Adsorption of messenger RNA of Novikoff hepatoma, normal liver, and regenerating liver on complementary DNA-cellulose affinity matrices.

Complementary DNA (cDNA)-oligodeoxythmidylate-celluloses were prepared from cDNA copies of polysomal messenger RNA (mRNA) of Novikoff hepatoma, normal rat liver, and regenerating rat liver. cDNA synthesis with reverse transcriptase was approximately 46% with respect to input mRNA with oligodeoxythymidylate-cellulose primer. The cDNA's of normal liver, regenerating liver, and Novikoff hepatomas were used as affinity matrices for hybridization of different mRNA species. Under the conditions used, degradation of mRNA was not detected. After normalization for homologous hybridization efficiency, 53 and 65% of the Novikoff hepatoma mRNA bound to normal liver and regenerating liver cDNA's. Under these conditions an average of 82% of mRNA of normal liver bound to regenerating liver cDNA, and 92% of regenerating liver mRNA bound to normal liver cDNA. The bound and unbound mRNA's were analyzed by translation in the wheat germ system; 2-D gel analysis of the proteins synthesized in the wheat germ system indicated that the cDNA affinity columns selectively adsorbed some mRNA species.

Electrophoresis of human multiple myeloma and Waldenström's macroglobulinemia sera in sodium dodecyl sulfate polyacrylamide gels.

Sera from normal persons and patients with IgA, IgD, IgG, and IgM monoclonal gammopathies were electrophoresed in polyacrylamide gels containing sodium dodecyl sulfate. The gels were stained with Coomassie blue or were used for immunodiffusion. By this method IgG multiple myeloma and IgM Waldenström's macroglobulinemia sera were readily distinguished by electrophoresis alone, whereas IgA and IgD myeloma sera were distinguished by further immunodiffusion against anti-alpha-chain antibody and anti-delta-chain antibody.

Characterization of monosomes produced by aflatoxin B1.

A single injection of 1.5 mg aflatoxin B1 per kg body weight produced approx. 70% disaggregation of rat liver polysomes into monosomes within 18 h. Isolated monosomes dissociated into 40 S subunits during centrifugation in linear sucrose gradients containing 0.3 M KCI. The 4 S to 5 S molar RNA ratio of the monosomes was calculated to be 0.6, indicating 0.6 tRNA and/or aminoacyl tRNA molecule per ribosome; no peptidyl tRNA was present. These results suggest that a single injection of affatoxin B1 produces monosomes which resemble runoff ribosomes.

Electromechanical transduction: reduction-driven hydrophobic folding demonstrated in a model protein to perform mechanical work.

It has long been appreciated that hydrophobic folding is an important element of protein structure formation. Here it is demonstrated for the first time that the electrochemical or chemical reduction of a nicotinamide in a model protein, which increases hydrophobicity, can drive hydrophobic folding and assembly in such a way as to lift a weight or otherwise contract against a constant tensional force. The model protein, poly[0.73(GVGVP),0.27(GK(NMeN)GVP], can be gamma-irradiation cross-linked to form an elastic matrix which contracts on raising the temperature from below to above the transition range for hydrophobic folding and assembly. On reduction of the N-methyl nicotinamide, (NMeN), the transition temperature range is lowered from above to below 20 degrees C to drive contraction due to hydrophobic folding with the performance of mechanical work.

Electro-chemical transduction in elastic protein-based polymers: a model for an energy conversion step of oxidative phosphorylation.

A pair of functional moieties, the carboxyl of an aspartic acid (Asp, D) residue and an N-methyl nicotinamide (NMeN) formed on amide linkage to the epsilon-amino group of the lysine (Lys, K) residue, are coupled to perform energy conversion by means of controlling the transition temperature, Tt, of a common hydrophobic folding and assembly domain within the polytricosapeptide, poly[GDGFP GVGVP GVGVP GFGVP GVGVP GVGK(NMeN)P]. The input of electrochemical energy in the form of the reduction of nicotinamide results in a reduction-induced increase in pKa by 2.5 pH units which represents the performance of the chemical work of picking up a proton. The primary structure and the structures of the oxidized and reduced states are verified by two-dimensional nuclear magnetic resonance. Thus electrochemical transduction, the conversion of electrochemical energy into chemical energy, has been demonstrated for the first time in a designed, synthetic protein-based polymer.

