Use of Lilium longiflorum, cv. ace pollen germination and tube elongation as a bioassay for the hepatocarcinogens, aflatoxins.

Although various animal tissues are used for bioassay of aflatoxins (B1, B2, G1, G2), a rapid bioassay dependent upon a plant part's response does not exist. Both pollen germination (G) and tube elongation (TE) were enhanced in a 3.0 mM KH2PO4 (K)-containing but AFB1-lacking, modified Dickinson's medium. The B1 did not affect G when K was withheld but K supplementation impaired G above 15 micrograms/ml B1. Without K, 5-20 stimulated but 25 and 30 micrograms/ml B1 inhibited TE which was suppressed by every B1 conc tested in K-containing medium. Addition of NaH2PO4(N) instead of K to medium did not promote G. Slight G stimulation occurred at 16.6 micrograms/ml mixed aflatoxins (MA) in medium lacking either K or N but low G inhibitions were observed with K or N. The MA at 33.3 micrograms/ml reduced G 2.5% in K's of N's absence and 26 or 17% in their presence. While K did not stimulate TE without MA, N did 26%. At 16.6 and 33.3 micrograms/ml MA, TE was reduced 19, 6, 19% and 24, 25, 31%, respectively, in control, K- and N- media. Pollen G and TE were markedly sensitive to G1. Significant inhibitions of Zea mays seed G were observed at 5.8 and 11.6 micrograms/ml B1 but not root elongation (RE) from 0.4-11.6 micrograms/ml. The MA (31.5 micrograms/ml) administered for 72-240 hr did not influence either Arachis hypogeae seed G or RE. However, imbibing 5 cultivars each of Avena sativa (65-117 hr) and Hordeum vulgare (39-89 hr) inhibited RE 4/15-62%. Thus, except for Z. mays, pollen G and TE appear to be more B1-sensitive than seed G and RE. But, the pollen bioassay is less sensitive than both certain animal bioassays (0.025 micrograms/ml) and analytical methodologies (10 pg.)

Carbohydrate and protein contents of grain dusts in relation to dust morphology.

Grain dusts contain a variety of materials which are potentially hazardous to the health of workers in the grain industry. Because the characterization of grain dusts is incomplete, we are defining the botanical, chemical, and microbial contents of several grain dusts collected from grain elevators in the Duluth-Superior regions of the U.S. Here, we report certain of the carbohydrate and protein contents of dusts in relation to dust morphology. Examination of the gross morphologies of the dusts revealed that, except for corn, each dust contained either husk or pericarp (seed coat in the case of flax) fragments in addition to respirable particles. When viewed with the light microscope, the fragments appeared as elongated, pointed structures. The possibility that certain of the fragments within corn, settled, and spring wheat were derived from cell walls was suggested by the detection of pentoses following colorimetric assay of neutralized 2 N trifluoroacetic acid hydrolyzates of these dusts. The presence of pentoses together with the occurrence of proteins within water washings of grain dusts suggests that glycoproteins may be present within the dusts. With scanning electron microscopy, each dust was found to consist of a distinct assortment of particles in addition to respirable particles. Small husk fragments and "trichome-like" objects were common to all but corn dust

A study of overproduction and enhanced secretion of enzymes. Quarterly report

Wood decay within forests, a significant renewable photosynthetic energy resource, is caused primarily by Basidiomycetous fungi, e.g., white rot fungi. These organisms possess the ability to degrade lignin, cellulose and hemicellulose, the main organic polymers of wood. In the case of the white rot fungi, e.g., Coriolus versicolor, the capacity results from the fungus` ability to elaborate extracellular cellulolytic and ligninolytic enzymes. With regard to the latter, at least one of the enzymes, polyphenol oxidase (PPO) appears within a defined growth medium. This proposal focuses on the over-production and enhanced secretion of PPO, cellulase and lignin peroxidase. There are two major sections to the proposal: (1) overproduction of lignocellulolytic enzymes by genetic engineering methodologies and hyper-production and enhanced secretion of these enzymes by biochemical/electro microscopical techniques and (2) the biochemical/electron microscopical method involves substrate induction and the time-dependent addition of respiration and PPO enzymes

A study of over production and enhanced secretion of enzymes. Quarterly report 1

The current project is concerned with the over-production and enhanced secretion of PPO, cellulase and lignin peroxidase. The project is divided into two segments: over-production of lignocellulolytic enzymes by genetic engineering methodologies and hyper-production and enhanced secretion of these enzymes by biochemical/electron microscopical techniques. The former approach employs recombinant DNA procedures, ligation of appropriate nuclease generated DNA fragments into a vector and the subsequent transformation of Escherichia coli to yield E. coli harboring a C. versicolor DNA insert. The biochemistry/electron microscopical method involves substrate induction and the time-dependent addition of respiration and PPO inhibitors to elevate C.versicolor`s ability to synthesize and secrete lignocellulosic enzymes. In this connection, cell fractionation/kinetic analysis, TEM immunoelectron microscopic localization and TEM substrate localization of PPO are being employed to assess the route of secretion. Both approaches will culminate in the batch culture of either E. coli or C. versicolor, in a fermentor with the subsequent development of rapid isolation and purification procedures to yield elevated quantities of pure lignocellulosic enzymes. During the past year, research effort were directed toward determining the route of polyphenol oxidase (PPO) secretion by the wood-decay fungus, Coriolus versicolor. In addition, research activities were continued to over-produce and to purify PPO as well as define the time-dependent intra- and extra-cellular appearances of C. versicolor ligninases and cellulases

A study of over-production and enhanced secretion of enzymes. Quarterly report 2

This project is concerned with the over-production of ligno-cellulolytic enzymes which are relevant to the paper-pulp industry and agricultural community. Since ligno-cellulosics are components of wood, the project involves the forest, a renewable energy resource. Attention is focused on the following: over-production of polyphenol oxidase; establishment of the route of polyphenol oxidase secretion; regulation of polyphenol oxidase secretion; purification of extracellular oxidase

The role of RECK in the pathogenesis of nonalcoholic steatohepatitis

[EMBARGOED UNTIL 12/1/2023] Reversion-inducing cysteine-rich protein with Kazal motifs, or RECK, is a cell surface-anchored glycoprotein and a critical regulator of the extracellular matrix (ECM). It has been examined extensively as an anti-metastatic peptide in the field of oncology, though its role in regulating inflammation and fibrogenesis has recently been postulated. As both inflammation and fibrosis are inherent in the pathology of nonalcoholic steatohepatitis (NASH), this dissertation explores the potential of RECK to serve as a hepatoprotective agent. NASH is a growing medical concern across the globe, for which there are currently no therapeutics available. Studies here show RECK is downregulated in the liver tissue of individuals with progressive NASH. Furthermore, utilizing transgenic rodent models, RECK is demonstrated to play a regulatory role in the development of NASH. Mechanistically, RECK may be exerting these effects through the ADAM10/ADAM17-AREG-EGFR signaling axis, as alterations in this pathway correlated with experimental manipulations of RECK. Furthermore, experiments here revealed hepatic stellate cells (HSCs) to be downstream targets in this process, as exposure to exogenous AREG induced a pro-fibrotic activation phenotype in these cells. This dissertation thus demonstrates the importance of hepatocellular RECK and attempts to provide a mechanistic explanation for this novel finding. Thus, RECK is identified as a potential therapeutic target which may be utilized in the treatment of NASH.Includes bibliographical references