Herschel Sidransky

Identification and immunohistochemical localization of a tryptophan binding protein in nuclear envelopes of rat liver

A tryptophan binding protein which was identified by binding studies has been purified to apparent homogeneity from rat liver nuclear envelopes. Two affinity matrices, namely, concanavalin A-agarose and tryptophan-agarose, were utilized for purification of the binding protein. Findings with lectin affinity chromatography suggested that the binding entity was a glycoprotein since it could be eluted off the column with methyl alpha-D-mannopyranoside (0.2 M). Eluates from both columns, when electrophoresed separately (under denaturing conditions) on polyacrylamide gels, revealed the presence of a protein with an apparent molecular weight of approximately 33,000-34,000 which is the same as that observed when covalently bound (i.e., crosslinked) [3H]-tryptophan is analyzed on polyacrylamide gels under denaturing conditions and then autoradiographed. Polyclonal antibodies raised against the binding protein recognized polypeptides with molecular weights of 64,000 and 33,000-34,000 when analyzed by the Western blot technique, suggesting that the protein was probably a dimer. Immunohistochemical studies revealed that the antigen is localized in the nuclear membranes, thereby corroborating our biochemical premise that the binding protein was present in the nuclear envelopes.

In vivo and in vitro studies on the effect of tryptophan on translocation of RNA from nuclei of rat liver.

Abstract The effect of a single administration of tryptophan on the transport of nuclear RNA to the cytoplasm of rat liver was investigated. Using a cell-free system containing nuclei (RNA labeled in vivo with [14C]orotate) and dialyzed cell sap of liver, the isolated hepatic nuclei of tryptophantreated rats demonstrated an increase in the release of labeled RNA into the medium in comparison to hepatic nuclei of control rats. Also, liver cell sap of tryptophan-treated rats with added tryptophan to the medium was found to stimulate the release of labeled RNA (poly(A)-mRNA) from isolated control nuclei. Pretreatment of rats with cycloheximide or puromycin before tryptophan administration prevented the liver cell sap effect. However, the hepatic nuclei of puromycin- and tryptophan-treated rats demonstrated an increase in the release of labeled RNA into the medium incubated with control liver sap, similar to that observed with hepatic nuclei of rats treated with tryptophan alone. These results suggest that the tryptophan-induced enhancement of nucleocytoplasmic translocation of RNA may be related to alterations in the controls within the nucleus and cytoplasm that act to regulate the intracellular transport of RNA.

Effect of tryptophan on nuclear envelope nucleoside triphosphatase activity in rat liver.

Summary The activity of nuclear envelope nucleoside triphosphatase, an enzyme implicated to be involved in nucleocytoplasmic translocation of mRNA, was investigated in the livers of rats that received a single tube-feeding of tryptophan. Tryptophan administered to fasted rats or to rats pretreated with puromycin or actinomycin D caused significant increases in the activity of Mg2+-dependent nucleoside triphosphatase in the hepatic nuclear envelopes over that in controls. Concomitant with this rapid (10 min) increase in the enzyme activity, there was more release of RNA from isolated nuclei of livers of tryptophan-treated rats than from those of control rats.

Tryptophan and carcinogenesis: review and update on how tryptophan may act.

This review covers the historical developments of the consideration that tryptophan may influence the induction of cancer in experimental studies. Studies relating to stimulatory effects, as well as to inhibitory effects, of tryptophan or tryptophan-related compounds are described. Also the effects of pyrolysis products of tryptophan on carcinogenesis are covered. In consideration that new L-tryptophan-related contaminants may be involved in a recently described human disease, a description is given of the eosinophilia-myalgia syndrome, which has been attributed to the ingestion of L-tryptophan-containing related contaminants. Whether these new L-tryptophan-related contaminants alone or together with L-tryptophan may prove to be carcinogenic remains to be determined. Lastly, recent developments relating to regulatory effects of L-tryptophan on liver metabolism are reviewed and then considered as possibly playing a role in carcinogenesis.

Influence of L-alanine on effects induced by L-tryptophan on rat liver

Abstract This study was designed to determine if a nuclear tryptophan receptor-ligand relationship plays a role in the stimulatory effects of L-tryptophan administration on rat liver. Two compounds, L-alanine and DL-β-(1-naphthyl)alanine(β-NA), which competed for in vitro 3 H-tryptophan binding to hepatic nuclei similar to that of L-tryptophan but which when administered alone in vivo did not stimulate hepatic protein synthesis as did L-tryptophan, were used in this study. Rats treated with L-alanine or β-NA together or before L-tryptophan failed to demonstrate the stimulatory effects in the liver (protein synthesis, nuclear poly(A)polymerase activity, and nuclear RNA efflux) as observed in rats treated with L-tryptophan alone. The findings indicate that the enhanced hepatic protein synthesis that occurs following the in vivo administration of L-tryptophan appears to be related to L-tryptophans ability to bind to a specific nuclear tryptophan receptor.