Reduction-driven polypeptide folding by the delta Tt mechanism.

Poly(Gly-Val-Gly-Val-Pro), i.e., poly(GVGVP), exhibits composition and solute dependence of Tt, the temperature of the inverse temperature transition at which hydrophobic folding and assembly occur on raising the temperature. Importantly, a means whereby the value of Tt is lowered from above to below the working temperature becomes an isothermal means of driving folding and assembly, i.e., of achieving free energy transduction. Using poly[0.73(GVGVP),0.27(GK[NMeN]GVP)] where [NMeN] indicates N-methyl nicotinamide attached to the epsilon-NH2 of the Lys(K) residue, chemical and electrochemical reductions are found to remarkably lower the value of Tt; reduction can drive hydrophobic folding and assembly as effectively as decreasing ionization. Changing the redox state of a protein becomes yet another means of achieving free energy transduction by the delta Tt mechanism.

In vitro assessment of the effect of halogenated hydrocarbons: chloroform, dichloromethane, and dibromoethane on embryonic development of the rat.

Halogenated hydrocarbons are widely used in industry, the laboratory, and in the home. In the present study three of these solvents--chloroform, dichloromethane, and dibromoethane--were examined for embryotoxic/teratogenic potential using rat embryo culture. The results showed that each of the solvents had a concentration-dependent embryotoxic effect on the developing rat embryo in vitro. The effect and no-effect concentrations (expressed in mumol/ml culture medium), respectively, for each of the halogenated hydrocarbons tested were: dibromoethane--0.33, < 0.18; chloroform--2.06, 1.05; dichloromethane--6.54, 3.46. The levels of chloroform and dichloromethane found to be embryotoxic in the present study were compared to reported blood levels attained following controlled human exposure. In the industrial situation, if the current exposure levels are adhered to, chloroform and dichloromethane appear to have little potential for reproductive toxicity in the human. Fatal or near fatal solvent levels would be required in the mother for the embryotoxic level to be reached. For dibromoethane, there are no reports following controlled human exposure presumably due to its carcinogenicity. In an attempt to elucidate the mechanism of embryotoxicity, histological studies were performed after exposure of rat embryos to an embryotoxic level of each of the halogenated hydrocarbons studied, for increasing time periods up to the standard 40-hour culture. Marked cell death in the neuroepithelium of the developing neural tube was a prominent feature in all embryos exposed to an embryotoxic level of these solvents for periods of 16 hours of longer.

Use of computational models to reconstruct and predict trichloroethylene exposure.

In this study, a type frequently encountered by ATSDR, groundwater and surface-water contamination have occurred near the Gratuity Road site in the town of Groton, Massachusetts. A petitioned public health assessment for the Gratuity Road site identified the primary contaminants as trichloro-ethylene (TCE), 1,1,1-trichloroethane (TCA), hexavalent chromium (Cr+6), chromium (Cr), and lead (Pb) (ATSDR 1992). The health assessment also indicated that off-site residential groundwater wells had been contaminated with TCE and TCA. Because direct measures of historical exposure to TCE are unavailable for the Gratuity Road site, computational models were used to reconstruct and predict exposure to TCE. These computational models included environmental transport and exposure models. For the environmental transport models, numerical methods were used to approximate the equations of groundwater flow and contaminant transport. Results of using environmental transport models provided us with the spatial and temporal database necessary to conduct an exposure analysis. This database indicated that groundwater concentrations of TCE typically exceeded EPA's MCL of 5 ppb for TCE. The study demonstrated that although a hazardous waste site can be remediated, nearby populations may experience significant exposure because of historical contamination, which will not be captured by remediation activities. The exposure analysis used simulated concentrations of TCE predicted by environmental transport models. These concentrations were used to compare exposure to TCE from inhalation in a one-compartment model shower with exposure from ingestion of domestic water contaminated by TCE. The exposure model indicated that exposure to TCE by the inhalation route during showering is nearly identical to exposure by ingestion of domestic water supplies contaminated with TCE. As a result, entry by inhalation route is as important as entry by ingestion route when conducting exposure analyses of contamination from volatile organic compounds such as TCE.