Demonstration of cytoplasmic receptors for the molting hormones in crayfish

Summary From in vitro experiments using different binding assays it is in crayfish demonstrated that the cytosol of target tissues is able to bind both ecdysone and ecdysterone. The ability to bind ecdysteroids is destroyed by heating and by treatment with -chymotrypsin and N-ethyl-maleinimide (NEM) (Figs. 4, 5). In target tissues there is a strong positive correlation between protein content and binding (Fig. 6). The association of the hormone-protein-complex is rapid, taking only a few min even at 5° C (Fig. 3). The binding of the two hormones to the cytosol is both specific and saturable. The association constants for the cytoplasmic receptors from hypodermis, hindgut and midgut gland are in the range of 3–6×10⁷ M^–1 for ecdysone and 5–7×10⁸ M^–1 for ecdysterone (Fig. 8). The data suggest the existence of cytoplasmic ecdysteroid receptors.     

The effect of cycloheximide on Euglena gracilis phenylalanyl-tRNA synthetases

The effect of cycloheximide on the chloroplastic, cytoplasmic and mitochondrial phenylalanyltransferRNA synthetases of Euglena gracilis was studied by growing both logarithmic and stationary phase cultures in the presence of the antibiotic. Enzyme activity was measured relative to untreated control cultures. At very low concentrations of cycloheximide (1 g/ml), all three log phase enzymes showed an increase in activity of 40–50%. At slightly higher concentrations (2.5 g/ml), the phenylalanyl-tRNA synthetase activities were comparable to those of the control cultures. At a cycloheximide concentration of 5g/ml the enzyme activities from stationary phase cultures showed only very slight decreases (5–20%). The cytoplasmic and mitochondrial enzymes behaved similarly in log phase cultures at this concentration. However, the chloroplastic phenylalanyl-tRNA synthetase from log phase cultures treated with 5g/ml cycloheximide showed a marked decrease in activity (70%). A further increase in antibiotic concentration to 10g/ml resulted in significant losses of activity of all three enzymes, from both growth stages. The implications of the data with regard to identification of the site(s) of chloroplast enzyme synthesis are discussed.     

Effects of yohimbine and naltrexone in counteraction of the morphine straub tail and the amphetamine-potentiated morphine straub tail in mice

The alpha-adrenergic blocking agent, yohimbine, prevented the production of the morphine Straub tail reaction in mice, although on a mg dose basis it was only about $\text{1}\text{400}$ as potent as the narcotic antagonist naltrexone by subcutaneous injection. Likewise, yohimbine prevented the potentiation of the morphine Straub tail reaction by amphetamine, being about $\text{1}\text{170}$ as active as naltrexone. Preliminary studies with another alpha-adrenergic blocking agent, phentolamine, indicated that it also inhibited the production of the Straub tail reaction by morphine, although it appeared to be somewhat weaker than yohimbine in this respect. These results suggest the involvement of alpha-adrenergic mechanisms in the production of the morphine Straub tail reaction and in the potentiation of the morphine Straub tail reaction by amphetamine.     

A mutant of CHO-K1 cells deficient in two nonsequential steps of de novo purine biosynthesis

An unusual new purine-requiring mutant, Ade^?P_AB, of Chinese hamster ovary cells (CHO-K1) is described. Ade^?P_AB will grow in medium supplemented with hypoxanthine, adenine, or aminoimidazole carboxamide. Ade^?P_AB fails to show genetic complementation with either Ade^?A, defective in amidophosphoribosyltransferase (E.C. 2.4.2.14), or Ade^?B, defective in phosphoribosylformylglycinamidine FGAM) synthetase (E.C. 6.3.5.3.), but will complement all five of our other hypoxanthine-requiring Ade^? complementation groups. Analysis of purine synthesis in wild-type, mutant, and revertant cells and analysis of relevant enzyme activities in cell-free extracts prepared from these cells demonstrates that Ade^?P_AB is similar to Ade^?B in that it has lost FGAM synthetase activity, and is similar to Ade^?A in that it has lost glutamine-dependent amidophosphoribosyltransferase activity. Unlike Ade^?A, however, Ade^?P_AB retains the ability to synthesize phosphoribosylamine (PRA), the product of the amidophosphoribosyltransferase reaction, if NH₄Cl is substituted for glutamine as the nitrogen donor. Moreover, partial revertants of Ade^?P_AB can apparently synthesize sufficient purines for growth using the NH₄Cl-dependent reaction. The available evidence indicates that neither a double mutation nor a deletion is probable in Ade^?P_AB. We discuss the relevance of these observations for our understanding of both the regulation of purine biosynthesis in mammalian cells and the structural organization of the enzymes defective in Ade^?P_AB and the genes coding for these enzymes.     

