, long-lasting increases in nuclear envelope (and matrix) NTPase; this derangement may underlie the alterations in RNA transport associated with cancer and carcinogenesis.Two considerations for RNA transport studies are discussed: 1) RNA transportin vitro relates to nuclear swelling, and colloidally-active agents (such as polyvinylpyrrolidone) should be employed to prevent nuclear swelling; and 2) contamination of nuclear preparations by nucleus-absorbed cytoplasmic RNA is substantial, and a detergent-rinse procedure is required to remove this contamination.In considering trafficking of RNP through nuclear pores, conceptual difficulties are obvious. Flux studies with exogenous tracers have indicated patent pore diameters of 90–120 Å. However, the RNP which cross the nuclear envelope (presumably through pores) are all at least 200 Å in diameter. Further, evidence is presented showing that nucleoplasmin-coated gold particles (of diameter 200 Å) pass through central channels in nuclear pores. Whereas previous models of RNP transport have enjoyed the luxury of ascribing deformability (local unfolding) to these RNP, this clearly is not possible for the coated gold particles. These considerations suggest that facilitated translocation of RNP occurs in some manner, and that the mechanisms governing this translocation are not subject to the constraints suffered by exogenous tracers in a ‘rigid-channel’ model of the nuclear pore.