Molecular species of epidermal growth factor carrying immunosuppressive activity

The suppression of antibody formation to sheep red cells in mice by partially purified fractions of mouse submaxillary gland was shown to be caused by epidermal growth factor (EGF). Purification of EGF by the method of Savage and Cohen resolved three components referred to as EGF a, EGF b, and EGF c. All three induced premature eye opening in neonatal mice, but only EGF a (identified as EGF 1-53) had full immunosuppressive activity. EGF c was shown by micropeptide mapping of chymotryptic and thermolytic digests and amino-terminal analysis to differ from EGF a only by the presence of beta-aspartyl instead of an asparaginyl residue. EGF b differed from EGF a in that it lacked the N-terminal asparagine. EGF shortened enzymatically at its carboxy terminal by two or five amino acids did not have any immunosuppressive activity. These findings suggest that, in contrast to some other biological effects of EGF, intact amino and carboxy terminals are required for the expression of immunosuppressive activity.     

On the role of tryptophan in luteinizing-hormone-releasing hormone (luliberin).

The tryptophan residue of luteinizing-hormone-releasing hormone (luliberin) was chemically modified to produce the following analogs: [Trp(o)3]luliberin, Trp-(2,4-dinitrophenylsulfenyl)-luliberin, Trp-(2-hydroxy-5-nitrobenzyl)luliberin, (Trp-S-luliberin)2, Trp-CH3S-luliberin and Trp-formyl-luliberin. The luteinizing-hormone-releasing activity of those analogs was determined by bioassay in vitro and found to be 0.2%, 0.2%, 0.6%, 1.5%, 1.7% and 7% of that of the natural hormone, respectively. These results demonstrate that alterations in the indole moiety of tryptophan-3, which lead to a reduction in its electron density or sterically restrict its electron avaliability, are associated with a dramatic loss of biological activity.     

Divergent effects of cyanate on amino acid and phosphate uptake by liver and hepatoma.

The uptake of alpha-aminoiso[3H]butyric acid and 32Pi was observed to be inhibited by sodium cyanate in transplanted hepatomas but was increased in the livers of the tumor bearing rats. Incorporation of 32Pi into macromolecules in hepatomas was also inhibited by cyanate. Treatment with this drug did not influence circulating concentrations of isotope-labeled materials. There were relatively small effects on uptake of 36Cl- in cyanate-treated rats and the action was not tissue specific. The data were compatible with an inhibitory effect of cyanate on active transport in hepatomas which was not seen under the same conditions in host liver.     

Aryl sulfatase inactivation of slow reacting substance. Evidence for proteolysis as a major mechanism when ordinary commercial preparations of the enzyme are used

Type II B arylsulfatases are known to inactivate slow reacting substance (SRS), but the mechanism is unclear. In the present study, ordinary commercial preparations of Sigma limpet arylsulfatase largely inactivated the glutathionyl and cysteinyl-glycyl forms of SRS, but the cysteinyl form of SRS was largely resistant to the enzyme. Evidence is presented which established that a major mechanism for the inactivation of the glutathionyl and cysteinyl-glycyl SRS types, at least by the particular enzyme preparations we have studied, involves cleavage of the glycine moiety from the sulfur containing side chain. This was confirmed by digestion studies with glutathione itself. In addition, there is some evidence to indicate that the enzyme may destabilize the double bond structure of the SRS molecule, contributing to the overall inactivation.     

Effect of screening on the incidence of cervical cancer in Alberta.

The rates of registration of cases of in-situ and invasive cancer of the cervix in Alberta have fallen for women aged 35 and over since the introduction of screening in the early 1960s, as predicted by theory and described in Finland. However, for women aged 15 to 34 years of age the predicted pattern was followed only initially: the registration rate for in-situ and probably also invasive cancer increased after 1973. This could be due to an actual increase in the incidence of in-situ cancer of the cervix among younger women, as might be expected from the epidemiologic aspects of the disease, but it might also be due to increased recruitment of younger women to the screening program.     

The metabolism of benzyl isothiocyanate and its cysteine conjugate.

