Type A allatostatins from Drosophila melanogaster and Diplotera puncata activate two Drosophila allatostatin receptors, DAR-1 and DAR-2, expressed in CHO cells.

The type-A allatostatins A (AST-A) are a group of insect peptides with a common C-terminal motif Y/FXFGL-NH(2). The existence of at least four putative type A Drosophila melanogaster ASTs (called type A drostatins or DST-As) has been predicted from the sequence of a recently cloned DST-A preprohormone [C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 273, 126-1131]. SRPYSFGL-NH(2), (DST-3A), the only DST isolated from Drosophila so far, activated the first cloned DST-A GPCR (DAR-1) [N. Birgül et al. (1999) EMBO J. 18, 5892-5900]. A newly cloned orphan Dm GPCR, which shares 47% overall and 60% transmembrane region sequence identity with DAR-1, was classified as a second putative Dm DST-A receptor (DAR-2) [C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 273, 571-577]. Although activation of DAR-2 by DSTs has been postulated, no experimental evidence for that has been presented to date. In this study, we expressed both DAR-1 and DAR-2 in CHO cells and used a GTPgammaS and a Ca(2+) mobilization assay for pharmacological evaluation of the receptors. Synthetically prepared DST-As, as well as selected Diplotera punctata (cockroach) ASTs, activated DAR-1 and DAR-2 in both functional assays indicating ligand redundancy and cross species activity. Cell pretreatment with pertussis toxin led to some differences in the nature and magnitude of signaling pathways at the DAR-1 and DAR-2 receptors, suggesting possible differential coupling to cellular effector system(s) and distinct biological functions of each receptor in vivo.

Isolation of beta-lactamase from a penicillin-susceptible strain of Staphylococcus aureus.

It is generally accepted that strains of Staphylococcus aureus which are susceptible to penicillin G do not produce beta-lactamase. However, we have found that such a strain susceptible to 0.06 mug of penicillin per ml and 0.56 mug of methicillin per ml produces beta-lactamase(s) which hydrolyzes penicillin G, methicillin, 6-aminopenicillanic acid, and probably cephaloridine. The enzyme which is found only during very early log phase of the growth cycle is not inducible either by penicillin or methicillin and is cell bound and liberated only by disruption of the cell. The rate of enzymatic hydrolysis of methicillin was 60% that of benzylpenicillin. This finding suggests that the elaboration per se of beta-lactamase does not necessarily afford resistance to penicillin in this gram-positive-producing cell.

Endogenous, spontaneous formation of beta-lactamase in Staphylococcus aureus.

In a beta-lactamase-inducible strain of Staphylococcus aureus, the enzyme appears spontaneously in the absence of added inducer during lag and early log phases of growth and then declines rapidly to low levels. The endogenous inducer responsible for appearance of the enzyme has been isolated and purified and characterized as a peptidoglycan, containing muramic acid, glucosamine, glutamic acid, alanine, lysine, and glycine. The inducing compound could be isolated from the cells only during the lag and early log phases and from no other later periods. The data obtained are consistent with the thesis advanced earlier from this laboratory that beta-lactamase serves a cellular function in the producing cell more important and beyond its capability of hydrolyzing certain penicillins to the antibiotically inactive penicilloic acids.

Derepression of beta-lactamase (penicillinase in Bacillus cereus by peptidoglycans.

In Bacillus cereus 569 a cellular inducer of beta-lactamase was isolated which has the same constituents and basic structure as the soluble peptidoglycan found in sporulation, extracts from spores, and germination extracts, and which was previously called "spore-peptide." The material has been extensively purified and characterized. Two acid-soluble, high-molecular-weight peptidoglycan fractions containing muramic acid, glucosamine, diaminopimelic acid, d-aspartate, and d- and l-alanine, -lysine, -glycine, and -glutamate, distinguishable on the basis of size and different amino acid to amino sugar ratios, have been found to be responsible for the observed induction. Both fractions are capable of inducing high levels of beta-lactamase in concentrations lower than those of benzyl penicillin required for optimal induction. Several experiments also suggest that it is the accumulation of such soluble peptidoglycan in penicillin-treated cells which leads to induction of beta-lactamase and not the penicillin itself. The "spore-peptide" inducer becomes available during sporulation, and endogenous derepression of beta-lactamase activity occurs simultaneously. Such derepression also occurs in a strain of B. cereus very sensitive to penicillin and in which both uninduced as well as "spore-peptide"-induced beta-lactamase is a small fraction of that produced by the typical penicillinase producer. These results suggest that beta-lactamase in B. cereus functions in cell wall metabolism during sporulation.