Conversion of versiconal acetate to versiconal and versicolorin C in extracts from Aspergillus parasiticus

The primary product of hydrolysis of versiconal acetate catalyzed by porcine liver esterase and the 35–70% ammonium sulfate fraction from a soluble extract from mycelia of Aspergillus parasiticus was versiconal. Versiconal was stable at neutral pH for several hours and was rapidly converted to versi-colorin C by treatment with 0.4 M HCl. The addition of NADPH to the 35–70% ammonium sulfate fraction resulted in conversion of versiconal acetate to both versiconal and versicolorin C. The conversion of versiconal acetate to versicolorin C in the cell-free system is proposed to involve an esterase and an NADPH-dependent cyclase.     

In vitro effect of actinomycin D on human neutrophil function

The effect of actinomycin D (ACT-D) on human neutrophil chemotaxis, chemiluminescence (CL), superoxide (O2-) production, phagocytic uptake, and intracellular bacterial killing has been examined. The viability of the ACT-D-treated neutrophils was 98% even at a concentration of 10 micrograms/ml for 4 hr. Using fMLP as the chemotactic factor, depressed chemotaxis was demonstrated following ACT-D (1-10 micrograms/ml) pretreatment of neutrophils as compared with the non-treated controls. Similar ACT-D pretreatment produced the depressed responses in phorbol myristate acetate-induced CL and superoxide production by neutrophils. Moreover, using heat-inactivated human serum as an opsonin for Salmonella enteritidis (NCTC 6676), there was a significant difference in intracellular killing (P less than 0.01) but no difference in phagocytic uptake between ACT-D-treated and non-treated neutrophils. These studies indicate that ACT-D profoundly impairs both intracellular bacterial killing by human neutrophil through an effect on respiratory burst activity and directed cell migration of human neutrophils.     

Extraction of amino acids by aqueous two-phase partition

Summary Amino acids, including lysine, glutamic acid, and phenylalanine, in pure solution or in fermentation broth, were extracted with the aqueous two-phase system consisting of polyethylene glycol and salts, giving a very sharp separation. The partition is influenced by the type and the amount of salts used, pH and components of the broth.     

RNA11 protein is associated with the yeast spliceosome and is localized in the periphery of the cell nucleus.

The yeast rna mutations (rna2 through rna10/11) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rna11 extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNA11-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNA11-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA11 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus.     

Role of the N-terminal region of the A chain in beta 1-bungarotoxin from the venom of Bungarus multicinctus (Taiwan-banded krait)

The N-terminal -amino groups of ₁-bungarotoxin (₁-Bgt) fromBungarus multicinctus venom were modified with trinitrobenzene sulfonic acid and the modified derivative was separated by high performance liquid chromatography. The trinitrophenylated (TNP) derivative contained two TNP groups at the -amino groups of A chain and B chain and showed a marked decrease in enzymatic activity. Methionine residues at positions 6 and 8 of the A chain were oxidized with chloramine T or cleaved with cyanogen bromide to remove the N-terminal octapeptide. Oxidation of methionine residues and removal of the N-terminal octapeptide caused a precipitous decrease in enzymatic activity, whereas antigenicity remained unchanged. The presence of dihexanoyllecithin influenced the interaction between ₁-Bgt and 8-antilinonaphthalene sulfonate (ANS) and revealed that ₁-Bgt consists of two types of ANS-binding sites, one at the substrate binding site of the A chain and the other might be at the B chain. The modified derivatives still retained their affinity for Ca²⁺ and ANS, indicating that the N-terminal region is not involved in Ca²⁺ and substrate binding. A fluorescence study revealed that the -amino group of the A chain was in the vicinity of substrate binding site and that the TNP -amino groups were in proximity to Trp-19 of the A chain. In addition, the study showed that the N-terminal region is important for stabilizing the architectural environment of Trp-19. The results, together with the proposal that Trp-19 of the A chain is involved in substrate binding, suggest that the N-terminal region of the A chain plays a crucial role in maintaining a functional active site for ₁-Bgt.