1. The corresponding cysteine conjugate was formed when the GSH (reduced glutathione) or cysteinylglycine conjugates of benzyl isothiocyanate were incubated with rat liver or kidney homogenates. When the cysteine conjugate of benzyl isothiocyanate was similarly incubated in the presence of acetyl-CoA, the corresponding N-acetylcysteine conjugate (mercapturic acid) was formed. 2. The non-enzymic reaction of GSH with benzyl isothiocyanate was rapid and was catalysed by rat liver cytosol. 3. The mercapturic acid was excreted in the urine of rats dosed with benzyl isothiocyanate or its GSH, cysteinyl-glycine or cysteine conjugate, and was isolated as the dicyclohexylamine salt. 4. An oral dose of the cysteine conjugate of [14C]benzyl isothiocyanate was rapidly absorbed and excreted by rats and dogs. After 3 days, rats had excreted a mean of 92.4 and 5.6% of the dose in the urine and faeces respectively, and dogs had excreted a mean of 86.3 and 13.2% respectively. 5. After an oral dose of the cystein conjugate of [C]benzyl isothiocyanate, the major 14C-labelled metabolite in rat urine was the corresponding mercapturic acid (62% of the dose), whereas in dog urine it was hippuric acid (40% of the dose). 5. Mercapturic acid biosynthesis may be an important route of metabolism of certain isothiocyanates in some mammalian species.     

Constancy of growth on simple and complex media.

An apparatus has been developed in which bacterial growth can be measured very precisely over short intervals of time. Its precision is presented and used to assess the constancy of growth in batch culture. Under certain conditions, i.e., Luria broth or 0.2% glucose-M9 medium at very low cell densities, the specific growth rate of Escherichia coli appeared to be constant within the measurement limits of the method. In succinate minimal medium, the growth rate increased gradually over several days and never became constant. With nutrient broth and with Luria broth, growth slowed progressively at moderate cell densities within the range considered to be in the logarithmic phase of growth. In addition, temporary slowdown in growth rate occurred in these two complex media at characteristic cell densities. These gradual increases in succinate minimal medium and temporary slowdowns in the complex media would be undetectable without precise measurements and may have been a source of variability in many bacteriological studies.     

The aminoacyl-tRNA synthetase-tRNA complex: detection by differential labelling of lysine residues involved in complex formation

The interaction of tRNA^Tyr with tyrosyl-tRNA synthetase from Bacillus stearothermophilus was studied by differential acetylation of lysine residues. The synthetase was trace-labelled in the free form and as the synthetase-tRNA^Tyr complex with [³H]acetic anhydride. In a second step the two ³H-labelled enzyme preparations were fully acetylated with cold reagent under denaturing conditions and were mixed with synthetase that had been homogeneously labelled with excess [¹⁴C]acetic anhydride. Peptides containing labelled lysine residues were isolated after chymotryptic digestion and their ${}^{14}\text{C}{}^3\text{H}$ ratios were determined. These ratios reflect the reactivity of primary amino groups towards acetic anhydride.Involvement of lysine side-chains in complex formation with tRNA^Tyr was suggested from altered ${}^{14}\text{C}{}^3\text{H}$ ratios. Out of the 22 primary amino groups of tyrosyl-tRNA synthetase at least three showed reduced reactivities towards acetic anhydride in the synthetase-tRNA^Tyr complex by factors of 1.6, 1.9 and 6.8, respectively. The sequences around these lysine residues have been determined enabling their placement when the primary and tertiary structure of the enzyme are available (G. L. E. Koch, to be published). No lysine residue of increased reactivity in the synthetase-tRNA^Tyr complex has been detected.Only one molecule of tRNA^Tyr binds to the dimeric synthetase molecule under the conditions of the differential labelling. If the binding site for the tRNA is on one of the two identical subunits, any observed decrease in chemical reactivity of a particular lysine residue should not exceed a factor of two. The detection of a lysine residue which reacts about seven times more slowly in the synthetase-tRNA complex could therefore indicate that the single binding site is formed by both enzyme subunits.