Effect of tryptophan on rat hepatic nuclear poly(A)polymerase activity

SummaryAddition of poly(A) to hnRNA in the cell nucleus is a post-transcriptional event and is presumed to be brought about by a specific poly(A)polymerase. Since it is known that tryptophan rapidly increases the cytoplasmic levels of polyadenylated mRNA, it was of interest to investigate whether the essential amino acid, tryptophan, affects the enzyme responsible for polyadenylation. Tryptophan (300 mg/kg body wt.) tube-fed for 10 min elevated the hepatic nuclear enzymatic activities of both the chromatin-bound nuclear poly(A)polymerase (44%, n = 7) as well as that of the free solubilized form (48%, n = 7). Hepatic nuclear proteins separated under denaturing conditions, transferred to nitrocellulose sheets, and then probed with antibody raised against hepatic nuclear poly(A)polymerase showed no differences between the hepatic nuclei of control and tryptophan-treated rats.

The presence of thiols in the hepatic nuclear binding site for L-tryptophan: Studies with selenite

Abstract This study investigated the effects in vitro of selenite and selenate on nuclear L-tryptophan receptors of rat liver. Sodium selenite at 10 −6 M inhibited the in vitro 3 H-tryptophan binding to rat liver nuclear receptors. No inhibitory effect of selenite on 3 H-tryptophan binding to nuclear receptor was found in the presence of 10 −4 M dithiothreitol, a protective agent for sulfhydryl groups. Selenate as well as sulfite or sulfate did not exert an inhibitory effect on the tryptophan receptor. The results based upon in vitro studies suggest that selenium in the form of selenite may reversibly affect the L-tryptophan binding at the sulfhydryl sites in the nuclear receptor protein. In vivo administration of high (toxic) levels of selenite before or along with L-tryptophan inhibited the L-tryptophan-induced stimulation of hepatic protein synthesis.

Inhibitory effect of demoxepam on tryptophan binding to rat hepatic nuclei

Since some patients with eosinophilia-myalgia syndrome ingested tryptophan along with benzodiazepines, we investigated whether demoxepam, the N-desalkylated compound of chlordiazepoxide, would influence the binding of tryptophan to hepatic nuclei. L-Tryptophan has been shown to bind (saturable, stereospecific, and of high affinity) to rat hepatic nuclei and nuclear envelopes. We report that demoxepam has an inhibitory effect on in vitro [3H]tryptophan binding to rat hepatic nuclei and has an apparent KD approximately 22 microM.

Protective effect of tryptophan and cysteine against carbon tetrachloride-induced liver injury.

The effects of the administration of tryptophan and/or cysteine on carbon tetrachloride (CCl4)-induced hepatic injury were investigated. Rats received CCl4 (1 ml/kg ip) followed 6 hr later by tryptophan (300 mg/kg) and/or cysteine (950 mg/kg) via stomach tube and rats were killed after 24 hr. Treatment with tryptophan, cysteine, or both reduced the degree of hepatic necrosis observed histologically. While CCl4 caused polyribosomal disaggregation and decreased [14C]leucine incorporation into liver proteins in vitro and in vivo, treatment with tryptophan, cysteine, or both caused a shift in polyribosomes toward heavier aggregation and protein synthesis was increased. Serum activities of lactic dehydrogenase (LDH), glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and gamma-glutamyl-transpeptidase were markedly increased after CCl4 alone but after subsequent treatment with cysteine or with tryptophan and cysteine appreciable decreases occurred. Glutathione concentration decreased but total amount remained constant in the livers of CCl4-treated rats while subsequent treatment with cysteine alone or together with tryptophan elevated both levels of glutathione. Using isolated hepatocytes, CCl4 caused decreases in cell viability, in release of LDH, and in [14C]leucine incorporation into protein. Treatment with CCl4 and tryptophan and/or cysteine revealed that cysteine alone or with tryptophan improved cell viability and decreased LDH release of the cells, while tryptophan alone or with cysteine improved protein synthesis. Upon cytologic evaluation, the isolated hepatocytes revealed membrane distortions after CCl4 alone but these were less marked after CCl4 plus tryptophan, cysteine, or both (most improvement). Thus, tryptophan and cysteine act in a beneficial manner against CCl4-induced hepatic injury in the rat.

Effect of tryptophan on hepatoma and host liver of rats. Influence after treatment with hypertonic sodium chloride and carbon tetrachloride.

Abstract Female inbred Buffalo rats bearing intrahepatically transplanted hepatoma 5123 were subjected intraperitoneally to the acute administration of hypertonic NaCl or CCl 4 followed by a tube-feeding of l -tryptophan. The responses in terms of changes in polyribosomal aggregation and protein synthesis ( in vitro ) of host liver and hepatoma were evaluated. While treatment with hypertonic NaCl or CCl 4 caused disaggregation of polyribosomes and inhibition of protein synthesis in both host liver and hepatoma, the subsequent administration of tryptophan caused some improvement in both parameters in host liver but not in hepatoma. Administration of hypertonic NaCl alone caused a decrease in [ 14 C]orotate incorporation into poly(A)-mRNA of host liver and hepatoma, whereas administration of tryptophan after hypertonic NaCl caused a significant improvement in host liver alone. Following the tryptophan administration, the activities of nuclear DNA-dependent RNA polymerases I and II, and of nuclear-envelope nucleoside triphosphatase, as well as labeled nuclear RNA release in vitro were slightly elevated in host liver but not in hepatoma. Tryptophan-related compounds, 5-hydroxy- dl -tryptophan, 5-fluorotryptophan, indole, and 3-hydroxyanthranilic acid, when administered in place of l -tryptophan, did not appreciably affect polyribosomal aggregation or protein synthesis in vitro in host liver or hepatoma.