Antimicrobial defense mechanisms in the horseshoe crab, Limulus polyphemus: preliminary observations with heat-derived extracts of Limulus amoebocyte lysate.

Heat-derived (60°C) extracts of Limulus amoebocyte lysate (LAL) were found to contain potent “broad-spectrum” antimicrobial activity. Additional heating of the LAL extracts to 100°C for 30 min completely inactivated the antimicrobial activity and served as a control. Antimicrobial activity was observed over a temperature range of 0° to 37°C (higher temperatures not tested) with greatest activity at 37°C. Antimicrobial activity of LAL extracts was variable when tested against Gram-negative bacteria of the family Enterobacteriaceae. A twofold concentration of the extracts resulted in a significant decrease in antimicrobial effectiveness. Dialysis of single- and double-strength LAL extracts against deionized water produced a marked and significant enhancement of antimicrobial activity against both resistant and sensitive species, confirming the presence of a dialyzable inhibitor(s). Dialyzed LAL extracts were active against 13 of 14 species of Enterobacteriaceae tested. Two strains of Pseudomonas aeruginosa were susceptible as were two of three Gram-positive cocci tested. Highly sensitive bacterial species were rapidly killed with a greater than 90% reduction in viable counts occurring within the first 30 min of reaction time. Dialyzed LAL extracts also possessed considerable antifungal activity. The role of the Limulus polyphemus amoebocyte in defense against microbial invasion and dissemination is discussed.     

Soluble polyvinylpyrrolidine and bovine serum albumin adsorb polyphenols from soybean suspension cultures

Soybean (Glycine max [L.] Merrill, cv. Dare) suspension cultures grown in Gamborg B5 medium became discolored and the cells began aggregating after 1 week in culture, especially in the absence of 2,4-dichlorophenoxyacetic acid (2,4-D). The addition of either soluble polyvinylpyrrolidine (PVP) or bovine serum albumin (BSA) to cultures grown in Gamborg B5 medium with 2,4-D prevents discoloration and cell aggregation by adsorbing excess polyphenols from the cells. Transfer of the PVP-treated cultures to fresh medium without 2,4-D stimulated the recurrence of excess polyphenols. Cultures pretreated with BSA did not develop excess polyphenols when transferred to fresh 2,4-D-free medium. Addition of either PVP or BSA to cultures grown in the absence of 2,4-D was found to inhibit growth.     

Tissue specific nuclear antigens in the germinal vesicle ofXenopus laevis oocytes

Summary A library of hybridoma cell lines has been established which produce monoclonal antibodies against antigens from the germinal vesicle ofXenopus laevis oocytes. Many of the antigens are also found in the nuclei ofXenopus embryonic cells in culture. The fate of two of these antigens during embryogenesis was traced by immunofluorescence on embryo and tadpole sections. Early in development these antigens appear to be evenly distributed in the nuclei of all cells. In later stages they gradually disappear from most embryonic structures but are strongly accumulated in the nuclei of some specific cell types and organs.     

Relaxed circular SV40 DNA as cleavage intermediate of two restriction endonucleases.

We have determined the mode of cleavage of superhelical SV40 DNA (Form I) by restriction endonucleases EcoRI and HpaII at 37 degrees C. By analysis with agarose gel electrophoresis and direct examination with dark field electron microscopy, we found that a large amount of the single-nicked circular DNA (Form II) was produced before the linear SV40 DNA (Form III) appeared. Thus, both restriction enzymes cleave only one strand of the superhelical DNA first. The second cleavage on the complementary strand occurred after a lag period. The first order rate constant for the second cleavage by EcoRI endonuclease was determined and a kinetic reaction scheme for both enzymes is proposed.     

Irreversible zinc ion inhibition of (Na+ -K+)-adenosinetriphosphatase, Na+ -phosphorylation, and K+-p-nitrophenylphosphatase of Electrophorus electricus electroplax.

Zinc ion in micromolar concentrations is an irreversible inhibitor of Electrophorus electricus electroplax microsomal (Na⁺-K⁺)-ATPase. The rate of inhibition is dependent on [ZnCl₂] and the extent of inhibition varies with the ratio of ZnCl₂ to microsomal protein. The same kinetics are observed for inhibition of K⁺ -p-nitrophenylphosphatase and steady-state levels of Na⁺ -dependent enzyme phosphorylation. The observations suggest that a Zn²⁺ -sensitive conformational restraint is important to both kinase and phosphatase activities. The fact that inhibition is irreversible has implications for models seeking to relate zinc effects in tissue to inhibition of (Na⁺-K⁺)-ATPase.     

The reversion of trp frameshift mutations in mut,polA, lig and dnaE mutant strains of Escherichia coli

Mutator mutations mutL25, mutR34, and mutU4 had similar effects on the reversion of 4 trp frameshift mutations of known sequence. The mutation trpE9777, which resulted from the addition of an A–T base-pair to a run of 5 A–T base-pairs, was most strongly reverted by the 4 mutators. Reversion of trpE9777 was also increased by mutation polA1 (DNA polymerase I) and dnaE486 and dnaE511 (DNA polymerase III). No effect was found with the ligase mutations, lig-4 or lig-ts7. Mutations polAex1 and polA107, both deficient in the 5′ → 3′ exonuclease activity of DNA polymerase I, had different mutator effects; the factor increase in reversion of trpE9777 was 28-fold for polAex1, 6-fold for polA107, and 21-fold for polA1. The trpE9777 mutation is a useful indicator of frameshift mutator activity.     

Differential effects of deuterium oxide on the growth and morphogenesis of Trichoderma viride

Trichoderma viride can be grown in deuterium oxide (D₂O) concentrationsas high as 99.7%. Increasing concentrations of D₂O (25–90%)progressively extend the lag phase in growth, but do not greatlyaffect the linear growth rate itself. The minimum age and sizeof colony required for photoinduction of the conidiation processis also increased. In 95% D₂O, although growth rates are stillrelatively high, both photomorphogenesis and dark conidiationare completely blocked. This block does not appear to involvethe photoprocess itself, but rather post photoinductive processessuch as differentiation of conidiophores. These observations point to an alternative, or at least additional,hypothesis to the one frequently cited—that D₂O acts throughstabilizing or "temperature lowering mechanisms. (Received January 6, 1978; )     

Production of Epoxides from alpha,beta-Halohydrins by Flavobacterium sp

The relative activity of Flavobacterium whole cells on the enzymatic synthesis of epoxides from alpha,beta-chlorohydrins, -bromohydrins, and -iodohydrins is described.