T cells in a suppressor circuit and non-T: non-B cells bear different 1-J determinants

T cells involved in the generation of suppressor activity bear an I-J-subregion controlled determinant (e. g., J₁) which is distinct from that (e. g., J₁) found on non-T: non-13 accessory cells. T-cell subsets examined include Ly-1 inducer and Ly-1,2 acceptor cells which collaborate to generate suppressor activity in the in vitro sheep red blood cell antibody system. Non-T:non-B accessory cells examined include accessory cells involved in concanavalin-A induced, T-cell proliferative responses and in in vitro antibody responses to sheep red blood cells. These results provide evidence for serologic and genetic complexity of the I-J subregion of the murine H-2 gene complex.     

Inhibition of platelet thromboxane synthetase by L-1-tosylamido-2-phenylethyl chloromethyl ketone

L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) was found to inhibit several aspects of arachidonic acid (20:4) metabolism in human platelets; the primary effect being inhibition of thromboxane synthetase. Thromboxane B₂ (TxB₂) formation from exogenous 20:4 or PGH₂, or from endogenous 20:4, was inhibited by TPCK at concentrations between 0.1 and 0.5 mM. Formation of malondialdehyde (MDA) and 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT), products which also arise from PGH₂, was inhibited to a similar extent. Inhibition of formation from 20:4 of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), the product of the lipoxygenase pathway, was observed; although the extent of this inhibition was less than that of TxB₂ formation. A small inhibitory effect of TPCK on the release of 20:4 from platelet phospholipids was also observed. This evidence indicated that while a number of reactions are inhibited by TPCK, the primary effect appears to be inhibition of thromboxane synthetase.     

Environmentally-induced changes in the neutral lipids and intracellular vesicles of Saccharomyces cerevisiae and Kluyveromyces fragilis

Saccharomyces cerevisiae, grown aerobically or anaerobically under conditions which induce a requirement for a sterol and an unsaturated fatty acid, synthesized approximately the same amounts of neutral lipid and intracellular low-density vesicles, although the neutral lipids in aerobically-grown cells contained more esterified sterol and less triacylglycerol than those in anaerobically-grown cells. Kluyveromyces fragilis synthesized much less neutral lipid and a smaller quantity of low-density vesicles than S. cerevisiae whether grown at 30°C (generation time 1.1 h) or 20°C (generation time 2.1 h). Both yeasts synthesized highly saturated triacylglycerols, relatively unsaturated phospholipids, and esterified sterols with an intermediate degree of unsaturation irrespective of the conditions under which they were grown. Free sterols in the yeasts were rich in ergosterol and 22(24)-dehydroergosterol, while the esterified sterol fractions were richer in zymosterol.     

Control of the production of orthophosphate repressible extracellular enzymes in Neurospora crassa

Summary N-1, a plasmid isolated from a strain ofShigella flexneri in Japan more than 10 years ago, mediates the phage inhibition phenotype which has recently been found to be characteristic of plasmids of the H₂ incompatibility group. Using the criteria of phage inhibition, surface exclusion and incompatibility, the N-1 plasmid is shown to be closely related to H₂ plasmids isolated from non-typhoid salmonella and distantly related to H₁ plasmids isolated fromSalmonella typhi. Plasmids of other incompatibility groups did not show the H₂ type of phage inhibition.     

Opportunities for natural selection on DNA and protein at the Adh locus in Drosophila melanogaster

A study was made of environmental and genetic factors affecting the quantity and disposition of the alcohol dehydrogenase (ADH) protein in Drosophila melanogaster. It was found that the amount of enzyme per fly is greatly influenced by the environmental conditions in which it develops. A critical factor is the concentration of yeast in the medium. A high concentration of yeast can double the quantity of ADH. The yeast appears to act through the provision of protein, and the protein to act through the provision of threonine, which is already known to induce ADH in fungi. Various genetic factors affect the quantity of enzyme. Males have more ADH than females. Files homozygous for the Fast allele have more ADH than those homozygous for the slow allele, and the difference is greater in females than in males. One particular line (ve), homozygous for Slow, has approximately half the normal quantity of enzyme, and the quantity segregates with the electrophoretic allele. Lines differ in the relative amounts of ADH in the gut (including Malpighian tubules) and the fat body. In general it seems that slow lines have relatively more enzyme in the fat body. In a cross between ve and a line homozygous to Fast, the difference in tissue distribution segregated with the electrophoretic allele. It is argued, but not demonstrated, that the differences in quantity and tissue distribution are due to nucleotide substitutions in noncoding regions close to, or within, the structural gene. It seems likely that the observed environmental and genetic differences in the quantity and disposition of ADH will influence the relative selective values of the electrophoretic genotypes.     

An improved medium for the detection of Aspergillus flavus and A. parasiticus

An effective selective medium for the enumeration of Aspergillus flavus and Aspergillus parasiticus has been developed by modification of Bothast and Fennell's Aspergillus Differential Medium. Results can be obtained with the new medium, Aspergillus flavus and parasiticus Agar (AFPA), after 42 h incubation at 30°C. The medium is thus suitable for use in quality control as a guide to the presence of A. flavus and, potentially, of aflatoxins. AFPA has been extensively tested on peanuts and soils. Results were reproducible and comparable with those on standard fungal enumeration media incubated for much longer periods. A very low percentage of false positives or negatives was found.